Enzymes of serine metabolism in normal, developing and neoplastic rat tissues

ENZYMES OF SERINE METABOLISM IN N O R M A L , DEVELOPING AND NEOPLASTIC RAT TISSUES KEITH SNELL Department of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH, England INTRODUCTION

The purpose of the present review is to prompt a reappraisal of the involvement of serine in cellular metabolism. In particular, studies to elucidate the changing pattern of serine metabolism in rat liver during organ differentiation and to elucidate some aspects of the reorientation of serine metabolism that may accompany neoplastic transformation will be described. For the most part the work that will be discussed has involved the measurement of maximal activities of key enzymes of the various pathways under consideration and the assumption is made that changes in the activities of such enzymes reflect, at least qualitatively, alterations in metabolic pathway flux. Clearly, proof of this ultimately rests on the measurement of actual pathway flux rates in intact cellular systems. Nevertheless the use of enzyme activities as a guide to maximum pathway flux is a valuable approach to the study of metabolism and one that has been applied with success and at various levels of sophistication in a number of laboratories (1-6). In this context it is necessary to know the regulatory properties of the particular enzymes under study, not only for the validation of optimal enzyme assays but also to aid further interpretation of the physiological significance of the findings. This review will therefore draw attention to such regulatory features as appropriate. A scheme of the major pathways of serine metabolism in mammalian liver is shown in Figure 1. The scheme emphasises the de novo biosynthesis of serine from carbohydrate precursors and its utilization by way of 3 major routes of dissimilation. The utilization of serine for protein synthesis, for phospho- and sphingolipid biosynthesis, and for cystathionine/cysteine biosynthesis has not been included. Since serine is a non-essential (or dispensable) amino acid in the nutritional sense, it follows that pathway(s) for its synthesis from endogenous precursors must be active. Arnstein and Neuberger (7) attempted to estimate the rates of biosynthesis of both serine and glycine in tile rat in vivo from isotope dilution studies following the feeding of [3-14C]serine or [2-14C]glycine. While certain assumptions regarding the mixing of tissue pools and regarding isotope exchange reactions are unlikely to be completely 325

326

KEITH SNELL

~olytic enzymes 13-I'HOSPHOGLTCERATE[ ~ pho~phoglyceratedehydrogenase IPtlOSPI~OHYDROX'gPYRLWATE1 pkosphoseri~caminotrznsferase I PHOSPHO~ER]NE J pho~phescrinephosphatase SERINE

serine~-dehydrataae lipogenic gluconeogenic er~ymo~ezymes

r

serine aminot

~

[HYDROXYPYRUVATE J D-glycerate dehydrogenase

~erine ethyltransferase +

*INOC~ICACm~I

D-GLYCERATE] I D glyceratekiaa~e [.-PHOSPHOGLYCEI &TE]

ghmoneogenic enzyme~

FIG. 1. Major pathways of hepatic serine metabolism. See Table 1 for maximal activities in rat liver of the enzymes involved.

valid, the calculated figures are probably of the right order of magnitude and indicate a substantial rate of serine synthesis in the rat of about 370 rag/100 g body wt./day, In vivo studies with the lactating cow (8) and the rat (9) showed that ~4Clabeled glucose was readily incorporated into serine. Analysis of labeling patterns in serine after administration of labeled glycerol, glycerate or pyruvate to rats suggested that serine must be derived from a 3-carbon precursor that precedes pyruvate in the glycolytic sequence (10-12). Further elaboration of the intermediates involved suggested 2 possible pathways for serine biosynthesis: that involving D-glycerate, hydroxypyruvate and serine, referred to as the 'non-phosphorylated pathway' (13); and that involving D-3phosphoglycerate, phosphohydroxypyruvate, L-phosphoserine and serine, referred to as the 'phosphorylated pathway' (14). In 1969 it was independently proposed by Sallach and colleagues (15), Rowsell and colleagues (16), and Lardy and colleagues (17) that, rather than these 2 pathways representing alternatives for serine biosynthesis, the 'non-phosphorylated pathway' might subserve, in its reverse direction, a role in gluconeogenesis from serine, with the 'phosphorylated pathway' having a primary role in serine biosynthesis. This indeed is the scheme represented in Figure 1, and the main lines of

327

REGULATION OF SERINE METABOLISM

evidence on which this is based are: (I) only the 'non-phosphorylated pathway' is able to function in the reverse gluconeogenic direction, because no kinase for the conversion of free serine to phosphoserine has been demonstrated in mammalian tissues [in contrast, of course, to the enzymic phosphorylation of protein-bound serine which has seemingly reached endemic proportions (18)!]; (2) there is evidence for gluconeogenesis from serine via the "non-phosphorylated pathway'; (3) the adaptive responses of enzymes involved in the 'non-phosphorylated pathway' to hormonal and dietary influences would implicate this pathway in a gluconeogenic role, whereas the responses of enzymes in the 'phosphorylated pathway' are more appropriate to a role in serine biosynthesis; (4) feedback regulation by serine operates on the 'phosphorylated pathway' but not on the 'nonphosphorylated pathway'. This evidence is further elaborated in the discussion below. The 3 alternative pathways of serine utilization depicted in Figure 1 are: that initiated by L-serine dehydratase leading to pyruvate formation for glucoor glyconeogenesis, for energy generation via oxidation in the 'citric acid cycle', or for lipogenesis; that initiated by L-serine-pyruvate aminotransferase leading to hydroxypyruvate formation for gluconeogenesis; and that initiated by L-serine hydroxymethyltransferase which generates glycine and 5,10TABLE 1, MAXIMAL ACTIVITIES OF ENZYMES OF SERINE METABOLISM IN ADULT RAT LIVER HOMOGENATES

Enzyme Serine biosynthesis o-3-Phosphoglycerat e dehydrogenase

EC number

Enzyme activity (~zmol/min/g of liver)

Apparent Km (raM)

Ref.

I. 1.1.95

0.05

19

L-Phosphoserine aminotransferase

2.6.1.52

0,27

20

L-Phosphoserine phosphatase

3.1.3.3

0.46

21

4,2.1.13

3.2*; 5.9t

(64-72)

22 (22a)

2.6.1.51

0.20

(16)

22 (21a)

D-Glycerate debydrogenase

1.1.1.29

0.23

I9

D-Glycerat e ki nase

2, 7.1,31

0.30

23

L-Serine hydroxymethyltransferase

2.1.2. i

2.75

Serine utilization t-Serine dehydratase t-Serine aminotransferasc

(0.54)

20(20a)

*Assayed in August-September. tAssayed in February-March. K~ values recorded are for the purified enzymes with L-serine as variable substrate.

328

KE[TH SNELL

methylene tetrahydrofolate, both of which may be utilised as precursors for de novo nucleotide biosynthesis. The properties and adaptive characteristics of these enzymes of serine utilization and of serine biosynthesis are discussed below. Table 1 lists the maximal activities of the enzymes measured in rat liver, together with the Km values with respect to serine of the enzymes of serine utilization. One particular difficulty in predicting activities in vivo from these data is in knowing the concentration of serine present at the intracellular location of the various enzymes of utilization. Serine dehydratase is predominantly cytosolic, whereas serine aminotransferase is predominantly mitochondrial and serine hydroxymethyltransferase is distributed between the mitochondrial and cytosolic subcellular compartments. There have been no studies to determine if mitochondrial-cytosolic concentration gradients exist for L-serine. Assuming a uniform intracellular distribution of the amino acid, it would appear that under normal physiological situations, where the concentration of serine in rat liver is reported to be 1-2 mM (24-26), the flux through serine hydroxymethyltransferase predominates. However, this interpretation is far from certain, in so far as an unspecified proportion of the cytosolic activity has been reported to be involved in a multi-enzyme complex with other tetrahydrofolate utilising or generating enzymes (27). Thus the activity may be governed more by the availability of the tetrahydrofolate co-factors, as determined by competion within this multi-enzyme complex, than by the availability of serine as a substrate.

MATERIALS

AND METHODS

Biological Systems Neoplastic tissues. Solid rat tumors of different growth rates were maintained by serial subcutaneous transplantation in an inbred isogenic Kx strain (formerly designated NEDH) (submaxillary gland carcinoma CCC-3, monocytic lymphoma RNC-290); a Buffalo strain (Morris hepatomas 7800, 7777, mammary carcinoma 270D, and renal carcinoma MK-1); an ACI strain (Morris hepatoma 3683F); and a Sprague-Dawley strain (Novikoff tumor). Morris hepatomas and the renal carcinoma were obtained originally from Dr. H. P. Morris (Howard University, Washington, D.C.) and were maintained, as were the other tumors, at the Cancer Research Institute of the New England Deaconess Hospital (Boston, Mass.). The Novikoff solid tumor was derived by subcutaneous implantation of the JL-1 and C-3 Novikoff cell lines. The characteristics and growth rates of the various tumors have been previously documented (28, 29). Normal tissues. Albino rats of an inbred isogenic Kx strain (New England Deaconess Hospital Breeding colony) or an albino inbred Wistar strain (University of Surrey Animal Breeding Unit) were used. Developing rats were

REGULATION OF SERINE METABOLISM

329

obtained after timed matings and fetal ages were determined from the weights of the embryos at the time of use within confidence limits of +0.24 day (30). AduIt rats were 60-90 day-old males, and pre-weanling rats were used without regard to sex. Animals were weaned at 23 (New England Deaconess Hospital) or 21 (University of Surrey) days post partum. Hormonal treatments. Hormonal agents were administered intraperitoneally and at doses as previously described (22). Human lymphocyte cultures. Lymphocytes were prepared from fresh, heparinized, human peripheral blood by a Ficoll-Triosil method, cultured in medium RPMI-1640 and subjected to mitogenic stimulation with phytohemagglutinin (PHA) as previously described (31).

Biochemical Studies Tissue fractionation.

Subcellular fractions designated cytosol and particulate refer to the supernatant and resuspended pellet, respectively, derived from liver homogenate in 0.4 ra sucrose after centrifugation for 105,000 × g for 60 rain. Enzyme assays. Assays for serine dehydratase, serine aminotransferase (22), serine hydroxymethyltransferase, phosphoserine aminotransferase (20), and allothreonine aldolase (32) were as detailed elsewhere. In all cases it was established for every biological system that enzymes were assayed under conditions where activities were proportional to amount of enzyme added and to time of incubation over the ranges employed. All enzyme incubations were at 37~C. Enzyme activities are expressed in units (/~mol/min) per g wet weight of tissue unless stated otherwise. Serine dehydratase multiple forms. Methods for the separation and preparation of multiple forms of serine dehydratase were as previously described (22). DNA synthesis in cultured lymphocytes. The measurement of incorporation of radioactivity from e-[3-14C]serine or [6-3H]thymidine into DNA was as previously described (31).

RESULTS

AND DISCUSS/ON

Pathway of Serine Biosynthesis Regulatory properties of enzymes of serine biosynthesis. From the values of enzyme activities recorded in Table 1, it might appear that the rate-limiting enzyme on the pathway ofserine biosynthesis is the first committed step of the pathway, phosphoglyeerate dehydrogenase. Indeed, in bacteria (33, 34) and peas (35), phosphoglycerate dehydrogenase is subject to feedback inhibition by L-serine. However, in animal tissues this is not the case and no inhibition by serine was observed in rat liver (36), chicken liver (37), mouse brain (38) and

330

KEITH SNELL

human KB cells in culture (39). An apparent inhibition of rat brain phosphoglycerate dehydrogenase only occurred at serine concentrations of 20 mM (40), far outside the physiological range. Feedback inhibition of the mammalian pathway by serine appears to be exerted at the level of phosphoserine phosphatase (41, 42), the final and irreversible step of serine biosynthesis. The low ~ value for this non-competitive inhibition by L-serine (0.3 mM; 21, 43, 44) suggests that the enzyme is substantially inhibited at normal liver concentrations of serine (by 80% according to Byrne, (45)) and may well be rate-limiting for the overall pathway. The equilibrium positions of phosphoserine aminotransferase and phosphoglycerate dehydrogenase will favor 3-phosphoglycerate formation when phosphoserine phosphatase is inhibited, thereby ensuring that this is an effective control point for the pathway as a whole. Adaptive changes in the enzymes of the serine biosynthetic pathway in rat and rabbit liver in response to dietary and hormonal influences have been reported 0 5 , 36, 41, 46-50). Phosphoglycerate dehydrogenase activity is sensitively and inversely related to the protein content of the diet in the rat (36, 41, 46, 49, 50) and rabbit (15). Phosphoserine phosphatase shows a similar, though less sensitive, dependence on dietary protein in the rat (36, 41, 46); whereas in the rabbit, although decreased by a high-protein diet, a low-protein diet had little effect on the enzyme (15). Phosphoserine aminotransferase behaves similarly to the phosphatase in the rabbit (15), whereas in the rat a protein-free diet leads to elevation of the aminotransferase and of the phosphatase (50). These observations, together with studies showing a proportionality between phosphoglycerate dehydrogenase activity and pathway flux [as measured by [3-~4C]phosphoglycerate (47) or [6-t4C]gtucose (50) incorporation into serine in liver supernatant extracts] in relation to variations in dietary protein status, have been advanced as evidence for the key regulatory role of phosphoglycerate dehydrogenase in the long-term adaptive changes in the pathway of serine biosynthesis. In this regard the role of the sulfur amino acids (methionine and cysteine) in mediating the effects of dietary protein variation have been emphasised (36, 41, 46, 49) and the changes in hepatic sulfur amino acid content are compatible with this role (46). The effects of dietary protein (and by implication the sulfur amino acids) on phosphoglycerate dehydrogenase activity have been shown to be due to changes in the rate of enzyme protein synthesis (36, 41, 47). Since serine can serve as a precursor for sulfur amino acid biosynthesis, this phenomenon may conceivably constitute an example of feedback repression in animal cells. However, other mechanistic explanations are also tenable, including a translational action related to the availability of certain amino acids in the free amino acid pool (49), or effects of protein a n d / o r amino acids which are secondary to coincident changes in hormone levels or other intracellular metabolites. Whatever the mechanistic interpretation of the observations the

REGULATION OF SERINE METABOLISM

331

physiological appropriateness of reducing the rate ofserine biosynthesis when dietary protein (and amino acid availability) is plentiful, and of increasing the pathway when protein is lacking, is apparent and lends support to the role of these enzymes in a pathway of serine biosynthesis. Also supportive of this interpretation of the role of the enzymes in serine metabolism is their adaptive responses to endocrine changes that promote or reflect catabolic changes in hepatic amino acid metabolism. Again, phosphoglycerate dehydrogenase has been the most intensively studied enzyme and decreased activity was observed after glucocorticoid treatment of normally-fed rats (36, 48) or rats where enzyme activity had been increased by a protein-deficient diet (36, 47, 50). Phosphoserine aminotransferase and phosphoserine phosphatase activities were also reduced by glucocorticoids in rats previously induced by a protein-deficient diet (50) or, for the phosphatase, when naturally elevated in fetal life (51). Glucagon appeared to have no effect on control levels of phosphoglycerate dehydrogenase in normally-fed rats (48), but reduced enzyme activity induced by proteindeficient diets (48, 50). Alloxan diabetes decreased phosphoglycerate dehydrogenase in normally-fed (48) or protein-deficient rats (50), and phosphoserine aminotransferase and phosphoserine phosphatase in proteindeficient rats (50); the changes were reversible by insulin administration. Starvation for 24 or 48 hr also decreased phosphoglycerate dehydrogenase compared to the activity in normally-fed rats (48). In these catabolic situations the hepatic serine concentration is probably decreased (24) and the adaptive decreases in activities of the enzymes ofserine biosynthesis will be necessary at the very least to offset the deinhibition of phosphoserine phosphatase by the fall in serine concentration. Tissue distribution o f enzymes o f serine biosynthesis. Few systematic studies on the tissue distribution of the enzymes of serine biosynthesis have been reported and most of these record activities relative to unit weight of protein in dialysed or Sephadex G25-treated tissue supernatants. By taking into account the relative differences in soluble protein content of different tissues (5), it is possible at least to arrive at a ranking order of tissue enzyme activities per unit weight of tissue (Table 2). The enzymes of serine biosynthesis are widely distributed in rat tissues and there is rough agreement between different laboratories on the relative activities in different tissues. There is also a reasonable correlation between the various enzymes with regard to the rank order of activities in different tissues, with the kidney being the most active of the tissues investigated. Phosphoglycerate dehydrogenase has been reported (but without quantitation) to be active in kidney, brain, liver or intestine, but absent from skeletal muscle or heart (46), in agreement with other workers (50; see Table 2). Although values were not given, the enzyme is also reported to be very active in the pancreas and epididymal fat pad (50). Hayashi et al. (50) measured serine formation from [~"C]glucose in extracts of tissue

332

KEITH SNELL TABLE 2. RANK O R D E R OF ACTIVITIES OF ENZYMES OF SERINE BIOSYNTHESIS IN RAT TISSUES Rank order Tissue (ref.) Kidney Testis Brain Spleen Liver Lung Intestine Skeletal muscle Heart Mammary gland

Phosphoglycerate Phosphoserine dehydrogenase arninotransferase (50)* (50)* Table 3 2 1 3 4 6 7 5 n.m.

1 2 3 4 8 5 6 7 n.rn.

1 n.m. n.m. 4 2 6 3 n.m. 5 n.m.

Phosphoserine phosphatase (50)* (21) 1 6 4 2 3 5 9 7 8 n.m.

1 n.m. 5 3 2 4 n.m. n.m. 6 7

Activities were expressed or *re-calculated in terms of units per g tissue. -, activity absent; n.m., activity not measured.

supernatants supplemented with co-substrates and co-factors, and reported a ranking order (re-calculated per unit weight of tissue) of: kidney, testis, spleen, brain, liver, lung, and negligible activity in intestine, muscle or heart, which is in reasonable agreement with the enzyme data. The relative importance of the kidney in serine biosynthesis, indicated by its formation from [14C]glucose in vitro and the enzyme data (Table 2), is not apparently confirmed by observations indicating negligible serine biosynthesis from [14C]glucose by rat kidney in vivo (52). Pitts et aL (52) presented evidence that the major precursor for serine formation in the kidney is glycine. While confirming that the mitochondrial glycine dehydrogenase complex (also referred to as 'glycine cleavage' system) is the predominant route of glycine metabolism in kidney and is coupled to serine formation, Rowsell et al. (53) make the point that extraction of glycine from the circulation by the kidney and its total conversion to serine could not sufficiently account for the observed serine output in vivo from this organ. The relative quantitative importance of serine biosynthesis in the kidney by the de novo pathway (from glycolytic intermediates) and from glycine remains to be established. Unlike the liver enzymes, it appears that the enzymes of serine biosynthesis in other tissues do not show adaptive responses to variations in dietary protein content (46, 50). However, phosphoserine phosphatase of brain and kidney, like the liver enzyme, is subject to feedback inhibition by 10 mM serine (46). The unique sensitivity of the liver to long-term enzyme adaptation in response to dietary variation is perhaps not unexpected in view of its anatomical relationship to the gastrointestinal tract via the portal circulation. The same phenomenon has been described for other enzymes involved in amino acid metabolism (54).

333

REGULATION OF SERINE METABOLISM

Enzymes of serine biosynthesis in developing rat tissues. The pattern o f neonatal d e v e l o p m e n t o f each o f the enzymes ofserine biosynthesis in rat liver has been reported: p h o s p h o g l y c e r a t e d e h y d r o g e n a s e (19), p h o s p h o s e r i n e aminotransferase (20), and p h o s p h o s e r i n e p h o s p h a t a s e (51). The developmental curve for p h o s p h o s e r i n e aminotransferase established in this Laboratory is s h o w n in Figure 2A, and the other enzymes s h o w a very similar pattern: high activity in fetal life, a sharp fall in the perinatal period, and then a further fall to very l o w adult values (phosphoglycerate dehydrogenase), or to adult values which are 50% [phosphoserine aminotransferase (20)] or 35% [ p h o s p h o s e r i n e p h o s p h a t a s e (21)] o f fetal activity. Together these results suggest an increased capacity for serine biosynthesis from glycolytic intermediates in the fetal-perinatal period, although no direct flux measurements have been made in this regard. In part, this enhanced biosynthetic capacity can be viewed in terms o f an increased provision o f

B

250 300 200 200

150

100

.-o~ °

100

50 "~

0

I B

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I

I0

20

tilt 30 ' A

-5

B

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20

B

10

20

30

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D

i00

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so

~

6o

200

40

I00

20 -5

B '

10 '

20'

~o 3 ~~

-5

Age (days)

FIG. 2. Developmental patterns of key enzymes of serine metabolism in rat liver. (A) Lphosphoserine-2-oxoglutarate aminotransferase; (B) L-serine dehydratase, showing electrophoretograms of crude liver cytosoi fractions at different developmental ages (22) (bands correspond to: serine dehydratase-Form I; serine dehydratase-Form II; and L-homoserine dehydratase-cystathionase activity, in order of increasing electronegativity); (C) total L-serine hydroxymethyltransferase (O)and cytosoli¢ L-serine hydroxymethyltransferase (L-allothreonine aldolase) (o); (D) t-serine-pyruvate aminotransferase. Except where noted, activities were determined in whole homogenates of rat liver (as referenced in Methods), recorded as units per g of liver, and shown here as percentages of adult liver values. Points indicate mean values of 5 to 8 determinations at each age on animals from 3 to 4 different litters. B, birth; A, adult.

334

KEITH SNELL

serine for incorporation into proteins during the rapid phase of fetal liver growth. However, the fact that such growth is essentially hyperplastic in nature suggests that serine may also be synthesized at this time to provide nucleotide precursors for RNA and DNA biosynthesis (see below). Indeed developmental peaks of activity of many of the enzymes involved in the biosynthesis of pyrimidines and purines de novo are also found in the perinatal period (55). In addition, several key glycolytic enzymes are elevated in the fetal period (56), so that the provision ofglycolytic intermediates for hepatic serine biosynthesis should also be enhanced. Developmental patterns of the enzymes of serine biosynthesis in other tissues have not been reported, except for phosphoserine phosphatase in the kidney (51). Here the pattern is strikingly different from the liver, with very low fetal activity that increases rapidly during the Iate fetal period and then continues to rise slowly over the first 40 days of postnatal life to reach the adult level. This pattern perhaps emphasizes that the capacity for serine biosynthesis in the kidney is an adult attribute and one that is related to the functioning of the mature organ. In this regard it would be of interest to investigate if phosphoserine phosphatase, or the other serine biosynthetic enzymes, show adaptive changes in acidotic situations. In fetal brain and lung, phosphoserine phosphatase activity was 45% and 32% respectively of the adult values (51). Activities of phosphoserine aminotransferase in adult and fetal tissues are presented in Table 3. As with phosphoserine phosphatase, fetal kidney activity of the aminotransferase was lower than in the adult (57% of the adult value), but in all other tissues investigated fetal activity exceeded that of the adult. No corresponding studies have been made of phosphoglycerate dehydrogenase, so that our view of serine biosynthetic capacity in tissues other than the liver is as yet incomplete. There is little information on the regulation of the developmental changes in the enzymes of serine biosynthesis. During the time-period over which enzyme activities are declining most rapidly (the perinatal period) there is a decrease in total liver cell number that is largely accounted for by a loss of hematopoietic cells, which constitute a high proportion of the fetal liver cell population (5). However, the decrease in the number of total cells per g of liver (57) would not account, either temporally or in magnitude, for the perinatal decline of the enzymes of serine biosynthesis. Thus, it is unlikely that this is a factor regulating the developmental changes observed, or indeed that the enzyme activities are associated to any great extent with the metabolism of non-parenchymal liver tissue in fetal life. For phosphoserine phosphatase, attempts have been made to identify the stimuli responsible for the prenatal and early postnatal decline in activity by the administration of agents which might prematurely evoke the natural changes (51). Hydrocortisone was effective (but not the administration of serine, thyroxine or glucagon) in accelerating the decline in the phosphatase activity when administered to fetal

OF ENZYMES

0.495 +_0.027( 10) 0.283 +- 0.017(6)

0.081 f 0.014(3) 0.175 +_0.021(3)

0.089 + 0.020(3) 0.128 ? 0.018(3)

0.049 f 0X06(3) 0.182 + 0.020(3)

0.029 + 0.004(3) 0.040 2 0.006(3)

Adult kidney Fetal kidney

Adult spleen Fetal spleen

Adult intestine Fetal intestine

Adult heart Fetal heart

Adult lung Fetal lung 0.04 + O.Ol(3) <0.02(3)

0.12 + 0.03( 3) <0.02(3)

0.06 + 0.03(3) <0.02(3)

<0.02(3) <0.02(3)

0.35 + 0.02(6) 0.03 + 0.01(4)

31.9 + 2.53(8)* 0.14 + 0.02(6)

Serine dehydratase (pmol/min

IN ADULT

RAT

0.16 + 0.03(3)
<0.01(3) <0.01(3)

0.18 If: 0.02(3) 0.49 * 0.04(3)

0.43 rt_0.03(3) 0.31 k 0.03(3)

0.95 + 0.08(6) 0.20 t- 0.06(6)

2.75 + 0.19( 12) 1.41 + 0.04(12)

Serine hydroxymethyltransferase

AND FETAL

0.034 + 0.012(3) <0.01(3)

0.029 + 0.002(3) <0.01(3)

<0.01(3) <0.01(3)

0.036 + 0.008(6) 0.015 + 0.002(S)

0.245 + 0.014(X) 0.080 + 0.006(6)

Serine aminotransferase per g of tissue)

OF SERINE METABOLISM TISSUES

Values are means + s.e.m. with numbers of determinations in parentheses. Animals were an inbred Kx (formerly NEDH) strain; fetal ages were 20.5 days gestation, adult ages were 60-90 days. *There is no ready explanation for the high value measured in this strain of rats in the U.S.A.; other measurements in the same strain and in the same laboratory at an earlier time gavea mean value of 8.63 pmol/min per g (5). Values from the author’s laboratory in the U.K. range from 2-8 pmol/min per g depending on the month of assay (see also, 22).

0.275 + 0.021(10) 0.554 * 0.021(8)

Phosphoserine aminotransferase

3. ACTIVITIES

Adult liver Fetal liver

Tissue

TABLE

336

KEITH SNELL

rats. Since the natural secretion of glucocorticoids is known to increase around day 18 of gestation, this may well be the physiological stimulus for the decrease in the enzyme (51). However, the mechanism of this adaptive decrease in enzyme activity has not been established. In the kidney, the phosphoserine phosphatase activity of adult male or female rats was increased by estradiol administration, but this did not occur in neonatal rats and is therefore unlikely to be involved in the perinatal upsurge of the kidney enzyme (51). Hydrocortisone was not tested for possible effects on the developmental formation of kidney phosphoserine phosphatase activity (51). Enzymes of serine biosynthesis in neoplastic rat tissues. A number of laboratories have investigated the activities of enzymes of serine biosynthesis in neoplasms of the rat. Phosphoglycerate dehydrogenase was assayed in four transplantable hepatomas of the Morris series (7794B, 7793, 9121 and 5123TC) and found to be increased by 1.7- to 10.6-fold compared to the liver of control rats of the same strain (58). The magnitude of the increase was related to the growth rate of the hepatoma. Interestingly, feeding host animals on a low-protein (2%) diet which induces adaptive increases in liver phosphoglycerate dehydrogenase in control animals (and in the tumorbearing host liver) failed to have any effect on the hepatoma enzyme (58). The observation may suggest a loss of mechanisms of enzyme adaptation in the neoplastic liver compared to the tissue of origin. Alternatively, it may simply reflect the independence of the intramuscularly implanted hepatomas from the intimate association with the portal circulation enjoyed by the normal liver tissue. A more extensive survey of neoplastic tissues has been made with respect to phosphoserine phosphatase activity, and included not only transplanted hepatomas but also other transplanted tumors and some primary (dimethylbenz(a)anthraeene-induced) mammary gland tumors (21). Activity in 6 transplantable hepatomas varied from 50% to 280% of that in normal liver and was not related to the growth rate of the tumors. However, in both primary and transplanted mammary gland tumors, activities were 5- to 20fold greater than in virgin mammary tissue, and up to 3-fold greater than in lactating mammary tissue, and activity was related to the growth rate of the tumors (21). Of the remaining tumors studied, enzyme activity in a slowgrowing renal cell carcinoma (Morris series, 9789K) was 76% greater than in normal adult kidney, activity was high in an osteogenic sarcoma compared to its absence in normal adult bone, and significant activity was detected in a rapidly growing fibrosarcoma compared to very low activity in normal adult skeletal muscle (21). In general then phosphoserine phosphatase activity is found to be persistent in all tumors and to be increased, compared to the cognate normal tissue, in many of the tumors. No information has previously been published on the activity of phosphoserine aminotransferase in neoplastic tissues, but some initial studies

0.316 + 0.024(3)

0.304 ± 0.010(5)

0.248 + 0.024(3)

Submaxillary carcinoma CCC-3

Lymphoma RNC-290

Mammary carcinoma 270D <0.02(3)

0.03 + 0.01(6)

0.05 ± 0.01(3)

0.04 + 0.0t(3)

26.0 ± 2.06(6) <0.02(3) <0.02(6) <0.02(3)

<0.01(3)

0.019 + 0.001(6)

<0.01(3)

<0.01(3)

0.031 + 0.002 <0.01(3) <0.01(6) <0.01(3)

Serine Serine dehydratase aminotransferase ( # m o l / m i n per g of tissue)

Values are means _+ s.e.m, with numbers of determinations in parentheses. Hepatomas are listed in order of increasing growth rate. For normal tissue activities see Table 3.

0.400 +_ 0.047(3)

0.041(6) 0.032(3) 0.030(9) 0.036(3)

Renal carcinoma MK-I

+ + + +

0.475 0.218 0.259 0.282

Hepatomas: 7800 7777 3683F Novikoff

Tissue

Phosphoserine aminotransferase

+ 0.08(6) _+ 0.05(3) +_ 0.04(9) _+ 0.21(3)

1.99 ± 0.08(3)

2.56 ± 0.06(5)

1.31 + 0.14(3)

1.72 _+ 0.08(3)

1.16 0.55 2.09 2.42

Serine hydroxymethyltransferase

TABLE 4. ACTIVITIES O F ENZYMES O F SERINE M E T A B O L I S M IN T R A N S P L A N T E D N E O P L A S T I C RAT TISSUES

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KEITH SNELL

by the author are presented in Table 4. Activity in 4 transplantable hepatomas varied from 80% to 173% of that in normal liver and was not related to the growth rate of the tumors. Of the remaining tumors studied, enzyme activity in the renal cell carcinoma was 81% of that in normal adult kidney, and in the other tumors was 80% to 115% of that in normal adult liver. Although some tumors do have elevated activity of phosphoserine aminotransferase, in general the pattern is one of selective retention or persistence in tumors. Of the tumors investigated in these various studies only 2 (hepatomas 7793 and 5123TC) have been the subject of more than one of the studies. Thus, in both these hepatomas phosphoglycerate dehydrogenase is increased compared to normal adult liver (58), whereas phosphoserine phosphatase was 63% and 93% of normal liver activity (21). In the former study enzyme activity was recorded per g of supernatant protein (58) and it is possible that differences in supernatant protein content between normal liver and hepatoma tissue may exaggerate the magnitude of the differences in activity. For 1 of the hepatomas (5123TC) it was shown that the overall rate of serine biosynthesis from [~4C]phosphoglycerate in co-substrate and co-factor supplemented supernatant fractions correlated closely with the activity of phosphoglycerate dehydrogenase, suggesting that the enhanced activity of this enzyme in the 4 hepatomas in the study is responsible for increased serine biosynthetic capacity. This conclusion needs to be treated with caution in view of the above comment on protein content and, in any case, only relates to a very limited number and range of tumors (all hepatomas). Taking a broader view of all 3 studies discussed, it would appear that at the very least neoplastic tissues of various origins have selectively retained the enzymic capacity for serine biosynthesis and in some cases may even have an increased capacity. This conclusion needs to be evaluated in the light of changes in the activities of the various enzymes of serine utilisation and will be discussed further below.

Pathways of Serine Utilization: L-Serine Dehydratase and L-Serine-Pyruvate Aminotransferase. Regulatory properties of serine dehydratase. Serine dehydratase is probably one of the most intensively studied enzymes of amino acid metabolism (along with tyrosine aminotransferase) from a regulatory standpoint. Its physiological role is problematical because of the very high Km with respect to serine: values up to 150 mM have been recorded in this Laboratory with crude liver extracts and values up to 300 mM have been quoted elsewhere (59). The apparent K~ is affected by enzyme dilution, by monovalent cations and by the ionic strength, pH and nature of the incubation buffer. The actual activity in vivo is therefore difficult to predict but is almost certainly at least an order of magnitude lower than that measured as maximal activity in vitro. The enzyme purified from rat liver has been reported to have an apparent Km value of 52-72 mM (22a, 60-63). Evidence from the more recent purification

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procedures has established that L-serine and L-threonine dehydratase activities are properties of the same enzyme molecule (22a, 64) (and indeed the same catalytic site; 62, 63), which does not possess cystathionine synthetase activity (61, 63). L-Serine dehydratase activity is also exhibited by the Lhomoserine dehydratase-cystathionase enzyme, but the relative activity is very low and can be distinguished from "true" serine dehydratase multiple enzyme forms after separation of partially purified preparations by Sephadex G-200 chromatography (65) or after resolution of crude liver extracts by cellulose acetate electrophoresis (22). Homoserine dehydratase and serine dehydratase were also distinguished as separate enzymes in this Laboratory by their different developmental patterns during neonatal rat liver differentiation (66). The possibility that L-serine dehydratase occurs as multiple molecular forms in rat liver cytosol was first suggested by the observation of 2 peaks of activity after running a purified, crystallized enzyme preparation on a starch zonal electrophoresis system (62). Pitot and co-workers purified and characterized 2 forms of the enzyme by buffer gradient elution from a DEAEcellulose column and by polyacrylamide gel electrophoresis (22a, 67) and this was confirmed by Simon et al. (68). The purified enzyme preparations in these studies were from adult rats with activity induced to a high level by feeding a high protein diet (see below). In this Laboratory, crude liver supernatants from neonatal rats in which activity is naturally induced at 2 and 15-20 days post partum also contained 2 enzyme forms after separation by DEAEcellulose column chromatography or cellulose acetate electrophoresis (22) (see also Fig. 3). The properties of the adult multiple enzyme forms after purification have been examined: both enzyme forms are dimers with identical sub-unit relative molecular masses of 34,000-35,000 daltons, giving a native enzyme relative molecular mass of about 65,000 daltons (61, 68); both contain 2 molecules of pyridoxal phosphate co-factor per molecule of native dimer (62, 67); both forms have identical absorption spectra and are immunochemically indistinguishable (22a, 67); the 2 forms have the same optimum pH, kinetic constants for L-serine and L-threonine, and similar heat inactivation properties (22a). Form II of the enzyme is the more electronegative form and contains one additional residue of lysine which may account for this behaviour (22a). Apart from their differing electrophoretic mobilities, the only other distinguishing feature of the 2 molecular forms of the enzyme is the apparent differential regulation in the adult (see below). The characteristics of the multiple forms of the partially purified enzyme from postnatal rat liver have been investigated in this Laboratory (Table 5). The properties examined were virtually identical to those found for the purified multiple forms of the adult enzyme induced by a high protein diet (22a). Again, the characteristics of the two enzyme forms were indistinguishable from one another. AER 22-L

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KEITH SNELL A. t.5

0,5 az

16

15

20

Postaatal

25

age (days)

][3.

5O

I I -~

o, 6

3

/ I

J

g ~

t

20

0.3

,$ L) 10 m

I 48

I 50

I

52

I 54

s 56

I 58

0 60

F r a c t i o n rtumbet-

FIG. 3. Developmental formation of rat liver cytosolic L-serine dehydratase activity at weaning (A), and the resolution of multiple enzyme forms by DEAE-cellulose column chromatography (22) of crude liver cytosol fractions from five 19-day-old rats (B). Enzyme activity was determined as referenced in Methods. For the developmental profile (A), points are the means of values from 4 animals at each age, with bars showing s.e.m. (K. Snell and P. Kwasowski, unpublished observations).

The subcellular localization of L-serine dehydratase is of some relevance in relation to its metabolic function (see later). Work in this Laboratory has established that the enzyme is entirely confined to the cytosol in both adult and neonatal rat liver (113) and this localization has been confirmed by others

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TABLE 5. PROPERTIES OF SERINE DEHYDRATASE MULTIPLE FORMS FROM 19DAY-OLD RAT LIVER Property % of each form Molecular weight (SDS-PAGE) pH optimum Km for serine Heat inactivation 50°C for 60 min) -PLP +PLP (10-3M)

Form I

Form II

39

61

33,000

32,000

8.6

8.6

7.1 × 10-2M

80% 16%

6.5 × 10-2M

72% 19%

Serine dehydratase was partially purified as far as the acetone fractionation stage (61) and multiple forms separated by DEAE-cellulose chromatography (22). PLP, pyridoxal Y-phosphate.

(87). Although serine dehydratase occupies a key position at a metabolic branch-point and, at least in principle, may be involved in a number of alternative metabolic functions (see Fig. 1), it is remarkable that no credible allosteric effectors of the mammalian enzyme have been described. The enzyme is insensitive to regulation by adenine nucleotides (K. Snell, unpublished observations; 22a, 36, 41, 62) or glycine (36, 41). Although cysteine was reported to be a competitive inhibitor of the enzyme (Ki of 0.54 mM) in crude liver extracts (36, 41), it has been suggested that this is an artifact of sulfur liberation in the crude system (62, 75) and is not physiologically meaningful. Substrate inactivation by L-serine has also been reported but this only occurs in the absence of pyridoxal phosphate as co-factor and at alkaline pH and is unlikely to have any physiological significance (69). Similarly, the activating effects of K÷ and NH4 + seem to be related to protection against cofactor dissociation and enzyme sub-unit dissociation promoted by enzyme dilution in vitro and are without physiological relevance (68). Presumably sufficient 'fine control' is provided by fluctuations in the concentration of serine as substrate, given the very high K~ value for the enzyme in comparison to liver serine concentrations. Although this is a reasonable argument when considering control of enzyme flux in relation to variations in dietary protein (and presumably portal serine concentrations), it is not valid when we come to consider enzyme regulation in gluconeogenic situations, when the liver serine concentration is actually decreased (24). Presumably in these situations it is the very marked and rapid adaptive increases in enzyme amount which exert control over metabolic flux through the enzyme. Because o f the relatively short half-life of serine dehydratase (5.2 hr, 70), significant adaptive changes in

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enzyme amount, due to effects on the rate of synthesis or degradation of the enzyme protein, can occur rapidly within a few hr of the regulatory stimulus. Many studies have shown that dietary protein depletion reduces the activity of serine dehydratase to very low levels, but that above a dietary content of about 10% protein there is a proportionate increase in activity with increasing protein content (15, 36, 41, 46, 49, 64, 71-76). This response is quite the opposite of that which occurs with enzymes of serine biosynthesis (see above) and illustrates the catabolic role of serine dehydratase in serine metabolism. The response of serine dehydratase to dietary protein is not dependent on or secondary to the release of adrenal hormones since it occurs with equal magnitude in adrenalectomized rats (64, 71, 72, 76, 77). On the other hand there is a synergistic interaction between the inductive effects of protein and of hydrocortisone in intact rats which is dependent on the level of dietary protein (72, 73, 77). Glucagon is a major factor involved in the induction of serine dehydratase (see below) and it may be that high-protein feeding acts, at least in part, through increased glucagon secretion (78). That the induction of serine dehydratase by intubation of an amino acid mixture was abolished in pancreatectomized rats (79) gives support to this view. Many of the studies involving variation in dietary protein content have employed carbohydrate as a replacement nutrient. Thus many of the low protein diets are in effect high carbohydrate diets. Since glucose is known to depress enzyme activity in normally-fed animals (74) and to suppress the induction by intubated amino acid mixtures (see below), the effects of protein feeding may not be wholly accounted for simply by the variation in the protein dietary component. It is clear, however, that the forced feeding of amino acids alone (either made up as a synthetic mixture or derived from a casein hydrolysate) acts as a potent inducer of serine dehydratase activity in rats that have been fed on a protein-depleted diet for a number of days to lower the basal enzyme activity (49, 63, 70, 74, 80-82). The induction has been shown to be due to an increase in the rate of synthesis of enzyme protein (70, 80) and to be suppressed by the administration of glucose which acts primarily to inhibit enzyme synthesis and to increase enzyme degradation (70, 81, 83). The induction by casein hydrolysate or amino acid mixtures and the suppression by glucose occur in adrenalectomized rats (70, 80), but not in pancreatectomized rats (79) which suggests that glucagon may be the mediator of the effects of amino acid feeding. The effects of some single amino acids on serine dehydratase have also been investigated, although none are capable of producing the full response obtained with the amino acid mixture and the various experimental conditions employed make the physiological significance difficult to evaluate. Thus, serine (or to lesser extents glycine, glutamate, aspartate or alanine) was shown to be an inducer, but only when present as the sole source of nonessential amino acid nitrogen in a synthetic diet which reduced serine dehydratase to very low basal levels (75, 84); the induction was apparently due

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to increased synthesis of enzyme protein (84). Alanine (but not serine, glutamate or proline) stimulated serine dehydratase activity when force-fed to normally-feeding or 26 hr starved rats (85). When protein-depleted rats were fed synthetic amino acid mixtures, only mixtures of essential amino acids were effective in stimulating serine dehydratase activity (49, 70, 82); and when the essential amino acids were deleted singly from the mixture the most effective were found to be tryptophan, threonine, methionine and valine (49). Other studies also singled out tryptophan as being more effective than any other single amino acid in increasing the activity of serine dehydratase previously decreased to low levels by feeding a protein-depleted diet (70, 74, 77, 82). The effect of tryptophan was through an increased rate of synthesis of enzyme protein (70) and appears to be mediated by adrenal hormones, since it was abolished in adrenalectomized rats (77). In this respect tryptophan alone differs from the complete amino acid mixtures which appear to act through glucagon secretion. Glucagon secretion is also a factor in the induction ofserine dehydratase by starvation since in protein-depleted rats adrenalectomy does not abolish the response (70), whereas in either normally-fed young rats or protein-depleted adult rats the induction by fasting was abolished by pancreatectomy but not by adrenalectomy (79). The effect of fasting (24-48 hr) on serine dehydratase appears to be a very variable phenomenon which occurs in normally-fed young rats (body weights about 100 g or less) (48, 74, 79), but in adult rats only after feeding protein-depleted diets to reduce the enzyme activity (70, 79, 86). This phenomenon may be related to an effect of age and dietary status on the sensitivity of the pancreas to glucagon secretagogues, including the effects of fasting. In addition there appears to be a strain difference with respect to inducibility of serine dehydratase by fasting (79), and indeed basal enzyme levels appear to vary remarkably over a 10-fold range depending on the strain of rat (64, 87; K. Snell, unpublished observations) or the season when assayed (22). Alloxan- or streptozotocin-induced diabetes also leads to a stimulation of serine dehydratase activity and this can be partially reversed by insulin administration (22a, 48, 71, 75, 88-90). Here again the major determinant of the enzyme response may not be insulin-lack but glucagon, which is elevated about 10-fold in the plasma of alloxan-diabetic rats (91). A direct action of insulin is suggested, however, by the effect of glucose to suppress enzyme induction by amino acids and glucagon in either intact or adrenalectomized rats (70, 74, 81, 92). It seems probable that the glucose effect is mediated by increased insulin release (48, 70, 74, 89), although an additional effect of glucose (or a metabolite thereof) has not been completely excluded. More than likely the induction of serine dehydratase in diabetic animals (and in other situations) is a direct consequence of the level of hepatic cyclic AMP, which in turn is determined mainly by glucagon but may be modified by insulin (or agents which alter insulin secretion in vivo). Indeed glucagon or cyclic AMP

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are able to partially reverse the action of glucose on serine dehydratase (81,

92). The effect of glucagon to stimulate serine dehydratase has been reported in a number of studies, but only when the enzyme activity has been decreased by feeding a protein-depleted diet (70, 74, 75, 81,92-94) and not in normally-fed adult rats (22, 88, 95) unless the hormone is administered repetitively at frequent intervals (48, 79). Pancreatectomy markedly reduces the activity of the enzyme in normally-fed adult rats and can be restored to near normal levels by glucagon treatment (79). These observations suggest that glucagon is a major factor in maintaining serine dehydratase activity in normally-fed rats. The stimulatory effect of glucagon is attenuated, but not abolished, in adrenalectomized (48, 70) or pancreatectomized-adrenalectomized (79) rats, suggesting that adrenal hormones are exerting a 'permissive' action on the glucagon response. The effects of glucagon on enzyme induction are mediated by cyclic AMP (48, 93) and the hormone and its second messenger act by stimulating the synthesis of enzyme protein (70, 92, 93). Although the induction probably involves increased mRNA formation (92, 96), observations involving the transcriptional inhibitor actinomycin D are not entirely clear-cut (97) and the direct demonstration of increased levels of functional mRNA for serine dehydratase after glucagon or cyclic AMP treatment has not as yet been reported. The question as to whether or not adrenal hormones, such as hydrocortisone, directly affect serine dehydratase activity is a vexed one. The influence of dietary protein on the hydrocortisone-mediated induction of serine dehydratase has already been referred to above. On protein-free diets the effect of hydrocortisone is either abolished (74, 77, 83, 93) or minimal (72, 73), in contrast to induction by amino acid mixtures, fasting or glucagon (see above). In normally-fed animals hydrocortisone may induce serine dehydratase (48, 64 71 but cf. 22, 95), but in adrenalectomized rats it is only effective if animals are simultaneously fasted (48, 77, 79). Paradoxically, hydrocortisone was reported to be effective in protein-depleted rats if the animals were also adrenalectomized (70, 77). In general, the above studies suggest that hydrocortisone may not be effective by itself, but only when glucagon secretion is taking place. This is supported by the absence of induction of serine dehydratase by hydrocortisone in fasted, pancreatectomized-adrenalectomized rats (79). In these circumstances hydrocortisone was shown to potentiate the action of glucagon (79), but in animals with the pancreas intact the effect of hydrocortisone and glucagon (or cyclic AMP) together was no greater than that with glucagon (or cyclic AMP) alone (48, 93). From the above discussion it seems that adaptive changes in serine dehydratase can occur in a variety of circumstances. The primary and common effector in these various situations appears to be hepatic levels of

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cyclic AMP with hydrocortisone exerting a potentiative or 'permissive' action by an as yet obscure mechanism. Many of the unresolved features of the inductive and repressive actions inherent in studies on the whole animal may become clearer with the development of responsive liver cell culture systems which allow for studies in vitro. Thus, in both adult (98, 99) and fetal (100) hepatocytes in primary cell culture, neither glucagon, dibutyryl cyclic AMP nor dexamethasone alone were capable of inducing serine dehydratase activity, whereas the combination of steroid hormone with glucagon or dibutyryl cyclic AMP induced the enzyme 24 hr later. Insulin partially suppressed the induction by dexamethasone and glucagon (98, 99) or dibutyryl cyclic AMP (100). In a study from Pitot's laboratory involving adult rat hepatocytes in culture the presence of insulin throughout the culture period was shown to be necessary to maintain cellular integrity (90). In these circumstances, again, neither glucagon nor dexamethasone were effective as inducing agents (although the combination was not tested), but in view of the suppressive action of insulin in the other studies above these results are equivocal. When insulin was not present in the medium, dexamethasone or glucagon, either singly or together, increased serine dehydratase activity above that observed in cultures with insulin. However, in the absence of control values without insulin, it is not clear that the increased activity actually represents enzyme induction. Interestingly, in this study glucose (8 m s ) in the presence of insulin was shown to reduce enzyme activity below that in cultures with insulin alone. This leaves open the question as to whether the glucose effect on the enzyme observed in vivo is due to a combination of insulin action and a direct hepatic effect by glucose. Recently it has been shown that epinephrine (adrenalin), like insulin, is able to suppress serine dehydratase induction by dexamethasone and glucagon in cultured adult hepatocytes (99), but to stimulate activity in cultured fetal hepatocytes (100). The suppressive effect of epinephrine was related to its at-adrenergic action in the adult whereas the/3-effects, which predominate in fetal hepatocytes, act via cyclic AMP to induce the enzyme (99). The induction of serine dehydratase by dexamethasone and glucagon and its suppression by insulin or epinephrine in adult hepatocytes are due to effects on the synthesis of enzyme protein (98, 99). Furthermore, the inhibitory effects of actinomycin D and cordycepin on enzyme induction suggest the involvement of increased mRNA levels, at least in the fetal hepatocyte (100). In general the work with cultured hepatocytes confirms the conclusions from experiments in vivo that steroid hormones and glucagon act directly on the liver to induce serine dehydratase. While not yet resolving the mechanistic roles played by the 2 classes of hormone, the studies are at least consistent with a primary enzyme inducing effect ofglucagon, with steroid hormone having an essential but perhaps permissive action. Although it has been demonstrated that serine dehydratase may occur as multiple enzyme forms, only a few studies (and all from Pitot's laboratory)

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have addressed this in relation to enzyme adaptation (22a, 67, 77). DEAEcellulose column chromatography was used to separate the 2 forms of serine dehydratase from crude liver supernatants prepared from rats subjected to various inductive treatments. In normally-fed rats all the enzyme is in Form II (the more electronegative form) and the induction by glucagon, or in alloxandiabetic rats, is confined to this molecular form. Similarly increasing activity in animals fed 0%, 12% and 20% (normal) protein diets is confined to changes in F o r m II; but feeding a 90% protein diet, while increasing F o r m II still further, also leads to an increase in Form I (which represents about 50% of the total activity)~ Hydrocortisone, which has no or little inductive effect in intact rats on 0% or 12% protein diets, induces serine dehydratase in adrenalectomized rats to similar extents regardless of the diet and this involves an increase in F o r m II, but also the appearance of Form I (representing about 30% of total activity). Tryptophan, which has no effect in adrenalectomized rats, induces enzyme activity to a greater extent in intact rats on the 12% protein diet and this is reflected by a greater induction in Form II whereas the increase in F o r m I is similar for both diets. Thus glucagon-related responses involve changes in Form II, whereas adrenal hormone-related responses involve both enzyme forms. Two possibilities suggest themselves to account for the observations: each form may be the product of a separate gene (isoenzyme) which is under independent and differential control; or the 2 forms may be derived from the product of a single gene which then undergoes rapid post-translational modification to generate a second form. This post-translational processing may then be under the control of particular hormones which would influence the steady-state ratios of the two forms. Pitot and co-workers (67) would appear to favor the second of these possibilities. One possible posttranslational mechanism that immediately suggests itself is protein phosphorylation. The enzyme is under the control of glucagon which owes many, if not all, of its effects to the activation of cyclic AMP-dependent protein kinases with broad substrate specificities for protein phosphorylation. Remarkably few enzymes of amino acid metabolism have been shown to be subject to this type of reversible covalent modification, even though it is a widespread regulatory phenomenon in carbohydrate and lipid metabolism (18). One hypothesis might be that the newly translated serine dehydratase gene product may be rapidly phosphorylated (perhaps as a co-translational process) to generate F o r m II, but that this process may be attenuated by steroid hormones so that the lesser-phosphorylated F o r m I would accumulate. Tryptophan could also modify the phosphorylation process in the presence of steroid hormones, perhaps by a direct interaction with the enzyme protein itself or through an interaction with the polysomes engaged in the synthesis of the serine dehydratase polypeptide. Recent studies in this Laboratory provide no indication that phosphorylation, should it indeed occur, would lead to any change in catalytic

REGULATION OF SERINE METABOLISM

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properties of the serine dehydratase enzyme forms (101). Liver cytosol fractions from normally-fed rats (containing Form II only) or rats fed on a 90% protein diet for 5 days (containing equal amounts of Forms I and II) were incubated at 37°C for up to 4 hr in the presence of 0.05 Mpotassium phosphate buffer (pH 7.2), 0.15 M KCI, 1 mM EDTA, I mM dithiothreitol, 0.4 mM pyridoxal 5'-phosphate, 0.1 M NaF, 4 mM MgC12, 2 mM ATP, 2 mM phosphocreatine, 10 I.U. creatine phosphokinase and either 0.2 mM cyclic AMP, or 1 I.U. cyclic AMP-dependent protein kinase catalytic subunit (from rabbit muscle, dissolved in 0.05 M potassium phosphate buffer, pH 7.2, containing 4 mM cyclic AMP and obtained from Sigma (U.K.) Ltd., London), or 1 I.U. alkaline phosphatase (calf intestine, from Sigma (U.K.) Ltd., London). No significant change in Vm~ (7.1 to 7.8 mtzmol/min per 0.5 ml cytosol, normal diet; 34 to 35 m#mol/min per 0.5 ml cytosol, high protein diet) was observed under conditions promoting protein phosphorylation (ATP + ATP regenerating system + cyclic AMP, or ATP + ATP regenerating system + protein kinase catalytic subunit) or protein dephosphorylation (alkaline phosphatase) at any time over a 4 hr incubation period. Nor was there any significant difference in I ~ values with respect to L-serine after 2 hr of incubation under the above conditions (average of 66 mM for normal diet; average of 62 mM for high protein diet). In view of the similarity in characteristics of the 2 molecular forms of serine dehydratase in adult (22a) and neonatal (Table 5) rats, the above results are not surprising although they do provide evidence that modification of the phosphorylation state of either enzyme form (should this occur) has no discernible effect on the catalytic properties. Studies with radioactive-phosphorus labeled ATP and the purified multiple enzyme forms in vitro under the above incubation conditions are required to establish whether the enzymes are subject to phosphorylationdephosphorylation. However, the chemical content of phosphate in the apoenzymes (without pyridoxal phosphate co-factor) derived from the purified molecular forms was negligible and this would argue against a role for this covalent modification (67). No significant change in the proportion of the multiple enzyme forms ofserine dehydratase from rats fed a high protein diet, after incubation for 2 hr under the conditions of phosphorylationdephosphorylation described above, was observed (after separation by cellulose acetate electrophoresis; K. Snell, unpublished observations). The hypothesis that Form II is a phospborylated derivative of Form I is not supported by these studies. An alternative hypothesis that Form I is a catalytically-active intermediate on the pathway ofserine dehydratase enzyme degradation is an attractive one, in view of the relation of this posttranslational process to the multiple molecular forms of tyrosine aminotransferase (102, 103). The relevance of enzyme degradation, or other covalent protein modifications, to the multiple forms of serine dehydratase and indeed the physiological significance, if any, of such multiple forms AER 2 2 - L ~

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KEITH SNELL

require further investigation. It is of interest that we find that serine dehydratase from rabbit liver is present as a single molecular species (on DEAE-cellulose chromatography), and that after induction of the enzyme by hydrocortisone all of the activity remains associated with this single form

(101). Tissue distribution ofL-serine dehydratase. In our survey serine dehydratase activity appears to be largely confined to the liver in the rat and this contrasts with the more ubiquitous distribution of the enzymes of serine biosynthesis (see Table 3). We found low levels of activity associated with kidney and heart, and other workers have also reported low activity in kidney and heart (71), kidney and brain (46, 150), or kidney (104). We have found activity in the rabbit to be largely confined to the liver [0.36 + 0.03 (5) ~ m o l / m i n per g], with kidney and spleen containing 17% and 15% respectively of the activity in liver. Activity was absent from brain, heart, intestine, lung, skeletal muscle and adipose tissue (K. Snell and C. M. Harris, unpublished observations). Unlike liver, the activity from rat kidney and brain is not responsive to changes in dietary protein content (46). It has been suggested that the low activities found in tissues other than the liver may be related to the presence of L-homoserine dehydratase-cystathionase which possesses weak L-serine dehydratase activity (65), or to L-amino acid oxidase activity (71) or cytosolic L-serine aminotransferase activity which would produce keto acids that would react with lactate dehydrogenase to re-oxidize N A D H in the coupled assay procedure commonly used to measure serine dehydratase activity. In fact Freedland and Avery report (71) that when this assay was substituted by the estimation of enzymically-produced pyruvate by the dinitrophenylhydrazine method, activity was only detectable in liver; this was also the case in the study by Rowsell et al. (87) which used the latter assay method. It seems that specific L-serine dehydratase activity is confined to the liver. Regulatory properties of L-serine-pyruvate aminotransferase and related pathway enzymes. L-Serine-pyruvate aminotransferase is an alternative enzyme initiating the utilisation of serine and channeling the amino acid carbon to glucose formation. The enzyme has been purified from rabbit (105), rat (106), mouse, dog and cat (21a) liver. With most species relative molecular mass determinations suggest a dimeric protein with a monomeric Mr of about 40,000 daltons by SDS polyacrylamide disc gel electrophoresis (21a, 106). However, the rabbit enzyme is reported by the same techniques to be a single polypeptide chain with Mr of about 41,000 daltons (105). Whether dissociation of subunits had occurred during the purification procedure and prior to estimation of the relative molecular mass by gel filtration in this study is not yet clear. Purification of the rabbit enzyme was from a KCl-phosphate bufferextracted supernatant fraction of liver from animals fed on a high-protein diet to induce enzyme (105). With the other species, mitochondrial fractions were used and animals were untreated (21a), except for the rat where the enzyme

REGULATION OF SERINE METABOLISM

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was glucagon-induced (106, 107). The purification of peroxisomal serinepyruvate aminotransferase from Triton-treated rats has also been reported with properties indistinguishable from those of the purified mitochondrial enzyme (108). The suggestion has been made that the mitochondrial and peroxisomal enzymes activities are the result of intracellular sorting of an enzyme synthesised in the cytosol (109). The purified enzymes from rat and mouse have a broad amino acid substrate specificity with pyruvate, glyoxylate or phenylpyruvate as keto acid substrates - - the major activities are transamination of serine-pyruvate and alanine-glyoxylate (21a, 107, 108, 110). In contrast the enzymes from the rabbit (105), cat and dog (21a, 110) were highly specific for serine transamination with pyruvate and glyoxylate and alanine transamination with glyoxylate and hydroxypyruvate. The alanine-glyoxylate aminotransferase activity was essentially irreversible (105, 110), as previously observed in crude extracts (16, 1 I1). The serine-pyruvate aminotransferase activity was reversible but the reaction in the direction of serine as amino donor exceeded the reverse reaction in rat and mouse (21a), whereas in the cat, dog (21a) and rabbit (105) alanine-hydroxypyruvate transamination exceeded the reaction in the opposite direction, again as observed previously in crude extracts (112). The apparent Km values for serine and pyruvate with the rat liver enzyme are 16 and 1.0 mM (21a). Physiological inhibitors have only been investigated with the purified rabbit enzyme and at 5 mM no inhibition was found with D-glycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoserine or phosphohydroxypyruvate (105). The subcellular distribution of L-serine-pyruvate aminotransferase has been investigated in a number of studies and for normal adult liver activity is present at 5-17% in the cytosol and 80-95% in a total-particles fraction (87, 106, 107, 113, 114). More repetitive differential centrifugation a n d / o r prolonged sucrose density gradient centrifugation leads to the appearance of an increased proportion of cytosolic activity (109, 114), which it is suggested could reflect leakage of the enzyme from peroxisomes (109). The distribution of serine-pyruvate aminotransferase activity between mitochondria and peroxisomes in the particulate fraction of liver homogenates is still controversial. Rowsell and co-workers (114) could find no evidence for any appreciable peroxisomal association of the enzyme and correcting the data in their enriched peroxisomal fraction for glutamate dehydrogenase (mitochondrial marker) and peroxisomal enzymes, a rough estimate of about 6% of total homogenate activity would be located in peroxisomes, A similar conclusion was reached in work in this laboratory on the subcellular distribution of serine-pyruvate aminotransferase in neonatal rat liver (113, 115). Correcting for marker enzymes in a peroxisomal-enriched preparation, a maximal value of about 11% of total homogenate activity could be located in this organelle. In contrast, Oda and colleagues (109) using similar corrections

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KEITH SNELL

for marker enzyme profiles in sucrose density gradients from various fractions prepared by differential centrifugation, obtained estimates of aminotransferase activity ratios of about 2:1 for mitochondria:peroxisomes, in a similar manner, using zonal centrifugation, Noguchi el al. (116) estimated an equal distribution of activity between mitochondria and peroxisomes. In the absence of recovery data it is difficult to interpret these latter studies in terms of distribution of total homogenate activity, but they seem to imply a greater association of serine-pyruvate aminotransferase with peroxisomes than do other studies. A further approach to assessing the subcellular distribution of specific serine-pyruvate aminotransferase activity has been to use the antibody prepared to the mitochondrial enzyme (purified from glucagon-treated rats) to precipitate enzyme activity in subcellular fractions obtained by density gradient centrifugation and pooled according to the distribution of cytosol, mitochondrial and peroxisomal marker enzymes (109). By this method the majority (90%) of apparent peroxisomal-associated aminotransferase activity is immunologically identical with the purified mitochondrial enzyme, confirming the conclusions reached on the basis of comparison of physical and catalytic properties of the purified enzymes (108). The relative distribution of immunoprecipitable enzyme activity in the fractions was in the ratio 1:1:1.25 for cytosol, mitochondria and peroxisomes (109). However only about 50% of the aminotransferase activity extracted from a whole homogenate was antibody-precipitable indicating that this method underestimates total tissue capacity for serine-pyruvate aminotransferase and may not accurately reflect the subcellular distribution of total transaminating capacity. The finding that 50% of the cytosolic fraction prepared by density gradient centrifugation was immunoprecipitable (109) suggests, but does not prove, that the higher cytosolic activities found in some studies (109, 114) may be the result of leakage of aminotransferase from peroxisomes (see above). Of the other enzymes involved in the aminotransferase-initiated pathway of serine gluconeogenesis (Fig. 1), D-glycerate dehydrogenase has been partially purified from rat liver (117) and more extensively purified from beef liver ( 118, 119), and D-glycerate kinase has been partially purified from rat liver (120). Although not extensively studied, glycerate kinase appears not to be sensitive to physiological regulators. On the other hand glycerate dehydrogenase activity appears to be modified by numerous metabolites, the most prominent of which are inhibitory glycolytic intermediates (2- or 3phosphoglycerate, 2,3-diphosphoglycerate, fructose 1,6-bisphosphate), noncompetitive inhibition by ATP and GTP, and by phosphohydroxypyruvate (118, 119). Serine and phosphoserine had no effect on the enzyme activity. The physiological role of the allosteric inhibition by the purine nucleoside triphosphates is not clear. However the inhibitory effect of phospho-

REGULATION OF SERINE METABOLISM

351

hydroxypyruvate on glycerate dehydrogenase is of possible importance in regulating the relative activity of the serine biosynthetic (phosphorylated) pathway and the gluconeogenic (non-phosphorylated) pathway. The inhibition of glycerate dehydrogenase by glycolytic intermediates suggests that the pathway may be viewed as operating in the gluconeogenic direction and is subject to feedback inhibition by gluconeogenic end-products. The lack of effect of serine (or phosphoserine) is consistent with such a role. The enzyme operates preferentially in the direction of glycerate formation from hydroxypyruvate (equilibrium constant of 3 × 10-12 M) and this is consistent also with the functioning of the pathway in a gluconeogenic direction. The beef liver enzyme is able to use either hydroxypyruvate or glyoxylate as substrates for reduction and in this respect behaves like the glyoxylate reductase (glycerate dehydrogenase) of plant origin. A rat liver glyoxylate reductase (glycerate dehydrogenase) has been reported having activity with hydroxypyruvate or glyoxylate, and in addition a weaker activity with pyruvate as substrate (121); the regulatory properties of this enzyme have not as yet been characterized. With respect to subcellular distribution, D-glycerate dehydrogenase has been reported to be largely cytosolic (122), but the ambiguity of the assays used makes such an assignment equivocal. In particular a major mitochondrial complement was dismissed because of the ability to reduce pyruvate, whereas a hydroxypyruvate reductase with such a substrate specificity has been reported to be distributed between mitochondrial and peroxisomal fractions (121). The relation of these various activities to one another clearly requires further investigation, particularly in view of the major mitochondrial localisation of the other enzymes on the aminotransferase gluconeogenic pathway. The rat liver D-glycerate kinase activity, which produces 2-phosphoglycerate as a product, appears to be largely (about 70%) associated with the mitochondria (123, 124). Adaptive responses by enzymes of the serine-pyruvate aminotransferase pathway to numerous stimuli have been reported. With respect to variations in protein feeding in rabbits, serine aminotransferase is the most responsive showing a 70-fold increase going from 2% to 88% protein content in the diet (15). Glycerate kinase also shows a marked increase with increasing protein content, but glycerate dehydrogenase is unaffected by changes in protein intake (15). Essentially the same observations have been made in the rat (36, 41, 50, 109, 124, 125). In rabbit (15) and rat (109) evidence with protein synthesis inhibitors and specific enzyme antibodies suggests that new enzyme protein synthesis is required for the increased serine aminotransferase activity. For both serine aminotransferase (109) and glycerate kinase (124), it has been shown that the increased activity on high protein diets is largely, though not entirely, due to increases in the mitochondrial activity. The direction of the enzyme changes with variation in protein content is the same as that observed

352

K E I T H SNELL

for serine dehydratase but the inverse of that found for the serine biosynthetic enzymes. With starvation the general coincidence of response with serine dehydratase no longer seems to hold. Starvation of rats for 24, 48 or 72 hr has no effect on serine aminotransferase activity (48, 59, 109, 126), unless enzyme activity is first reduced by feeding rats on a low-protein diet for 2 days (125). The reason for the lack of response of serine aminotransferase during starvation has not been adequately investigated, although it seems likely that it is related to the increased secretion of adrenal steroid hormones, in view of the suppressive effect of cortisone on enzyme induction (see below). Glycerate dehydrogenase shows a small increase (by 44%) in activity compared to fed controls after 48 hr starvation, but not after 24 hr starvation (48). In contrast, in al|oxandiabetic rats serine aminotransferase activity is increased in mild-diabetic rats (48, 50, 88) (though not in severely-diabetic animals) but this increase, unlike that of serine dehydratase, was not greatly diminished by insulin treatment (48, 50). Similarly, glycerate dehydrogenase showed a small increase in activity in mild-diabetic, but not severely-diabetic, rats (48, 50). No information on glycerate kinase in these situations is available. These two conditions (starvation and diabetes) are accompanied by enhanced gluconeogenesis and increased liver cyclic AMP concentrations; yet, despite the responsiveness of serine aminotransferase to this inducing agent, the responses in these two conditions are quite small in comparison with those seen with serine dehydratase. The response of the aminotransferase to steroid hormones is similar to but smaller than that observed with serine dehydratase: no effect on serine aminotransferase was found after a single injection of cortisone to intact (22) or adrenalectomized (48) rats, but a small increase was seen after 5 days of cortisone treatment in intact rats (48), or after triamcinolone treatment for 4 days in protein-depleted rats (50). Glycerate dehydrogenase showed a small decrease in activity in these same studies (48, 50). Again, no information is available on glycerate kinase. The greatest hormonal response of serine aminotransferase is to glucagon administration, which we first reported in 1969 (16), and in this situation the magnitude of the response exceeds that of serine dehydratase. As shown in Figure 4, the effect of glucagon is mimicked by its second messenger dibutyryl cyclic AMP and the induction is blocked by actinomycin D (22), cordycepin or cycloheximide, suggesting a requirement for continued m R N A production and showing that the induction involves the increased synthesis of new enzyme protein. The effect of glucagon and cyclic AMP has been confirmed by other workers in normally-fed rats (48, 88), protein-depleted rats (48,127), and fasting intact or adrenalectomized rats (48, 127). In addition it has been shown that cortisone can apparently partially suppress the glucagon- or dibutyryl cyclic AMP-mediated induction of serine aminotransferase (127, 128); the

353

REGULATION OF SERINE METABOLISM Relativa e~yme 41

3

2

I

0

i

|

I

i

i

activity 0 I ~

i r

2

3

4

I

I

l

5

corti$ol thyroxine

I

I glucagon I

I

Bt2cAMP ,

Bt2eAMP + act D + cord A. FETAL

+ cyclohex

B . ADU L'r

FIG. 4. Hcrmc~aal regulation of hepatic L-serine-pyruvate aminotransferase in (A) fetal and (B) adult rats. Fetal rats were injected intraperitoneally in utero on day 20 of gestation with hydrocortisone (cortisol), thyroxine, glucagon, dibutyryl cyclic AMP (Bt2 cAMP), actinomycin D (act D), cordycepin (cord), or cycloheximide (cyclohex) at dosages of 0.125, 0.003, 0,05,0.5, 0.005, 0.15, 0.01 rag/fetus, respectively, in a vol of 0.05 ml. Adult rats were injected intraperitoneally with hydrocortisone (cortisol), thyroxine, glucagon, dibutyryl cyclic AMP, actinomycin D, cordycepin, or cycloheximide at dosages of 10.0, 0.5, 0.5, 5.0, 0.1, 3.0, 0. i mg/100 g body wt., respectively. Actinomycin D and cordycepin were injected 30 rain prior to other co-administered agents. Enzyme activities were measured in liver homogenates (from 4 to 6 rats) 24 hr after injection, recorded as units/g of liver, and shown here relative to control values in saline-injected animals (as indicated by the broken line).

glucose suppression observed with serine dehydratase induction is not found with serine aminotransferase (128). We have confirmed that the increase in serine aminotransferase activity after induction by glucagon is almost entirely confined to the mitochondrial activity (K. Snell and W. C. Ng, unpublished observations), with only a small increase in the cytosol and perhaps the peroxisomes (106, 107, 109, 114, 116). Using the mitochondrial-enzyme directed antibody it was shown that glucagon increased the amount of enzyme protein and also increased pulse-labeling of the immunoprecipitable protein in homogenates or mitochondrial fractions, reflecting a 10-fold stimulation of enzyme protein synthesis (106). The amount of functional mRNA coding for serine aminotransferase was quantitated using heterologous in vitro protein synthesizing systems together with specific immunoprecipitation, and was found to be increased 30- to 40-fold in livers from rats treated 3.5 hr previously with glucagon (129). Interestingly the relative molecular mass of the immunoprecipitated translation product was about 2000 daltons larger than that of [3SS]methionine-labeled serine aminotransferase immunoprecipitated from the mitochondrial fraction of isolated hepatocytes. This work shows that glucagon induces serine aminotransferase by stimulating the synthesis of specific mRNA for the enzyme a n d / o r by inhibiting its degradation. Moreover, it also suggests that increased synthesis of enzyme protein on cytoplasmic ribosomes in glucagon-treated animals results in the production

354

KEITH SNELL

of a precursor polypeptide which undergoes post-translational proteolytic processing before, or more likely during, its transfer into the mitochondria. In view of the immunochemical and enzymological identity of the mitochondrial and peroxisomal forms of serine aminotransferase and the mitochondrial specificity of the glucagon induction, there is the intriguing aspect of increased cytoplasmically-synthesized serine aminotransferase precursor protein being directed specifically to a mitochondrial localisation under the influence of the hormone. D-Glycerate dehydrogenase was induced slightly (by 18%) in normally-fed rats after repeated glucagon injections for 4 days, but was unchanged 12 hr after 2 injections to 24 hr-starved adrenalectomized rats (48). D-Glycerate kinase has not been investigated for glucagon inducibility. It appears that although D-glycerate dehydrogenase is the only enzyme with allosteric properties appropriate to participation in a pathway of gluconeogenesis from serine, it is serine aminotransferase which displays the greatest adaptive responses to stimuli associated with gluconeogenesis. Indeed in terms of enzyme adaptation, D-glycerate dehydrogenase shows little association with gluconeogenesis. It may be that the role of this enzyme requires re-evaluation in connection with the aminotransferase-initiated gluconeogenic pathway and that evaluation of other hydroxypyruvate reducing activities (particularly in the mitochondria) is required. D-Glycerate kinase responds less strongly, but in parallel, with serine aminotransferase, although information on adaptive regulation of this enzyme is still incomplete. Tissue distribution of L-serine-pyruvate aminotransferase and related pathway enzymes. As shown in Table 3, serine-pyruvate aminotransferase activity in the adult rat is essentially confined to the liver, with low activities in kidney (15% of liver activity), heart (13%) and intestine (12%), and no detectable activity in the other tissues tested. In the adult female rabbit, kidney activity was only 10% of that in liver (0.690 +_0.023 (6) t~mol/min per g), and activity was undetectable in brain, heart, intestine, lung, spleen, skeletal muscle or adipose tissue (K. Snell and C. M. Harris, unpublished observations). Using slightly different assay conditions, Rowsell et al. (87) report kidney activities of 33% and 4% of liver activities in the rat and rabbit, respectively, and no detectable activity in brain, heart, intestine, spleen, or skeletal muscle in either species. Measuring the aminotransferase in the reverse direction, Walsh and Sallach (13) report activity in liver, kidney (50% of liver) and heart (33%) in the rat, and liver and kidney (5%) in the rabbit, but not in other tissues. In general, whole body activity of the aminotransferase seems to be confined to the liver, given the relative masses of the liver and other organs investigated. For D-glycerate dehydrogenase, activity was equally divided between liver and kidney, but was negligible in brain, heart, intestine, spleen, lung, or skeletal muscle (13, 46, 50). No tissue distribution surveys of D-glycerate kinase have been made. The tissue distributions of the

REGULATION OF SERINE METABOLISM

355

enzymes of the aminotransferase-initiated pathway are similar to that of serine dehydratase and contrast with the widespread occurrence of the enzymes of serine biosynthesis in the body. Metabolic role o f L-serine dehydratase and L-serine pyruvate aminotransferase. Serine is a good gluconeogenic precursor in the perfused rat liver giving a maximal rate at least as good as alanine (130, 131). The rate of hepatic gluconeogenesis from serine in vivo, however, is less than that of alanine in 24 hr-starved rats (132). In 48 hr-starved dogs the rate of body serine turnover is 40% less than that of alanine, and consequently the rate of body glucose formation in vivo is also 40% less from serine compared to alanine (133). Sandoval and Sols (59) estimated the contribution of serine to glucose formation to be about 7% in the livers of 30 hr-starved rats perfused with physiological concentrations of glucose precursors and [~4C] serine. This agrees well with the maximal contribution of serine metabolism to glucose formation of 4-6% calculated from hepatic metabolite balances measured in 20 hr-starved rats in vivo (134). This contribution was 56-60% less than that possible from alanine (9-15%) (134). Nevertheless, it is clear that the involvement of serine in hepatic gluconeogenesis from amino acids is second only to alanine in importance. Unlike alanine, however, it appears that there are 2 alternative and separate pathways by which serine may be converted into glucose (Fig. 5), and their relative contributions have been the subject of much controversy. The tissue distributions of the enzymes involved in both pathways are consistent with a role for either in gluconeogenesis. Serine dehydratase and serine aminotransferase activities are essentially restricted to the liver. Although either liver or kidney can carry out gluconeogeneis, rat kidney is unable to use serine as a gluconeogenic precursor (53, 135, 136). It is noteworthy that pyruvate, which serves as an intermediate in gluconeogenesis via serine dehydratase, has a number of alternative metabolic fates, at least in principle (see Fig. 1). On the other hand hydroxypyruvate, the intermediate in gluconeogenesis via serine aminotransferase, has no obvious metabolic fate other than glucose formation. Both D-glycerate and hydroxypyruvate, on the aminotransferase pathway, have been shown to be readily converted to glucose in vivo and in vitro (59, 137-139). This experimental evidence led to the first credible proposal of a route for gluconeogenesis from serine via transamination (Dickens and Williamson, 135). Some 10 years later, the same route was proposed by ourselves (16), and independently by Sallach and coworkers (15), on the circumstantial evidence of an association between increased activity of serine-pyruvate aminotransferase with situations linked to enhanced gluconeogenesis. In general though, as noted above, the adaptive increases in serine aminotransferase occur under the same conditions as adaptive increases have also been observed with serine dehydratase. The suppressive effect of glucose on serine dehydratase induction could be viewed

356

K E I T H SNELL

as a feedback repression phenomenon involving glucose as an end-product of the gluconeogenesis pathway. However, it should also be recalled that Dglycerate dehydrogenase, on the serine aminotransferase-mediated pathway, is subject to inhibition by glycolytic metabolites, again consistent with a feedback control phenomenon by gluconeogenic pathway end-products. The stimulatory effect of a high-protein diet on the activity of both enzymes poses a problem in physiological interpretation, since a requirement to dispose of an excess dietary amino acid intake need not necessarily include an enhancement of hepatic gluconeogenesis. Since most of the high-protein diets employed in enzyme adaptation studies are also deficient in carbohydrate, this compounds the difficulty of interpretation (but see further discussion below). Evidence consistent with a specific role of dietary protein in the acceleration of gluconeogenesis in rats fed a high-protein diet has been presented (140, 141), but serine was not the glucogenic precursor used. More recently an attempt was made by Remesy et aL (134) to correlate enzyme adaptation with changes in metabolic flux in response to dietary changes. Rats were fed for 21 days on a 13% casein - - 78% starch diet or a 50% casein - - 40% starch diet (% of total calories) and then starved for 20 hr. Serine dehydratase activity was increased 5-fold in the high-protein adapted rats, and gluconeogenic capacity from serine in isolated hepatocytes in vitro was increased 4-fotd (as was urea production). However, despite the increase in hepatic gluconeogenic capacity measured in vitro, metabolite balances measured across the liver in vivo revealed no change in net glucose output and only a small, but statistically insignificant, increase in the net rate of hepatic serine utilization (134). Perhaps more telling in this study were measurements made in fed animals adapted to the same diets: serine dehydratase activity was increased 9-fold in the high-protein fed rats and net hepatic serine utilization was increased 6fold. No measurements were made of the gluconeogenic capacity from serine in this situation, although liver metabolite balance measurements in vivo showed a switch from net glucose uptake to net glucose output in rats on the high-protein diet. No measurements of serine aminotransferase activity were made in this study, so despite the correlations that emerged (with the reservations indicated above) it is not possible to unequivocally account for the changes in gluconeogenic flux solely in terms of the adaptive changes in serine dehydratase activity. In any case evidence of this kind can only really be circumstantial in nature, persuasive though that sometimes may be. A more direct approach to assessing the relative contribution of the 2 pathways is the use of inhibitors specific to metabolism by one or other route. The use of quinolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase, to block the serine dehydratase-mediated gluconeogenic pathway via pyruvate (Fig. 5) was introduced by Lardy and co-workers and formed the basis of their independent proposal of a pathway for gluconeogenesis from serine involving transamination (17). Much of the

REGULATION OF SERINE METABOLISM MITOCHONDR IA

CYTOSOL

A%'r%o o?

357

s ~ r ta¢ - p y r ~ v a t e aminotransferase

deficiency

f~

q~liao linate m vrcapto-pi~llnate

.

Phosp pyruv~ I

2 Pho

FIG. 5. Mitochondrial-cytosolic interrelationships in the pathways of gluconeogenesis from serine and the loci of specific inhibitory actions. (?) The presence of a specific translocase for 2phosphoglycerate has not been demonstrated. Figure adapted from (113).

controversy over the role of the serine aminotransferase pathway in gluconeogenesis from serine stems from the use of concentrations of quinolinic acid which may not completely block glucose synthesis from precursors known (or presumed) to proceed via pyruvate as an intermediate (lactate or alanine). A dose-response curve shown by SOling et al. (142) indicated 90% inhibition of gluconeogenesis from 10 mM alanine at 2.4 mM quinolinate in the perfused livers of 48 hr-starved rats. We have found a similar degree of inhibition from 10 mM alanine at 5 mM quinolinate (143), but at this concentration of inhibitor other workers have reported only 40% inhibition of gluconeogenesis from 10 mM alanine (144) or 48% inhibition from 10 m M lactate (145). Nevertheless, under conditions where a reasonable degree of inhibition of gluconeogenesis from precursors proceeding via pyruvate has been established (about 70% inhibition or greater), it has been consistently shown that gluconeogenesis from serine is much less affected (17, 143, 144, 146). The introduction of 3-mercaptopicolinic acid, which is a more potent inhibitor of phosphoenolpyruvate carboxykinase and gluconeogenesis (147), prompted us to reinvestigate this problem (Table 6). In these experiments, using isolated hepatocytes from 20 hr-starved rats, 5 mM quinolinate only inhibited gluconeogenesis from alanine by 60%; even so, gluconeogenesis from serine was inhibited to a much lesser extent (by 30%). With 1 mM mercaptopicolinate, inhibition was essentially complete from alanine but only 68% inhibition was found with serine. Thus, a small but finite proportion of

358

KEITH SNELL T A B L E 6. E F F E C T O F Q U I N O L I N A T E (QA) A N D 3M E R C A P T O P I C O L I N A T E (MPA) ON G L U C O N E O G E N E S I S IN I S O L A T E D H E P A T O C Y T E S F R O M S T A R V E D RATS Rate of gluconeogenesis 0~mol/min per g wet wt)

% inhibition

10 mM alanine + 5 m~ QA

0.430 + 0.008 0.171 + 0.004

60

10 mM serine + 5 mM QA

0.344 + 0.021 0.237 + 0.017

31

10 mM alanine + 1 rr~ M P A

0.424 + 0.009 0.016 + 0.002

96

10 mM serine +1 mM MPA

0.335 _+ 0.011 0.108 +0.008

68

Additions

80-100 g male rats were starved for 20 hr and isolated liver cells prepared (148). Inhibitors were present for 15 min prior to the addition of substrate and incubations were continued for a further 45 rain. Glucose production was measured over the time period 30-60 rain. Measurements were made in triplicate on hepatocyte preparations from three separate animals and rates of glucose production in the absence of substrate (0.123 for the QA experiments and 0.090 for the MPA experiments) have been subtracted to give the values recorded +_ s.e.m. Interference by M P A in the determination of glucose by certain methods (not employed here) is a potential complicating feature of this inhibitor (147).

gluconeogenesis (perhaps 30% of the maximal flux) may proceed by way of the serine aminotransferase-initiated pathway in 20 hr-starved rats. Using isolated hepatocytes from 48 hr-starved rats, Beliveau and Freedland have reported somewhat greater inhibition of gluconeogenesis from serine (as assessed by radioactivity incorporation form 14C-labeled substrate into glucose) in the presence of 1 mN mercaptopicolinate (146). The starvation status of the rat is an important factor in such experiments, since it is well established that serine aminotransferase activity does not change with progressive starvation, whereas serine dehydratase activity continues to increase up to 4 days of starvation, as does the maximal rate of gluconeogenesis from serine measured in vitro (145, 126). Therefore it would not be surprising to find that the inhibition by quinolinate increases with increasing duration of starvation and this indeed has been shown (126). Unfortunately in this study (126) no assessment of the effectiveness of quinolinate (4.8 mM) inhibition of gluconeogenesis from pyruvate-mediated precursors was made. This makes interpretation difficult, particularly as the interesting observation was made that in the fed rat the low rate of gluconeogenesis from serine (assessed by radioactivity incorporation into glucose) was not inhibited at all by quinolinate in the perfused liver. Support for the inference that in the fed rat gluconeogenesis proceeds predominantly

REGULATION OF SERINE METABOLISM

359

via the serine aminotransferase pathway came from experiments in vivo showing no difference in the specific activity of blood glucose after injecting [3-14C]serine or [U-~4C]serine in the fed rat, whereas in the fasted rat there was a predictable decrease in specific radioactivity from [U-~4C]serine in comparison with the alternatively labeled substrate (126). This conclusion was not entirely confirmed by the study of Beliveau and Freedland (146) who found that inhibition of gluconeogenesis by 5 mM quinolinic acid in isolated hepatocytes from fed rats was essentially the same with either lactate or serine as substrates (61-69%). With 1 mM mercaptopicolinate, inhibition from lactate was 100% and from 10 mM serine it was virtually complete (97%). However, using a more physiological concentration of serine (1 mM), gluconeogenesis was inhibited by only 53% with 1 mM mercaptopicolinate, indicating a substantial contribution by the serine aminotransferase-mediated pathway. This is a significant finding since, given the relative K m values of serine dehydratase and serine aminotransferase, the use ofsupraphysiological concentrations of serine would exaggerate the contribution of serine dehydratase to gluconeogenesis if, as seems likely (see below), the initiation of serine metabolism is rate-limiting for glucose formation. However, since gluconeogenesis in the normally-fed rat is of minor quantitative importance, the route in this dietary state is perhaps of mainly academic significance. More significant are the findings with progressive starvation, where there is a clear physiological role for increased gluconeogenesis, and here the evidence strongly supports the route for glucose formation from serine initiated by serine dehydratase as being the predominant pathway. Alternative approaches to the assessment of the relative contributions of the 2 pathways in starvation have been based on: inhibition at the level of pyruvate carboxylase by using biotin-deficient rats (59); and inhibition at the level of the transport of pyruvate across the mitochondrial membrane by the monocarboxylate translocase inhibitor o~-cyano-4-hydroxycinnamate (149) (see Fig.5). Biotin deficiency is a rather non-specific inhibition since many carboxylation reactions may be affected (though none are implicated in the serine aminotransferase-mediated pathway), and in that study the inhibition of pyruvate carboxylase activity was incomplete (65%). Nevertheless, perfused livers from 24 hr-starved, biotin-deficient rats showed an 84% inhibition of gluconeogenesis from alanine and a somewhat lesser, but still substantial, inhibition (by 72%) from serine (59). The use of cyanohydroxycinnamic acid to distinguish between the 2 pathways of serine gluconeogenesis is open to certain criticisms regarding the involvement of mitochondrial pyruvate transport in both routes and the assumption that mitochondrial transport of hydroxypyruvate (also susceptible to inhibition), in relation to the subcellular localisation of D-glycerate dehydrogenase, is not necessary for the aminotransferase pathway (113). Nevertheless, despite these reservations, under conditions where inhibition of lactate gluconeogenesis in

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KEITH SNELL

isolated rat hepatocytes was virtually complete (92%), gluconeogenesis from serine was inhibited to a lesser extent (78%) (149). Incidentally, in this same study gluconeogenesis in isolated hepatocytes from starved mice was inhibited completely from alanine and lactate by tryptophan (which acts mainly through its metabolism to quinolinate), whereas gluconeogenesis from serine was inhibited by a lesser amount (75%) (149). These alternative approaches largely confirm the conclusions that in starvation the serine dehydratasemediated pathway of gluconeogenesis predominates, but that there is evidence for the existence and minor contribution of an alternative pathway initiated by serine aminotransferase. Inhibitors have also been used as a more direct approach to the role of alternative serine gluconeogenic pathways in dietary protein-mediated changes in glucose formation from serine (see above). Diets with high or normal protein contents were used, but additionally variations in carbohydrate content within each protein level were also employed (146). With a normal diet (normal protein, high carbohydrate), gluconeogenesis from serine (measured by incorporation of radioactivity into glucose) in isolated rat hepatocytes was low with 10 mM serine as substrate, and was doubled by lowering the carbohydrate content of this diet. With the highprotein diet gluconeogenesis from I0 mM serine (but not from lactate) was increased 13- to 15-fold regardless of any variation in carbohydrate content (146). This indicates a primary role of protein content in the stimulation of gluconeogenesis by the high-protein diet. It also implies that, since gluconeogenesis from lactate was not affected by the high-protein diet, the enzymes initiating serine metabolism are rate-limiting for glucose formation. With complete inhibition of gluconeogenesis from lactate by 1 mM mercaptopicolinate, it was found that 17-25% ofgluconeogenesis from serine was resistant to inhibition (at either 1 mM or 10 mM serine) in the high-protein fed rats and this represents a conservative estimate of the proportion of glucose formation from serine proceeding via the serine aminotransferasemediated pathway (146). Interestingly, the proportion of serine metabolized via the aminotransferase-mediated pathway was virtually identical for rats on the normal protein-low carbohydrate diet compared to rats on the highprotein diet with similar carbohydrate content and yet the overall rate of gluconeogenesis from serine was increased nearly 7-fold. This must mean that metabolic flux through the aminotransferase-mediated pathway is increased by a similar factor and this is consistent with the effect of high-protein feeding on serine aminotransferase activity (at least as observed in the rabbit (15)). A comparative survey of serine aminotransferase and serine dehydratase activities in different animal species has provided further clues to the relative role of the enzymes in metabolism (87, 112). Relatively higher serine aminotransferase activity was associated with all flesh-eating animals, compared with omnivorous or herbivorous species, suggesting a metabolic

R E G U L A T I O N O F SERINE M E T A B O L I S M

361

role related to the increased dietary protein intake of the carnivorous species. Such an association is not observed for serine dehydratase (87, 150). The conversion of protein-derived amino acids to glucose must be of particular importance in relation to glucose homeostasis in carnivorous animals and the comparative survey would suggest that the pathway initiated by serine aminotransferase plays a predominant role in these species. This has been directly confirmed for the cat, where gluconeogenesis from [14C]serine is insensitive to inhibition by quinolinate or mercaptopicolinate in isolated hepatocytes (151). Moreover, isotope incorporation into [~4C]glucose in these experiments was diluted by the addition of i0 mM hydroxypyruvate to the incubation but not by 10 mM pyruvate, providing further evidence for the operation of the serine aminotransferase-mediated pathway of gluconeogenesis (151). The increase in the serine aminotransferase-mediated pathway in rats fed a high-protein diet (see above) is further evidence for the association of this pathway with glucose formation in relation to protein intake. The teleological question arises as to why this pathway should be involved at all given the adequate gluconeogenic role of serine dehydratase in other circumstances of enhanced gluconeogenesis (such as starvation). I have argued elsewhere (55, 111, 113, 115, 143, 152) that in considering the physiological significance of the alternative routes of glucose synthesis from serine, it is important to take account of the mitochondrial localization of the initial steps of the aminotransferase pathway compared to the cytosolic intracellular site of serine dehydratase. Taking this viewpoint it was suggested that the aminotransferase pathway served as part of an intramitochondrial system for efficiently coupling the mitochondrial metabolism of certain amino acids, whose pathways relate to serine formation, to a pathway solely involved in glucose synthesis. The amino acids, apart from serine itself, that are considered significant in this regard are hydroxyproline and glycine and a scheme was proposed that emphasised these features (see Fig.6) (143). It is relevant that collagen, which is particularly rich in glycine and hydroxyproIine, is likely to be especially prevalent in the diet of meat-eating carnivores. In support of this proposal I found that gluconeogenesis from hydroxyproline was less sensitive to inhibition by quinolinate (comparable to serine in fact) than alanine in perfused livers (143). [The increased involvement and particular relevance of this metabolism to the neonatal rat are discussed in the next section.] In addition, glycine has been shown to be at least as good a precursor for glucose formation as serine in cat hepatocytes, compared to its negligible rate in rat hepatocytes (151). Interestingly, this negligible rate of gluconeogenesis from glycine in rat hepatocytes becomes substantial after adaptation of the rats to a high-protein diet (134). The stoichiometry of glucose production and urea production from glycine in hepatocytes from high-protein adapted rats (134) is consistent with the

362

KEITH SNELL

COLLAGEN

P HYDROXYPROLINE

I EXCRETION

Hydroxyprollne -1 ---~n y d r oxyoxoglutar ate Glycine i I 4 liver rnitochondria

3

Glyoxylate

~-.Ser ille

I

5 GLUCOSE

FIG. 6. Proposed carbon salvage pathway for hydroxyproline released from body collagen turnover. Hydroxyproline not excreted in the urine is shown to be metabolised by enzymes in rat liver mitochondria: 1, enzymes of hydroxyproline breakdown (154); 2, 4-hydroxy-2-oxoglutarate aldolase (EC 4.1.3.16); 3, L-alanine-glyoxylate aminotransferase (EC 2.6.1.44); 4, glycine dehydrogenase complex (EC 2.1.2.10); 5, gluconeogenic pathway initiated by L-serine-pyruvate aminotransferase (EC 2.6.1.5.1), see Figs. 1 and 5.

conversion of glycine to serine (by the combined actions of the glycine dehydrogenase complex and serine hydroxymethyltransferase) and metabolism to glucose by the serine aminotransferase-mediated pathway. A similar conclusion was reached on the basis of a comparison of radioactivity incorporation from [2-14C]glycine or [U14C]serine into glucose in cat hepatocytes (151). The conversion of glycine to serine in rat liver and kidney is apparently catalyzed by a mitochondrial glycine dehydrogenase enzyme complex linked to the mitochondrial isoenzyme of serine hydroxymethyltransferase(53, 150), a subcellular localization appropriate for the proposed coupling to the serine aminotransferase-mediated pathway of gluconeogenesis. No comprehensive study of a possible association of these enzymes with gluconeogenesis has been carried out to date (see later) but the glycine dehydrogenase complex is reported to be twice as active in the dog as in the rat (87), to be very much more active in rat liver and kidney than in other tissues (53,150), and to be elevated in the neonatal rat (153) where gluconeogenesis is enhanced (see next section). The metabolism of hydroxyproline in rat liver also takes place in the mitochondria (154) leading in the final stages, via a mitochondrial 4-hydroxy2-oxoglutarate aldolase (155), to glyoxylate formation and conversion to glycine by a mitochondrial L-alanine-glyoxylate aminotransferase (111, 156). Direct studies on hydroxyproline as a gluconeogenic precursor have been confined to my own findings that the gluconeogenic rate in the perfused liver is elevated in the neonatal rat (143). Of the enzymes involved in tbe pathway of hydroxyproline metabolism to glycine: hydroxyproline oxidase and 4hydroxy-2-oxoglutarate aldolase (153), and L-alanine-glyoxylate aminotransferase (16, 111, 156-158), have high activities in carnivorous animals; have activities largely confined to the liver and kidney in the rat; have elevated activities in neonatal rat liver; and have increased activities in certain conditions associated with enhanced gluconeogenesis.

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Accepting this specialized role for serine aminotransferase in a gluconeogenic pathway for intramitochondrially-metabolized amino acids, the question still remains as to whether the metabolic role of serine dehydratase is entirely accounted for by gluconeogenesis. In principle other metabolic fates of the pyruvate carbon are possible (see Fig. 1), although there is little evidence to associate adaptive enzyme changes with energy generation or lipogenesis. It is perhaps significant that the enzyme is one of the few that directly liberates ammonia from an amino acid. There is a possibility that the enzyme could function as a terminal deamination system for a number of amino acids, particularly in view of the doubts cast upon the operation of glutamate dehydrogenase in the direction of oxidative deamination in vivo. However, the low activity of the enzyme would not provide much support for its general role in amino acid deamination. From the comparative survey of serine dehydratase activity in 15 animal species already referred to (87), an inverse correlation between body weight and liver enzyme activity was shown. The authors considered the possibility that serine deamination might be linked to thermogenesis and thus contribute to the basal metabolic rate which is known to be species-size dependent. This may be an aspect of the 'specific dynamic action' of dietary amino acids and, since it is not shared by other nutrients, implies that ammonia liberation from amino acids is involved in the stimulation of futile cycles resulting in an increased ATP turnover. The precise loci of the specific dynamic action are not known but could include, for example, a stimulation of the turnover of glutamine via glutamine synthetase and glutaminase in the liver by ammonia liberated from serine by serine dehydratase. Glutamate dehydrogenase does not show any dependence on species body size (87) and is unlikely to be involved in ammonia generation for this purpose. It is conceivable that gluconeogenesis itself may show a relationship with species body size; however no comparative animal surveys on glucose turnover rates have been reported. Thus as a consequence of limited carbohydrate storage capacity and an increasing metabolic rate with diminishing species size, it may be necessary to have the metabolic rate coupled to the capacity for glucose synthesis from amino acids to prevent an inadvertent depletion of carbohydrate reserves (87). L-Serine dehydratase and L-serine-pyruvate aminotransferase in developing rat tissues. The first complete developmental profiles of rat liver serine dehydratase and serine aminotransferase were published from this Laboratory (22). We found a biphasic pattern of development of serine dehydratase with peaks of activity in the neonatal and weanling periods (Fig.2B). The separate peaks had been identified in some previous studies, but not in others, due to the need to follow the developmental pattern at very close time intervals (see 22). The biphasic developmental pattern is typical of enzymes involved in amino acid metabolism and some of the enzymes of urea biosynthesis (55, 152, 159). The trough of activity in the mid-suckling period,

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when gluconeogenesis from other non-amino acid precursors (lactate, glycerol) is at its height, has been interpreted by the author as signifying a low general capacity for glucose synthesis from serine at a time when the involvement of amino acids in protein synthesis for rapid growth is at its greatest (55, 160). The neonatal peak of activity may be viewed as a response to the glucose-deprived state at birth, before re-orientation of energy metabolism towards lipid oxidation has become fully established (161). The second postnatal peak at weaning is associated with a dietary repletion of carbohydrate and an increasing amino acid intake allowing for the conversion of amino acids to storage carbohydrate by hepatic glyconeogenesis (55). Notwithstanding these arguments, our observations on the maximal metabolic flux from serine to glucose in the postnatal rat liver in vitro indicate an enhanced capacity in the mid-suckling period (55, 115, 143, 152). This pattern closely matches that observed for serine aminotransferase activity (Fig. 2D) (22, 88) and for o-glycerate dehydrogenase activity (19) during postnatal development. I have argued that rather than reflecting the capacity for gluconeogenesis from serine per se, this signifies the operation of a pathway from hydroxyproline and glycine to glucose involving mitochondrial serine as an intermediate (55, 143, 152). The elevation of the relevant enzymes in the neonatal rat has already been described (see above). I have shown that gluconeogenesis from hydroxyproline is increased in the perfused liver of the neonatal rat and is relatively insensitive to inhibition by quinolinate (35%), as is gluconeogenesis from serine (38%), compared to a 74% inhibition from alanine (143). This again indicates the operation of the gluconeogenic pathway involving serine aminotransferase at this stage of development. The particular relevance of this overall pathway to the metabolism of the neonatal rat lies in the turnover of body collagen. NormaIly collagen in the nongrowing animal has a rather low rate of turnover, but in the growing neonatal rat where considerable extension and remodeling of anatomical structures are taking place, particularly with the skin which in young rats is about 22% of the total body weight, there is a greatly increased turnover of collagen protein. Probably much of the glycine, which is prevalent in collagen (27%), can be reincorporated back into the newly-synthesized protein after collagen breakdown, but hydroxyproline cannot be reincorporated and is either excreted or catabolized in the liver. There is an increased urinary excretion of free hydroxyproline in young mammals signifying an accelerated turnover of collagen (162). The hepatic metabolism of hydroxyproline and glycine via a pathway leading to glucose synthesis (see Fig. 6) serves as a salvage pathway for reducing carbon loss by excretion, in the stringent metabolic economy imposed by the demands for growth and energy metabolism in the neonatal rat. A quite different, and entirely speculative, role for the serine aminotransferase-mediated gluconeogenic pathway which I have previously discussed (55, 111, 115, 152, 156) is that if 4-hydroxy-2-oxoglutarate (the

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intermediate on the pathway of hydroxyproline breakdown) could be formed by hydroxylation of 2-oxoglutarate (or hydroxylation of glutamate followed by transamination) and then undergo aldolase cleavage to glyoxylate, transamination to glycine and conversion to serine, then a complete sequence would exist to allow a citric acid cycle intermediate (2-oxoglutarate) containing acetyl CoA carbon to be converted to glucose. In effect this would enable the synthesis of glucose from fatty acids to take place in the neonatal rat, a metabolic function that would be entirely appropriate at this unique stage of development (55, 111, 152). The conventional enzymes of the 'glyoxylate cycle' are not present in neonatal rat liver (163), but there is some equivocal evidence that the neonatal rat is capable of carrying out such a scandalously heretical metabolic transformation (for refs. see 55, 152). The physiological stimuli which control the developmental adaptations of serine dehydratase and serine aminotransferase have been studied and their mechanism of action investigated, but there is still much that is not known. For serine dehydratase it is conceivable that given the existence of two multiple forms of the enzyme (as discussed above), the two peaks of activity observed during postnatal development could correspond to a differential, developmental sequence for the two forms. This attractive hypothesis was not supported by work in this Laboratory: DEAE-cellulose chromatography and cellulose acetate electrophoresis of crude liver extracts prepared from 2-dayold or 15-day-old rats (corresponding to the age at which peaks of activity were found in these rats) revealed the presence of both multiple forms at both ages in identical proportions (40%, Form I and 60%, Form II) (22). At least for the second, postnatal peak, the 2 forms had identical properties to those reported for the adult multiple forms (see above; Table 5). The electrophoretic patterns we found during development are shown in Figure 2B. At the trough of activity in the mid-suckling period only Form II was observed by electrophoresis, although at this low total activity Form I may have been below the sensitivity of detection. The neonatal peak of serine dehydratase is apparently elicited by the increased glucagon secretion that occurs naturally at birth and which leads to elevated hepatic cyclic AMP levels (166). We showed that enzyme activity was very low in fetal liver, when glucagon levels are low, and could be prematurely induced in utero by an injection ofglucagon or dibutyryl cyclic AMP (but not by hydrocortisone or thyroxine). The induction can be blocked by inhibitors of protein synthesis (cycloheximide) or mRNA production (actinomycin D) (Fig. 7A). This implies that the induction involves synthesis of new enzyme protein as a result of increased formation of mRNA coding for serine dehydratase. The natural rise in serine dehydratase activity after birth is also blocked by actinomycin D, and is suppressed by the administration of glucose or insulin which presumably counteracts the natural increase in hepatic cyclic AMP after birth and so prevents induction (Fig. 7B). The inhibition of the

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KEITH SNELL C.

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FIG. 7. Hormonal regulation of hepatic L-serine dehydratase in (A) fetal, (B) newborn (Caesarianderived term fetuses), and (C) postnatal (10-13 days post partum) rats. Fetal rats were injected intraperitoneally in utero on day 20 of gestation with agents and at the dosages indicated in the legend to Fig. 4. Animals were killed 24 hr after injection and the columns indicate the mean relative enzyme activity for 4 to 6 determinations. Newborn rats were injected intraperitoneally at the time of delivery or at 6 hr postpartum with actinomycin D (0.005 rag/rat) and killed at 12 hr post partum. Other newborn rats were injected intraperitoneally at the time of delivery and again at 3 hr post partum with glucose (25 rag/rat) or insulin (0.04 I.U./rat) and killed at 6 hr post partum. The columns indicate the mean relative enzyme activity for 6 to 8 determinations. Postnatal rats were injected intraperitoneally with glucagon, dibutyryt cyclic AMP, hydrocortisone (hydrocort), tryptophan or actinomycin D at dosages of 0.5, 5.0, 2.5, 50, 0.05 rag/100 g body wt., respectively, and killed 12 hr later. Some animals injected with hydrocortisone alone or hydrocortisone together with an intragastric intubation of glucose (625 mg of glucose/100 g body wt.) were killed 6 hr later. The columns indicate the mean relative enzyme activity for 4 to 6 determinations. Enzyme activities were recorded as units/g of liver, and are shown here relative to control values in saline-injected animals at zero time (as indicated by the broken lines).

natural increase at birth by actinomycin D has some interesting features in that if administration of the inhibitor is delayed until 6 hr after birth, although further enzyme induction is prevented, there is no decline in activity (despite the enzyme having a half-life of about 6 hr). One explanation is that during the period of induction after birth, enzyme protein degradation may be blocked so that activity is maintained for a while even when mRNA production is inhibited. The absence of degradation has also been reported for the induction of phosphoenolpyruvate carboxykinase at birth (165), and may be a general characteristic of developmental inductions. The effect of glucagon in utero on serine dehydratase was first described by Greengard (95), and the inhibition by actinomycin D and cycloheximide was also observed by Oliver (164). Using antibody raised against purified enzyme, Miura and Nakagawa (167) confirmed that the induction of serine dehydratase at birth involves at least the increased synthesis of new enzyme protein. However, consistent with the

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block in enzyme protein degradation proposed above, it can be seen in this study that immunoprecipitable-enzyme protein synthesis shows a large increase between birth and 6 hr post partum, in parallel with activity changes, but a much smaller increase between 6 hr and 48 hr when the greatest increase in enzyme activity occurs. The induction by glucagon and dibutyryl cyclic AMP, its partial suppression by insulin, and its prevention by the inhibitors actinomycin D or cordycepin have been recently demonstrated by Oliver in day 19 fetal hepatocytes in cell culture (100). Interestingly, induction required the presence of dexamethasone, even though the steroid alone had no effect. An increased secretion of corticosteroids would already have occurred naturally in vivo to exert this permissive effect on enzyme induction. The requirement for both corticosteroid and glucagon in the neonatal induction of serine dehydratase may account for the presence of both multiple forms of the enzyme at the peak of induction. The transient nature of the peak of activity induced in the neonatal rat is an unusual feature. The level of inducers, both plasma glucagon and hepatic cyclic AMP, remains elevated throughout the suckling period and other enzymes presumed to be induced by the same stimuli (e.g. serine aminotransferase, see below) remain at an induced level throughout this period. It is clear that a loss of responsiveness to the inducers occurs, because the ability of exogenous glucagon to induce serine dehydratase disappears early in postnatal life (22). It is known that during the early- to mid-suckling period the adrenal cortex becomes quiescent (6), and it may be that a decline in circulating corticosteroids will eliminate their permissive action so that glucagon (and/or cyclic AMP) is no longer able to elicit an inductive effect. The mechanism by which the steroid hormone exerts its action is of considerable interest and should be amenable to study in the hepatocyte cell culture system. The interactive effects of adrenal corticosteroids and glucagon are probably also involved in the adaptive changes reflected by the second peak of serine dehydratase activity around 15 to 20 days post partum. As indicated above, soon after birth serine dehydratase becomes refractory to induction by glucagon or cyclic AMP. However, we showed that at this time hydrocortisone was effective in inducing the enzyme prior to its natural developmental upsurge (22) (Fig. 7C). Tryptophan, which probably acts mainly via the release of adrenal corticosteroids in the whole animal (see earlier discussion), was not as effective as exogenous steroid hormone in the 10-day-old rat probably because of the quiescence of the pituitary-adrenal axis at this time. After adrenal function is re-established at about day 15post partum, tryptophan was as effective as hydrocortisone in inducing serine dehydratase (22). Induction by a combination of maximally effective doses of hydrocortisone and tryptophan was no greater than with hydrocortisone alone (Fig. 7C), supporting the view that tryptophan action is mediated by corticosteroid release in vivo. Together these findings implicate the adrenal

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corticosteroids as the natural developmental stimulus for serine dehydratase induction in weanling rat liver. We found that administration of hydrocortisone was effective in inducing serine dehydratase throughout postnatal life, but became progressively less effective after weaning (22). This pattern is associated with the natural decline in circulating glucagon and hepatic cyclic AMP concentrations after weaning, and this suggests that again it is the interaction between glucagon and adrenal corticosteroid which is responsible for enzyme induction. Supportive evidence for this hypothesis is provided by the suppressive effect of glucose on enzyme induction by hydrocortisone in the 10-day-old rat (Fig. 7C). The administration of glucose will result in a decrease in the elevated hepatic cyclic AMP levels (via effects on pancreatic hormone secretion) in the suckling rat, and as a result hydrocortisone becomes ineffective for induction. The interactive mechanism probably accounts for the transient nature of the natural postnatal induction and may possibly explain the appearance of both multiple enzyme forms at this time. As glucagon levels decrease around weaning, so the effect of corticosteroid and glucagon on induction will weaken and enzyme levels will decline to a basal value. The premature induction of serine dehydratase evoked by either hydrocortisone or tryptophan administration to rats prior to the natural developmental upsurge is inhibited by actinomycin D (Fig. 7C). This suggests that the induction is dependent, at least initially, on the increased production of mRNA for serine dehydratase. Interestingly, once natural induction of the enzyme is under way further induction by hydrocortisone (or tryptophan) appears to be insensitive to actinomycin D (22). Although a translational effect at the later stage of hydrocortisone induction may be suggested by this finding, interpretation is difficult in the whole animal in vivo. The use of cultured hepatocytes from postnatal rats may resolve some of the inherent ambiguities and enable a more precise definition of the mechanistic roles of steroid hormone and glucagon to be developed. Taking an overall view of the developmental adaptations of serine dehydratase, it appears that the cellular 'competence' to activate the gene involved in serine dehydratase production is ac quired at an early stage in fetal life [day 16 or 17 of gestation according to experiments with cultured hepatocytes by Oliver (100)]. The expression of gene activity and the appearance of serine dehydratase protein is then governed by the interactive action of adrenal corticosteroids and glucagon, and temporal fluctuations in the availability and secretion of each of these hormones can apparently account for the seemingly complex pattern of development of serine dehydratase during perinatal and postnatal life. The developmental pattern of serine aminotransferase presents a rather simpler analysis. A single induction occurs at the time of birth, enzyme is elevated throughout suckling and declines to a basal adult value at weaning. This pattern parallels the increased secretion of glucagon at birth (or more

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relevantly the marked increase in the plasma glucagon:insulin ratio) and the consequent rise in hepatic cyclic AMP, and the decline of these stimuli after weaning. This would implicate glucagon and cyclic AMP in the natural developmental induction and, in line with this prediction, we found that activity could be prematurely induced in fetal liver by the administration m utero of glucagon or dibutyryl cyclic AMP (but not hydrocortisone or thyroxine) (Fig. 4). The premature induction was blocked by actinomycin D (22), as was the natural increase at birth (K. Snell, unpublished observations). This suggests the involvement of increased mRNA production in the enzyme induction. The postnatal increase in serine aminotransferase is confined to the glucagon-inducible mitochondriaI enzyme (113) and experience with the adult enzyme (see earlier discussion) would support a role of increased cytoplasmic mRNA in the developmental change. Intracellular processing and specific organelle-directed enzyme protein translocation would also appear to be involved in the neonatal induction of serine aminotransferase. These aspects have still to be confirmed directly for the developmental enzyme adaptation. It also remains to be determined when cellular 'competence' to activate serine aminotransferase gene expression is acquired in fetal life. Sequencing of the purified enzyme protein and the construction of cDNA probes will allow the precise developmental timing of genetic competence and gene activation to be determined and to be related to the temporal sequence of appearance and functional coupling of the various components of the developmental stimulus complex (e.g. glucagon receptor-adenyl cyclase-cyclic AMP productionnuclear interaction). As is to be expected from the tissue distribution of serine dehydratase and serine aminotransferase in the adult, fetal tissues are devoid of the enzyme activities, apart from the very low activities in fetal liver noted above (Table 3). L-Serine dehydratase and L-serine-pyruvate aminotransferase in neoplastic rat tissues. In our study we found that serine dehydratase activity was undetectable in all the hepatomas investigated (apart from Morris hepatoma 7800) and was virtually absent from all other neoplastic tissues examined (Table 4). With respect to tumors of non-hepatic origin this is hardly surprising given the normal tissue distribution of the enzyme (Table 3). We have also found that serine dehydratase activity is absent from diethylnitrosamine-induced primary hepatocarcinomas (biological system as described elsewhere (170)), and from ethionine-induced transplantable hepatoma lines (designated UA and WDA (171)). We found a similar pattern for serine aminotransferase activity, which was absent from all neoplastic tissues studied with the exception of a low activity (13% of the normal rat liver activity) detected in hepatoma 7800. No previous studies of serine aminotransferase activity in neoplastic tissues have been reported. The results suggest that in general both of the enzymes involved in gluconeogenic pathways from serine are lost in neoplastic tissues and this is in line with

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previous work from Weber's laboratory showing that other gluconeogenic enzymes are decreased in activity or absent in hepatomas (3, 4, 4a, 174). Serine dehydratase has been studied previously in a series of transplantable Morris hepatomas (58, 168), in a Novikoffhepatoma and a primary hepatoma arising during azodye-induced carcinogenesis (169). Activity was undetectable in the primary tumor and the Novikoff hepatomas (169), and very low or absent in all 4 of the Morris hepatomas in 1 study (58) and in 5 of the 9 Morris hepatomas in another study (168). No correlation of enzyme activity (when present) with growth rate of the Morris hepatomas was observed but an apparent relationship with increasing chromosomal abnormality (number and morphology) in the hepatomas was noted (58, 168). Karyotypic aberration and instability evidently results in a variable expression of serine dehydratase since in the same hepatoma (7794B) activity was reportedly 8% (58) or 200% (168) of that in normal rat liver. This is also apparent in hepatoma 7800 which was found to have 20% of normal liver serine dehydratase activity, but this was noted to increase in activity with increasing generation times and increasing karyotypic abnormality (168). At a late generation time (generation 103) in our study (Table 4), activity in hepatoma 7800 was 80% of that in normal rat liver (Table 3). In contrast, in a tumor that is relatively stable and which has a normal chromosome number and morphology, activity was undetectable and this was independent of the generation time (58, 168). The variation in activity of serine dehydratase in Potter's study of Morris hepatomas was exactly paralleled by the activity oftyrosine aminotransferase (but not by other enzymes) (168). Since tyrosine aminotransferase and serine dehydratase are subject to adaptive regulation by the same stimuli, the changes in activity in the hepatomas might signify a variation in factors governing enzyme regulation. However, it was found that control of enzyme levels by dietary-protein variation was lost in the hepatomas (168) and there was no correlation between enzyme activities and the basal cyclic AMP level, or its responsiveness to hormones, in the hepatomas (172, 173). Thus the different serine dehydratase activities in certain hepatomas appear to represent a constitutive change in gene expression. Investigation of the distribution of the multiple forms of serine dehydratase in hepatomas 7800 and 5123 has shown that activity is mainly (80%) present as Form I (22a). This is different from the situation in liver from normal animals where activity is present as Form II (22a). If the multiple forms of serine dehydratase are the result of post-translational processing, it is conceivable that Form I might not be functionally active in vivo, despite its catalytic activity in vitro. Thus, the serine dehydratase activity assayed in certain hepatomas may not represent a functional capacity. Clearly further characterization of the multiple forms of the enzyme and their relationship to each other is required. If indeed protein phosphorylation is a relevant mechanism to the interconversion of the two

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forms, then the association of tyrosine-phosphorylating protein kinase activity with certain oncogene products might account for the predominance of Form I of serine dehydratase in hepatomas. Pathways o f Serine Utilization: L-Serine Hydroxymethyltransferase. Regulatory properties of L-serine hydroxymethyltransferase. The properties of mammalian serine hydroxymethyltransferase have recently been reviewed by Schirch (175) and will be dealt with only briefly here. The purified enzyme from rat or rabbit liver has a subunit relative molecular mass of about 50,000 and is composed of 4 identical subunits, each containing a single pyridoxal phosphate co-factor molecule (175, 176). The enzyme is able to catalyze a broad range of reactions covering aldolytic cleavage, transamination and decarboxylation. The reversible reaction involving cleavage of serine to glycine and formaldehyde, with the removal of the latter from the active site by transfer to tetrahydrofolate, appears to be the major physiological action of the enzyme, serving to generate 5,10-methylenetetrahydrofolate as a 'onecarbon unit' for biosynthetic purposes. Primary among these biosynthetic uses is the direct participation of 5,10-methylenetetrahydrofolate in the formation of the pyrimidine nucleotide thymidylate from uridylate and the indirect participation, via interconversion with other tetrahydrofolate onecarbon units, in the de novo pathway of purine biosynthesis. The enzyme is distributed about equally between the cytosol and mitochondria in rat liver (20a), but in rabbit (176) and monkey ( 181) liver it is mainly cytosolic (75-78%). It appears to exist as distinct isoenzymes which in the rat differ in immunochemical properties, amino acid composition, electrophoretic mobility, pH optimum and stability (20a, 176, 177). The isoenzymes in rabbit liver also differ in some molecular properties, although not as markedly as in the rat (178-180). The substrate affinities seem to be identical for both isoenzymes, regardless of species. The Km values reported are: L-serine, 0.5-0.6 mM (in the presence of tetrahydrofolate), tetrahydrofolate, about 0.01 raM; glycine, 1.2-1.8 mM; and methylenetetrahydrofolate, 0.13 mM (20a, 177, 180). These values suggest that the enzyme in vivo is probably near saturation. The similarity of the kinetic constants for both isoenzymes and the ready reversibility of the reaction suggest that the direction of the reaction catalyzed by each isoenzyme in vivo is largely dependent on the concentrations of reactants in the respective subcellular compartments, which are presently unknown. As regards other catalytic reactions, Palekar et al. (177) have reported that the rat cytosolic isoenzyme catalyzes aminomalonate decarboxylase and L-allothreonine aldolase activities, whereas the mitochondrial isoenzyme does not. However, in other studies with the rabbit (180) and rat (176) mitochondrial isoenzymes, low (10-20% of the cytosolic isoenzyme activity) allothreonine aldolase activity was detected. We have been exploiting the difference in reactivity of the 2 AEN 2 2 - M

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isoenzymes towards allothreonine in order to distinguish the mitochondrial and cytosolic serine hydroxymethyltransferase activities in rat liver homogenates, and we find that more than 90% of the total homogenate allothreonine aldolase activity is associated with the cytosolic fraction (K. Snell, unpublished observations). Unlike the purified rabbit liver isoenzymes (180), both the purified rat liver isoenzymes are devoid of threonine aldolase activity 076, 177). As regards regulatory features, the 2 isoenzymes show cooperativity between the binding of serine and tetrahydrofolate substrates. In addition competitive inhibition, with similar affinities to one another and for both isoenzymes, has been shown with 5-methyltetrahydrofolate and 5formyltetrahydrofolate (175, 180). Since the one-carbon tetrahydrofolate cofactors are present at higher concentrations in the cell than the substrate tetrahydrofolate, such inhibition can be considered to have physiological relevance in the interregulation of the components of the one-carbon tetrahydrofolate pool. Moreover such a feedback system on serine hydroxymethyltransferase will serve to attenuate activity when the onecarbon pool is replete and allow serine to be directed to alternative pathways of utilisation, such as the gluconeogenic pathways, lipogenesis or oxidation (Fig. 1). This is a significant regulatory mechanism because of the very high Km values and low maximal activities of the alternative enzymes of serine utilization (Table 1). A more controversial regulatory system for serine hydroxymethyltransferase relates to the positive homotropic cooperativity with respect to tetrahydrofolate reported by Rao and colleagues (181). N A D H and N A D + were reportedly positive and negative allosteric effectors respectively of the enzyme (181), but only at concentrations which were nonphysiological (10-50 raM). Schirch (182) has since accounted for the apparent cooperativity in this and other studies as an artifact in the assay method due to tetrahydrofolate instability; there was no evidence for an allosteric effect of NADH. With respect to enzyme adaptation, what few studies have been carried out with the liver enzyme (for other tissues see below) are difficult to interpret because of the failure to differentiate between cytosolic and mitochondrial activities of serine hydroxymethyltransferase. Discussing the regulation of the enzyme Schirch (175) comments that "this has been a relatively unexplored a r e a . . , only a few brief studies speak to questions of function and control." Suda (75) reports an increase in rat liver cytosolic serine hydroxymethyltransferase after hydrocortisone treatment, during starvation and in diabetic animals, but no quantitative data were presented. In a preliminary study, we have assayed serine hydroxymethyltransferase in a rat liver cytosol fraction prepared in 0.4 M sucrose and have further discriminated cytosolic activity by measuring altothreonine aldolase activity (see above). We found that starvation for 48 hr decreased cytosolic activity by 20% [0.308 _ 0.009 (4)

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for starved animals; 0.388 + 0.007 (6) for fed controls, in #mol/min per g of liver]. This response would appear to be physiologically appropriate for a situation in which serine is being utilized increasingly for gluconeogenesis rather than cellular nucleotide biosynthesis. It is noteworthy that a similar response is observed for the enzymes ofserine biosynthesis and this supports an argument to be advanced later in this article that the pathway ofserine biosynthesis and serine utilization via serine hydroxymethyltransferase may be metabolically coupled in certain situations. It is generally considered that the primary role of the cytosolic serine hydroxymethyltransferase is in the provision of methylenetetrahydrofolate, and consequently other one-carbon tetrahydrofolate co-factors, for nucleotide biosynthesis to support RNA and DNA formation. In this connection, it has recently been reported that an unspecified proportion of cytosolic serine hydroxymethyltransferase activity is associated in a multienzyme complex with other enzymes involved in the utilization and production of tetrahydrofolate co-factors for de novo purine biosynthesis (27). These reactions and the enzymes concerned are illustrated in Figure 8. Such a ROLE OF CYTOSOLIC SERINE HYDROXYMETHYLTI1ANSFERASE

I

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2

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FIG. 8. Role of cytosolic t-serine hydroxymethyltransferase in a putative multi-enzyme complex involved in folate co-factor interconversions and purine nucleotide biosynthesis. The enzymes involved are: 1, L-serine hydroxymethyltransferase (EC 2.1.2.1); 2, methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5); 3, phosphoribosylglycinamide formyltransferase (EC 2.1.2.2); 4, formyltetrahydrofolate synthetase (EC 6.3.4.3); 5, phosphoribosylaminoimidazoleearboxamide formyltransferase (EC 2.1.2.3); 6, methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9). F H , tetrahydrofolate; GAR, glycinamide ribonucleotide (phosphoribosylglycinamide); FGAR, formylglycinamide ribonucleotide (phosphoribosyl-N-formylglycinamide) AICAR, aminoimidazolecarboxamide ribonucleotide (phosphorlbosyl-5-amino-4-imidazolecarboxamide); FAICAR, formamidoimidazolecarboxamide ribonucleotide (phosphoribosyl-5-formamido-4imidazole-ca rboxamide).

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complex, which is analogous to the multienzyme complexes identified in the pyrimidine biosynthetic pathway (4a), provides an interacting system to increase the efficiency of co-factor transfer between catalytic sites and to coordinate the utilization and regeneration of the central tetrahydrofolate pool. It is interesting to note the participation of 5,10-methenyltetrahydrofolate cyclohydrolase in the multienzyme complex, which will contribute to an equalization of the production of the 2 purine biosynthetic pathway intermediates, a function enhanced by the observed inhibitory regulation of the cyclohydrolase by 10-formyltetrahydrofolate (Ki of 19 ~M (27)). It is noteworthy too that the inclusion of serine hydroxymethyltransferase in the complex not only serves to provide an essential tetrahydrofolate cofactor, but also provides glycine which is utilized in stoichiometric amounts in the purine biosynthesis pathway (in the reaction catalyzed by phosphoribosylglycinamide synthetase). An even more direct involvement of serine hydroxymethyltransferase in nucleotide biosynthesis is in the production of methylenetetrahydrofolate and its subsequent utilization via thymidylate synthase for the formation of thymidylate, the DNA-specific nucleotide. The folate co-factor produced in this latter reaction is dihydrofolate which is reconverted to tetrahydrofolate by dihydrofolate reductase. The 3 enzymic reactions constitute a "thymidylate synthesis cycle" (Fig. 9). There is evidence for the possible association ofthymidylate synthase and dihydrofolate reductase in a multienzyme complex involved in the channeling of UDP to dTTP (183), and it seems plausible that a part of the cytosolic serine hydroxymethyltransferase activity could be associated with this multienzyme complex. This would in effect generate a second, functionally compartmentalized, cytosolic pool of tetrahydrofolate distinct

ROLE OF CYTOSOLIC SEI/INE HYDI{OXYMETIIYI,TIUkNSFEIIASI,~

FIG. 9. Role of cytosolic L-serine hydroxymethyltransferase in the thymidylate synthesis cycle. The enzymes involved are: 1, L-serine hydroxymethyltransferase; 2, thymidylate synthase; 3, dihydrofolate reductase (EC 1.5.1.3). FH2, 7,8-dihydrofolate; FH4, 5,6,7,8-tetrahydrofolate; dUMP, uridylate; dTMP, thymidylate.

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375

from that involved in purine biosynthesis. A regulatory network for coordinating these folate co-factor pools would appear to be essential if a balanced production of purine and pyrimidine nucleotides for DNA synthesis is to be achieved. The role of the mitochondrial serine hydroxymethyltransferase isoenzyme is a much more controversial matter. It would seem unlikely that it participates in mitochondrial nucleotide biosynthesis. Apart from dihydroorotate oxidase, no other step in the de novo pathway of pyrimidine biosynthesis has been detected in mitochondria, including dihydrofolate reductase (183). The transport of cytoplasmic pyrimidine nucleotides a n d / o r the operation of the salvage pathway via a mitochondrial thymidine kinase appears to be necessary to preserve the distinct mitochondrial pyrimidine nucleotide pool. A role for mitochondrial serine hydroxymethyltransferase in nucleotide metabolism has been proposed by Cybulski and Fisher (184). Drawing an analogy with the involvement of isoenzymes of malate dehydrogenase and aspartate aminotransferase in the transfer of reducing equivalents across the mitochondrial inner membrane, they propose a shuttle system for the transfer of one-carbon units from the mitochondria to the cytosol involving serine hydroxymethyltransferase isoenzymes. The lack of folate co-factor transport across the mitochondrial inner membrane, implying the existence of separate cytosolic and mitochondrial folate coenzyme pools, is their main line of evidence to support this model. Serine formed from glycine and methylenetetrahydrofolate by the mitochondrial isoenzyme serves as a one-carbon unit carrier across the mitochondrial membrane. There it is reconverted to glycine by the cytosolic enzyme, with the production of the folate co-factor for use in nucleotide biosynthesis, and the transport ofglycine back into the mitochondrial matrix completes the shuttle cycle (184). They propose that the mitochondrial oxidation of sarcosine and dimethylglycine might provide the formaldehyde necessary to generate methylenetetrahydrofolate, although this would seem a quantitatively trivial source of onecarbon units. Alternatively, oxidation ofglycine by the mitochondrial glycine dehydrogenase complex could provide a source of methylenetetrahydrofolate, although this would upset the stoichiometry of a balanced shuttle cycle. Other proposals for a role for the mitochondrial serine hydroxymethyltransferase isoenzyme also link it with the glycine dehydrogenase complex. The glycine dehydrogenase complex (or "glycine cleavage system', or "glycine synthase system ~) has been thoroughly characterized by Kikuchi and his colleagues (150). It has similarities to the mitochondrial 2-oxoacid dehydrogenase complexes and is made up of 4 components: a glycine decarboxylase, an aminomethyltransferase, a 5,I0-methylenetetrahydrofolasynthesizing protein and a dihydrolipoyl dehydrogenase (P, H, T, and L proteins, respectively) (150), and is largely associated with the inner mitochondrial membrane (184). Although the mitochondrial serine

376

KEITH SNELL

hydroxymethyltransferase activity is largely (70-90%) located in the soluble matrix, a loose association of a small proportion with the mitochondrial inner membrane may occur (184). The association of serine hydroxymethyltransferase with the glycine dehydrogenase complex, possibly at the mitochondrial inner membrane, has been proposed by Kikuchi (150) as an enzymic mechanism to account for the oxidation of serine according to the equations: Serine + tetrahydrofolate ~---~glycine + NS,N ~°- methylenetetrahydrofolate Glycine + NAD ÷ + tetrahydrofolate ~-"~CO2+ NH4+ + NADH ÷ + NS,N z° -methylenetetrahydrofolate Formation of glycine from serine catalysed by serine hydroxymethyltransferase (first reaction) is followed by glycine oxidation catalysed by the glycine dehydrogenase complex (second reaction). The methylenetetrahydrofolate produced in both reactions could then undergo oxidation to CO2

ROLEOF MITOCHONDRIAL SERINEHYDROXYMETHYLTRANSFERASE

~

[rclethyle~

I methenyl- FHL~I

FIG. 10, Role of mitochondrial L-serine hydroxymethyltransferase in a putative glycine cycle for the interconversion of glycine and serine. The enzymes involved are: 1, glycine dehydrogenase complex (EC 2.1.2.10); 2, L-serine hydroxymethyltransferase (EC 2.1.2.1); 3, methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5); 4, methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9); 5, formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). FH4, tetrahydrofolate.

REGULATION OF SERINE METABOLISM

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via methenyltetrahydrofolate and N~°-formyltetrahydrofolate (see reactions 3-5 in Fig. 10), with the regeneration oftetrahydrofolate preventing depletion of the mitochondrial folate pool. This sequence seems unlikely on a number of grounds. The formation of methylenetetrahydrofolate in the serine hydroxymethyltransferase reaction is unlikely to be conducive to the operation of the reversible glycine dehydrogenase reaction in the direction indicated. More significantly, it has been shown that the formation of ~4COz from [2-t4C]glycine (which would label the methylene carbon of the tetrahydrofolate co-factor) is very low compared to the yield from [t~4C]glycine in isolated rat liver mitochondria (150, 185) or in rat kidney cortex slices (53). Noting my earlier proposal for a mitochondrial gluconeogenic pathway involving a transformation of glycine to serine, it can be proposed that the glycine dehydrogenase complex is coupled to serine hydroxymethyltransferase in the following sequence (see reactions 1 and 2 in Fig. 10): Glycine + NAD + + tetrahydrofolate ~-----~CO2+ NH4 + + N A D H + H + + NS,N ~°- methylenetetrahydrofolate Glycine + N~,N ~°- methylenetetrahydrofolate ~ Net:2Glycine+NAD +

~serine+CO2+

serine + tetrahydrofolate NH4 ~ + N A D H + H

÷

In this 'glycine cycle' formulation (Fig. 10), tetrahydrofolate is continuously regenerated by serine hydroxymethyltransferase. However, methylenetetrahydrofolate could be reconverted back to tetrahydrofolate in an oxidative sequence (reactions 3-5), which would thus effect the complete oxidation of glycine. As noted above the evidence suggests that this does not occur to any appreciable extent. The coupling of glycine metabolism to serine synthesis in the manner proposed would involve a stoichiometry of 1:1 for the liberation of t4CO2 and the formation of [laC]serine from [l-C]glycine, and this was observed experimentally in rat liver mitochondria (150). Furthermore, the predicted stoichiometry for glycine utilization, serine formation, ammonia production, and 14CO2 liberation from [1-~4C]glycine is 2:1:1:1 for the scheme as presented (Fig. 10), and this was demonstrated experimentally by Rowsell and colleagues using rat kidney cortex slices (53). The net reaction for the glycine cycle has a continuing requirement for the oxidative regeneration of NAD + and the stoichiometry of oxygen consumption in relation to [1-14C] glycine decarboxylation was confirmed in rat liver mitochondria by Olson and colleagues (185). The evidence for a requirement for N A D H reoxidation was supported by the inhibitory effect of respiratory chain inhibitors on glycine decarboxylation in liver mitochondria (185), and by the dependency on oxygen for ammonia liberation from glycine

378

K E I T H SNELL

in kidney slices (53). Olson and colleagues (185) have further demonstrated that glycine decarboxylation in intact rat liver mitochondria is sensitive to alterations in the NAD(H) or NADP(H) oxidation-reduction couples which may be brought about by the presence of other oxidizable substrates. Reducing substrates or increases in the proportion of reduced redox carrier NAD(P)H inhibited glycine metabolism. This effect of N A D H is the opposite to that reported for the alleged allosteric interaction with serine hydroxymethyltransferase (see above) and throws further doubt on the relevance of these latter observations. Propionate, at concentrations within the physiological range (and which did not uncouple mitochondria), stimulated glycine decarboxylation with a concomitant decrease in mitochondrial N A D P H (but not NADH) levels (185). The physiological significance of the regulatory effects on glycine metabolism has still to be elucidated, as indeed has the precise mechanism, but these recent studies are noteworthy for, as the authors point out (185), "little or no information has been reported on what mechanisms normally operate to regulate hepatic glycine catabolism." The formulation of radically different roles for the cytosolic and mitochondrial serine hydroxymethyltransferase isoenzymes proposed in this review (Figs. 8-10) emphasizes the need to distinguish the regulatory and adaptive responses of the isoenzymes separately. Tissue distribution ofL-serine hydroxymethyltransferase. In our survey of the tissue distribution of total serine hydroxymethyltransferase activity we found the enzyme to be fairly ubiquitous (Table 3), as with the enzymes of serine biosynthesis (Tables 2 and 3). Enzyme activity is highest in the liver and kidney, with appreciable levels in the spleen and to a lesser extent in intestine, heart and lung. The pattern of distribution agrees well with that found in rat tissue soluble extracts (186) and in monkey cytosol fractions (181). These studies also included assays in the testis, where activity was found at a similar level to that in the spleen. The relatively high activity in these 2 tissues with a high rate of cell turnover is consistent with a role for cytosolic serine hydroxymethyltransferase in nucleotide biosynthesis. It is noteworthy that spleen and testis are also the tissues with some of the highest activities of the enzymes of de novo serine biosynthesis suggesting an association between the synthesis of serine and its utilization via serine hydroxymethyltransferase for nucleotide formation (see below). No studies have been carried out on mitochondrial serine hydroxymethyltransferase, but glycine dehydrogenase activity ([1J4C]glycine decarboxylation) in tissue homogenates supplemented with co-factors (NAD +, tetrahydrofolate and pyridoxal phosphate) was relatively low in spleen and testis compared to, say, brain and heart which contained negligible activities of cytosolic serine hydroxymethyltransferase (180, 186). We investigated further the apparent association of cytosolic serine hydroxymethyltransferase with nucleotide biosynthesis and cell turnover by

379

R E G U L A T I O N O F SERINE M E T A B O L I S M

studies on the mitogenic stimulation of human lymphocyte proliferation in cell culture (131). Following the stimulation of cell proliferation by exposure to the mitogen phytohemagglutinin, increased DNA synthesis (assessed by incorporation from [6-3H]thymidine into nucleic acids) was accompanied by a parallel increase in incorporation from [3-'4C]serine into nucleic acids and by increased serine hydroxymethyltransferase activity (Fig. ll). Carbon-3 of serine provides the one-carbon unit which is used for thymidylate synthesis and subsequent DNA formation. Thus incorporation from [3-~4C]serine probably represents its utilization for DNA synthesis and this is in agreement with the simultaneous incorporation from [3H]thymidine into nucleic acid. Moreover incorporation of precursors into RNA precedes DNA synthesis S e r i n e i n c o r p o r a t i o n into nucleic a c i d s in PHA~stimulat~d lymphocytes

I

30

25

•

O

IX

×

2O

g

15

~ 10 o

10

20

0

4

50

60

70

Culture t i m e (hr)

FIG. 11. L-Serine hydroxymethyltransferase activity and serine incorporation into nucleic acids in phytohemagglutinin-stimulated h u m a n lymphocytes in culture. Cultures of h u m a n peripheral blood lymphocytes, containing 1 >< 106 initial cells per culture well at zero-time, were incubated and treated with phytohemagglutinin as referenced in Methods. At various times of incubation with the mitogen, cultures were pulse-labeled for 5 hr with 0.05 ~Ci of [ 6 : H ] t h y m i d i n e and 0.7 ~Ci of L-[3-~4C]serine and then harvested by freezing at -20°C for nucleic acid analysis and Lserine hydroxymethyltransferase assay as previously detailed (31). Points show the means of 3 observations (agreeing to within 10%). For sake of clarity, observations in unstimulated control lymphocyte cultures are not shown, but no changes in any of the parameters over the time-period shown were apparent. AER 22-~I~

380

KEII"H SNELL

considerably after mitogenic stimulation of lymphocytes (187, 188). Although the results are expressed on the basis of initial cell number, the increase in cell number in the 72 hr period (3- to 4-fold) was not sufficient to account for the magnitude of the increases observed. In any case the increase in cell number only occurs to any great extent after 47 hr of culture (K. Snell, unpublished observations), by which time an appreciable increase in incorporation from [3-~4C]serine into nucleic acids and in serine hydroxymethyltransferase activity has taken place (Fig. 11). The mechanism of the adaptive increase in serine hydroxymethyltransferase is not established, nor is it clear if this is the rate-limiting factor regulating serine utilization for nucleotide synthesis in vivo in this situation. The results of this study show that cell proliferation, at least in mitogenstimulated lymphocytes, is associated with an increased activity of serine hydroxymethyltransferase and an increased utilization of serine for nucleic acid precursor synthesis. A similar study with identical conclusions has been carried out independently by Thorndike et al. (189). The observations with serine hydroxymethyltransferase in proliferating lymphocytes are consistent with the hypothesis (see General Discussion) that in proliferating cells there is a coordinated response in serine metabolism involving an increased de novo synthesis of serine and an enzyme-directed utilization of serine for nucleotide biosynthesis. With respect to the latter point, there is no detectable activity of serine dehydratase or serine aminotransferase in resting or 72 hr mitogenstimulated lymphocytes (K. Snell, unpublished observations). Proliferating lymphocytes have markedly increased rates of aerobic glycolysis. Data consistent with a major function of this enhanced glycolysis in the maintenance of elevated levels of glycolytic intermediates as precursors for nucleotide biosynthesis have been reported (190, 191). Whether the increased provision of glycolytic intermediates includes their utilization in the pathway for the biosynthesis of serine as a nucleotide precursor remains to be established. Observations in other tissues during hormonally-induced stimulation of rapid growth lend support to the hypothesis that serine biosynthesis may be coordinated with a utilization of serine via serine hydroxymethyltransferase for nucleotide synthesis. Sanborn et al. (192) showed that castration of male rats led to a fall in the activities of enzymes of serine biosynthesis (3phosphoglycerate dehydrogenase and phosphoserine phosphatase) and of serine hydroxymethyltransferase in the atrophied prostate tissue. Tissue regeneration stimulated by testosterone increased the enzyme activities to control (non-castrated) levels. The hormonal induction of the enzymes was apparently due to increased enzyme synthesis, as the increases were blocked by cycloheximide. Herranen and Mueller (193, 194) showed that estradiol treatment of ovariectomized rats accelerated the incorporation from [3~4C]serine into purine bases by 5- to 6-fold in uterine segments, and

REGULATION OF SERINE METABOLISM

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significantly increased uterine serine hydroxymethyltransferase activity. No measurements of enzymes of serine biosynthesis were made in this study. The possible association between de novo serine biosynthesis and serine utilization for nucleotide biosynthesis will be pursued in the following sections. L-Serine h y d r o x y m e t h y l t r a n s f e r a s e in developing rat tissues. The patterns of development of total homogenate serine hydroxymethyltransferase and the cytosolic isoenzyme (allothreonine aldolase) are shown in Figure 2C. We determined the activities at different times and in different laboratories: the former activity in Kx albino rats at the Cancer Research Institute, New England Deaconess Hospital, Boston, Massachussetts, and the latter activity in an inbred Wistar strain at the Department of Biochemistry, University of Surrey, Guildford, U.K. Nevertheless, the 2 developmental patterns closely match each other and show a perinatal peak of activity, declining somewhat in the mid-suckling period, and rising to the adult value around weaning. The coincidence of the 2 activities suggests that the mitochondrial isoenzyme of serine hydroxymethyltransferase must show a very similar developmental pattern, although this will depend on whether the mitochondrial proportion of total activity is great enough to influence the overall pattern. Although the direct determination of the developmental profile of the mitochondrial isoenzyme has not been made, the glycine dehydrogenase complex, with which it is proposed the isoenzyme is functionally associated, has been investigated by K. V. Rowsell and T. K. S. AI-Saoudi (personal communication, quoted in 55). The enzyme shows low activity in fetal liver, rising to a peak in the mid-suckling period, and a final increase after weaning to the adult value. This pattern is consistent with an involvement in the mitochondrial pathway of neonatal gluconeogenesis from glycine and hydroxyproline, proposed in an earlier section, which is at a maximum in the mid-suckling period. The perinatal peak of cytosolic serine hydroxymethyltransferase activity is noteworthy in that elevations in liver DNA concentration, DNA polymerase activity, and the incorporation from [3H] thymidine into DNA by liver slices are also found at this age (see 55). The pattern is also shared by enzymes involved in the de novo pathways of purine and pyrimidine biosynthesis (for references, see 55). The coincidence of these developmental patterns supports the proposed role for cytosolic serine hydroxymethyltransferase in the provision of glycine and one-carbon units for nucleotide biosynthesis. Indeed, Sturman et al. (195) have shown an increased incorporation from [314C]serine into DNA in human fetal liver slices compared to adult liver slices which accords with this hypothesis. In view of the high rate of glycolysis of fetal liver, and the perinatal peaks of acitivity of the enzymes of serine biosynthesis (cf. Fig. 2A), the perinatal rat liver would appear to represent a situation where cell proliferation involves a coordinated increase in the de novo synthesis of serine from glycolytie intermediates with the utilization of

382

KEITH SNELL

serine for nucleotide biosynthesis via serine hydroxymethyltransferase (see previous section). This relationship does not hold for all fetal tissues however (Table 3). Whereas we found that phosphoserine phosphatase was greater than adult activity in all fetal tissues tested (except for kidney), serine hydroxymethyltransferase is at or above adult activities only in fetal spleen and intestine and is virtually absent from fetal heart and lung (Table 3). Serine biosynthesis in these latter fetal tissues apparently fufills some role other than providing precursors for de novo nucleotide biosynthesis. In fetal lung it may be suggested that the synthesis of phospholipids for surfactant generation might be a significant route of serine utilization, but no studies bearing directly on this question have been carried out. The increase in cytosolic serine hydroxymethyltransferase occurring around weaning (Fig. 2C) may be related to the phase of rapid liver growth that begins at this time and involves a 3.5-fold increase in organ weight in two weeks (196). This growth spurt is, in part, due to a phase of hyperplasia which results in a 2.5-fold increase in cell number and total liver DNA, and a 50% increase in liver RNA concentration (196). The developmental increase in serine hydroxymethyltransferase activity over this period could again be associated with a role in providing nucleotide precursors. Since, at the same time, there is an increase in dietary protein intake during weaning (158), the provision of serine from this dietary source might account for why a coordinated increase in enzymes of de novo serine biosynthesis is not observed in this situation. L-Serine hydroxymethyltransferase in neoplastic rat tissues. The results of our survey of serine hydroxymethyltransferase in transplantable rat tumors show that as with phosphoserine aminotransferase, but not serine dehydratase or serine aminotransferase, activity is retained in all neoplastic tissues (Table 4). For the hepatomas, serine hydroxymethyltransferase activity varies between 20% and 90% of the adult liver activity and, at least for this limited range of hepatomas, shows a positive association with tumor growth rate. Enzyme activity in the renal tumor was almost double that in normal adult kidney, while in the other tumors activity was 48-93% of the adult liver activity. Within the scope of the enzymes of serine utilization, it appears that serine hydroxymethyltransferase activity is selectively maintained. None of the measurements specifically distinguished cytosolic serine hydroxymethyltransferase activity. Nevertheless in view of the accelerated glycolytic rate observed in many tumors and the activities of the enzymes of de novo serine biosynthesis (see earlier section), it is tempting to suggest that neoplastic tissues represent another situation where cell proliferation involves a coordinated response of de novo serine biosynthesis metabolically coupled through serine hydroxymethyltransferase to the provision of carbon for nucleotide biosynthesis. We found the highest activity of serine hydroxymethyltransferase in

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383

neoplastic tissues in the lymphoma (RNC-290) and this is noteworthy in relation to our observations of the marked increase in activity in proliferating normal lymphocytes (see previous section). Other studies have also shown increased enzyme activity, relative to normal lymphocytes, in a human lymphoma (189). Serine hydroxymethyltransferase activity per cell is increased 5- to 10-fold in the lymphocytes of patients with chronic lymphocytic leukemia, and increased about 3-fold in the leukocytes of patients with acute lymphocytic or acute myelocytic leukemias (189, 197). Normal granulocytes have lower enzyme activity than lymphocytes, and in comparison the activity is slightly raised in patients with chronic myelocytic or monocytic leukemias (189). Whether the increased serine hydroxymethyltransferase activity involved in the neoplastic proliferation of leukocytes is Ossociated with an increased capacity to synthesize serine has not been investigated for all leukocyte cell types. However, it has been shown that granulocytes from patients with chronic granulocytic leukemia (as well as the largely stem cell population from normal bone marrow) are unable to proliferate in culture in the absence of serine and lack the capacity to synthesize [~4C]serine from [14C]glucose (198). This implies that other cell types (without this growth requirement) may well rely instead on de novo serine biosynthesis. General Discussion of Interrelationships in Serine Metabolism. It has been a major preoccupation of this review that there is a need to interpret the role of particular enzymes of serine metabolism in terms of the integrated operation of the pathways of synthesis and of utilization. In some cases such an integrated approach has been adopted but often this has been incomplete. The control mechanisms which govern the interrelation between the various pathways has still to be elucidated and indeed the regulation of the separate pathways of serine metabolism is still far from being established. The free concentration of serine within the cell is clearly a major influence, since it acts as a feedback inhibitor of the pathway of serine biosynthesis and a feedforward activator of pathways of utilization, where the initiating enzymes have K,~ values very much higher than the intracellular serine concentration (serine dehydratase and serine aminotransferase). However, this can not be the only consideration as activation of metabolic flux through hepatic serine dehydratase occurs under conditions of active gluconeogenesis when the intrahepatocellular concentration of serine is decreased. Attention has been drawn to enzyme adaptation as a major means of manipulating flux through the various pathways of serine metabolism. It is noteworthy that for the pathway of serine biosynthesis 3-phosphoglycerate dehydrogenase is the most sensitive in terms of adaptive change whereas more immediate feedback control operates at the level of serine inhibition of phosphoserine phosphatase. This is consistent with the contemporary view of control of a

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KEITH SNELL

pathway operating through shared responsibility at a series of regulatory loci each with its own particular 'control strength' which is variable so that its proportionate contribution to overall pathway control is flexible (199, 200). The concept of a single regulatory step catalyzed by a 'rate-limiting' or 'pacemaker' enzyme is no longer tenable and one must look instead to a number of 'key enzymes' (as defined by Weber; 3, 4a) for control of pathway flux. Of the various pathways of serine utilization, that involving metabolism via hydroxypyruvate (Fig. 1) contains tr-glycerate dehydrogenase which is sensitive to allosteric regulation (but note earlier reservations about its role) and serine aminotransferase which appears to be most sensitive as regards adaptive change. The problem of the control of the metabolism of serine by a number of alternative pathways of metabolism is solved in most tissues by the absence of serine dehydratase and serine aminotransferase (although one should be aware of other routes of serine metabolism, such as phospholipid synthesis, not covered by this review). The liver, and perhaps the kidney, as befits their metabolic importance and diversity, must contend with this regulatory dilemma. To some extent the control of the competing pathways of gluconeogenesis (initiated by serine dehydratase and serine aminotransferase) is facilitated by their segregation into different subcellular compartments. However, this merely shifts the onus for control to the level of the mitochondrial translocation of serine a n d / o r the intramitochondrial generation of serine, of both of which we are largely ignorant. The differing metabolic functions subserved by serine dehydratase and serine aminotransferase (as proposed in this review) and consequently their differing relative sensitivity to adaptive stimuli accomplishes control at one level, but the mechanisms of short-term regulation of the enzymes continue to be elusive. Relative metabolic flux through cytosolic serine hydroxymethyltransferase (which is in direct competition with serine dehydratase) is probably mediated via alterations in folate co-factor availability as well as by its functional sub-compartmentation within the cytosol in putative multi-enzyme complexes (Figs. 8 and 9). Investigations of the physiological role of serine hydroxymethyltransferase have been confounded to some extent by the necessity to distinguish between the cytosolic and mitochondrial isoenzyme activities. If their respective functions differ as radically as proposed (cf. Fig. 10), it is to be expected that both short-term regulation and adaptive responses would be quite distinct. Evidence to date has not revealed any major differences in regulatory properties of the isoenzymes, although previous studies have not involved approaches which specifically took into account differing metabolic roles. The occurrence of multiple enzyme forms of serine dehydratase in the cytosol is an enigmatic feature of the control of this enzyme. Again, no differences in enzymological properties are apparent and the lack of differential adaptation during neonatal development and its absence from rabbit liver might argue

REGULATION OF SERINE METABOLISM

385

against any physiological significance. Nevertheless, changes in the relative proportions have been described with certain adaptive stimuli, and between normal liver and the few hepatomas containing any detectabIe serine dehydratase activity, suggesting that the phenomenon may have a significance still to be determined. The multiple forms of serine aminotransferase, differing in subcellular location, introduce a potential for control at the level of enzyme segregation into intracellular compartments that needs to be taken account of in further investigations of the control of this enzyme. The physiological function(s) of the minor peroxisomal and cytosolic forms of the enzyme have not yet been identified. The adaptive patterns of enzymes during neonatal development is a useful indicator of physiological function, if they coincide with known patterns of metabolic flux (6, 55, 56, 152). Such an association was used as a part of the evidence for a gluconeogenic role for hepatic serine aminotransferase under particular dietary circumstances when serine might be an intermediate in the mitochondrial metabolism of hydroxyproline and glycine. It is noteworthy that when enzyme activity is at its height, in the mid-suckling period, serine dehydratase activity is very low and there is a trough in the developmental pattern of serine hydroxymethyltransferase. The enzymes of serine biosynthesis are rapidly declining to low levels at this time, since clearly there is no physiological advantage in synthesizing serine from glycolytic intermediates only to have it re-converted back to glucose again. Developmental adpative enzyme patterns also proved to be instrumental in settling a long controversy over the apparent alternative 'phosphorylated' and 'non-phosphorylated' pathways of serine biosynthesis. The former is presently considered the biosynthetic pathway and the latter pathway comprises the putative mitochondrial gluconeogenic pathway referred to above. An interesting finding that has emerged from the more recent work in this Laboratory is an association of high activities of the enzymes of serine biosynthesis with a peak ofcytosolic serine hydroxymethyltransferase activity in the perinatal liver when hepatocyte proliferation is high. A similar association was noted in other proliferative circumstances and in other tissues, and we propose the concept that cellular proliferation involves an accelerated rate of de novo synthesis of serine from glycolytic intermediates, which is metabolically coupled through serine hydroxymethyltransferase to the provision of glycine and one-carbon units for purine and pyrimidine biosynthesis. In some cases the data on enzyme activities are supported by measurements of pathway flux. The coordinated adaptations of the enzymes of the pathways are accompanied by low or negligible activities of the alternative enzymes of serine utilization, serine dehydratase and serine aminotransferase. However, the increased utilization of serine for nucleotide biosynthesis is not invariably coupled to an increased biosynthesis of serine. In the surge of postnatal hyperplastic growth in liver, and in granulocytes

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KEITH SNELL

undergoing replication in culture, serine biosynthesis is not increased. And in fetal heart and lung when phosphoserine aminotransferase activity is high, serine hydroxymethyltransferase activity is undetectable (see Table 3). However, these are exceptions to the more general rule. Neoplastic tissues provide a biological system of continual, if pathological, cellular proliferation in which to test this concept further. Clear evidence for the selective maintenance of the enzymes of serine biosynthesis and of serine hydroxymethyltransferase was obtained (with the cellular loss of serine dehydratase and serine aminotransferase). This is a further example of the specific adaptation of enzymes [according to Weber (3-4a), "the ordered reprograming of gene expression"] to subserve the "'biochemical commitment to replication" which characterizes the neoplastic state. It is often illuminating when considering adaptations in key enzymes from different pathways to perform a very simplified kind of correlation analysis (5) by expressing data in the form of enzyme activity ratios. This has been found particularly useful where the key enzymes lie on antagonistic or competing pathways of metabolism (3-4a). In the present case the ratio of serine hydroxymethyltransferase activity to either serine dehydratase or serine aminotransferase activities conveys the magnitude of the changes occurring simultaneously in both enzymes of the pair (Table 7). The activity ratios of serine hydroxymethyltransferase to serine dehydratase and of serine hydroxymethyltransferase to serine aminotransferase show that in both cases there is a T A B L E 7. ENZYME ACTIVITY RATIOS IN N O R M A L A N D NEOPLASTIC RAT TISSUES Enzyme activity ratio SHMT SHM'I" SHMT Tissue

SDH

SAT

PSAT

Adult liver Fetal liver Hepatoma: 7800 7777 3683F Novikoff

0.09 10.1 0.05 27.5 105 121

11.2 17.6 37.4 55.0 209 242

10.0 2.54 2.44 2.52 8,07 8,58

Adult kidney Fetal kidney Tumor MK-I

2.7 6.7 43

26.4 13.3 172

1.92 0.71 4.30

Salivary tumor Lymphoma 290 Mammary tumor

26.2 85.3 99.5

131 135 199

4.15 8.42 8.02

The relevant enzyme activities are taken from the data in Tables 3 and 4, using the upper limits of undetectable activity where appropriate. SHMT, serine hydroxymethyltransferase; SDH, serine dehydratase; SAT, serine aminotransferase; PSAT, phosphoserine aminotransferase.

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dramatic increase in neoplastic tissues compared to the normal adult liver or kidney. In the hepatoma series the activity ratio shows a positive correlation with increasing growth rate. In all the tumors it seems that the pattern of utilization of serine is being directed away from the pathways initiated by serine dehydratase and serine aminotransferase towards that involving serine hydroxymethyltransferase. Thus, there is an enzyme-specified direction of metabolism of serine towards the provision of nucleotide precursors for continual cell replication. The data in Tables 3 and 4 on the activities of the individual enzymes of serine utilization and biosynthesis are consistent with the concept of'fetalism' or 'retrodifferentiation' in neoplasia (5, 170): that the enzyme changes observed in neoplastic transformation are in the direction of reversion to an undifferentiated state, as a consequence of the reprograming of gene expression. That cancer may be conceptualized as a 'disease of differentiation' has been given fresh impetus in recent years as our knowledge of the nature and functions of oncogenes and their normal cellular counterparts has dramatically increased. However, it should be noted from Table 7 that although the activity ratios are indeed increased in normal fetal liver and kidney compared to the adult tissues, the magnitude of this increase is very much lower than that found in any of the tumors. This may simply reflect the fact that fetal tissue is at an intermediate stage on the retrogressive ontogenic path leading to a prototypic undifferentiated cell (5), and that neoplastic cells have travelled further along this path of cell lineage. It may, however, sound a note of caution in the too ready acceptance of the causality of superficial resemblances between fetal and neoplastic enzymic characteristics, attractive though such a relation might seem in conceptual terms. The activity ratio of serine hydroxymethyltransferase to phosphoserine aminotransferase is also given in Table 7. No dramatic changes in this ratio are apparent (the values in general lie between 2 and 10) and this relative constancy is consistent with the proposed coordinated role of the two enzymes. However, it should be noted that the de novo pathway of serine biosynthesis is by no means the sole source of intracellular serine for utilization by serine hydroxymethyltransferase. Other sources include serine taken up from the extracellular fluid and that derived from intracellular protein turnover. All of these factors will contribute to the increased free amino acid pool found in hepatomas. However, taken in conjunction with the findings that protein synthesis (including [~4C]serine incorporation) was increased in hepatomas whereas protein content is decreased (174), it seems that protein degradation must be increased in the tumors. Sauer et al. (201) have investigated amino acid extraction by transplantable Morris hepatomas and a Walker carcinoma in vivo by measuring afferent-efferent concentration differences in the blood supply across the tissue. Serine ranked second in quantitative importance after glutamine in 3 of the 4 tumors (and fourth in the

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remaining tumor). Therefore, these processes as well as de novo biosynthesis will contribute to intracellular serine formation. The acceleration of the de novo pathway ofserine biosynthesis is appropriate to the increased availability of glycolytic intermediates from the stimulation of glycolysis frequently observed in tumor tissues. Weber (3-4a) considered that a part of the role of the accelerated glycolysis was to furnish intermediates for phosphoribosylpyrophosphate formation, via the pentose phosphate pathway reactions, for purine and pyrimidine biosynthesis. Our current working hypothesis (shown in Fig. 12) is an extension of this role ofglycolysis to that of

PYRIMIDINES UMP

l dUMP

CTP dTMP

) IMP -

PURINES

DNA

CH2FH~

PRPP

T HF..~' S E R

RSP

[GiucosE

GBP

PSER

F6P

FBP

k

IIPYR

PHP

GI/~

3PG

2PG

Pimp ~ P/YR ~r '

...j.

FIG. 12. Integration of serine metabolism into carbohydrate and nucleic acid metabolism in neoplastic tissues. The broad arrows indicate metabolic pathways (and enzymes) retained or increased in neoplastic cells and the broken arrows indicate metabolic pathways (and enzymes) which are absent or deleted from neoplastic cells. Not all of the intermediates in the pathways are included in the outline, which is modified and extended from that shown by Weber (4), Abbreviations: G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6bisphosphate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; OAA, oxaloacetate; 6PG, 6-phosphogluconate; S7P, sedoheptulose 7-phosphate; R5P, ribose 5-phosphate; PRPP, 5'-phosphoribosyl pyrophosphate; UMP, uridine monophosphate; dUMP, uridylate; dTMP, thymidylate; CTP, cytosine triphosphate; IMP, inosine monophosphate; CI--12FH4, NS,Nl%methylenetetrahydrofolate; GLY, glycine; THF, tetrahydrofolate; SER, serine; PSER, O-phospho-L-serine; PHP, 3-O-phosphohydroxypyruvate; HPYR, 3-hydroxypyruvate; GLR, ~glycerate.

REGULATION OF SERINE METABOLISM

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furnishing intermediates for serine biosynthesis, which is then metabolically coupled via serine hydroxymethyltransferase to the provision of nucleotide precursors. The role of serine hydroxymethyltransferase is enhanced by the deletion of antagonistic pathways of serine metabolism and accords with the 'pathway antagonism concept' described by Weber (3-4a) and supported by work from that laboratory on carbohydrate and nucleotide metabolism. The value of the current hypothesis lies not only in providing a description of the metabolic reorganization characteristic of the neoplastic state, but also in providing a framework for the rational development of chemotherapeutic approaches to controlling the unremitting growth of tumors. It is my contention that serine metabolism plays a central role in the biochemical reorganization that is a part of the commitment of the neoplastic cell to continual replication. SUMMARY 1. The cellular pattern of serine metabolism was conceptualized into four main areas of metabolic sequences: the biosynthesis of serine from intermediates of the glycolytic pathway (the so-called "phosphorylated pathway"); and alternative pathways of serine utilization initiated by serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. 2. The known regulatory and adaptive properties of the enzymes involved in these pathways were reviewed in detail and key enzymes associated with each pathway (phosphoserine aminotransferase, serine dehydratase, serine aminotransferase, and serine hydroxymethyltransferase, respectively) were selected for further investigation. 3. Tissue distribution studies in the rat revealed that whereas serine dehydratase and serine aminotransferase activities were largely confined to the liver, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were more broadly distributed. In particular in tissues with a high rate of cell turnover, phosphoserine aminotransferase and serine hydroxymethyltransferase activities were coordinately increased. An increase in serine hydroxymethyltransferase activity coincided temporally with the incorporation of [3-14C]serine and thymidine into DNA in normal human lymphocytes during proliferation after mitogenic stimulation by phytohemagglutinin. The evidence suggested a primarily gluconeogenic role for serine dehydratase and serine aminotransferase. Serine hydroxymethyltransferase has a role in providing glycine and one-carbon folate co-factors as precursors for nucleotide biosynthesis and in some situations serves to metabolically couple the pathway of serine biosynthesis to utilization for de novo purine and pyrimidine synthesis. 4. Multiple enzymic forms were distinguished for serine dehydratase, serine aminotransferase and serine hydroxymethyltransferase. For serine dehydratase the two cytosolic multiple forms had no apparent functional

390

KEITH SNELL

significance; the multiple forms were catalytically unmodified by conditions promoting phosphorylation-dephosphorylation in vitro. The mitochondrial form of serine aminotransferase showed adaptive responses in gluconeogenic situations, and the hypothesis was proposed that the mitochondrial isoenzyme of serine hydroxymethyltransferase is associated together with serine aminotransferase in a pathway for gluconeogenesis from proteinderived amino acids such as glycine and hydroxyproline. 5. The adaptive behaviour of the enzymes during the neonatal development of rat liver revealed that serine aminotransferase reached a peak in the midsuckling period at a time when gluconeogenesis is known to be increased. Use of phosphoenolypyruvate carboxykinase inhibitors (mercaptopicolinate or quinolinate) supported a pathway via serine aminotransferase for gluconeogenesis from serine and hydroxyproline at this developmental stage. The concept of the involvement in a carbon salvage pathway to deal with increased body collagen turnover at this time was advanced. The developmental adaptation of serine aminotransferase at birth was shown to involve glucagon, acting via cyclic AMP, and to be dependent on transcriptional gene regulation. 6. Serine dehydratase showed a biphasic developmental pattern, similar to other enzymes involved in amino acid catabolism. The peaks of activity at the early neonatal and weaning developmental stages were shown to involve the joint action of glucagon, acting via cyclic AMP, and corticosteroid hormones. The inductions were dependent, at least initially, on transcriptional gene regulation but the precise mechanistic role of the two classes of hormone has yet to be defined. At both developmental peaks the distribution of serine dehydratase multiple forms was identical, and differential developmental regulation of the forms was not involved in determining the overall pattern of serine dehydratase development. 7. Phosphoserine aminotransferase and cytosolic serine hydroxymethyltransferase showed similar developmental patterns with a peak of activity in the perinatal period. This coincides with an active period of hepatocyte proliferation and of nucleotide biosynthesis. A further rise of serine hydroxymethyltransferase coinciding with a second postnatal surge of proliferative hepatocellular growth was independent of de novo serine biosynthesis and reflected increased provision of serine from dietary sources. 8. A survey of the key enzymes of serine metabolism in transplantable rat neoplasms revealed that, in general, serine dehydratase and serine aminotransferase were deleted from the cellular repertoire of metabolic capacities. In contrast, phosphoserine aminotransferase and serine hydroxymethyltransferase were selectively retained to varying degress in neoplastic tissues. 9. The pattern of serine metabolism displayed in normal, developing and neoplastic tissues revealed an integrated, genetically-programed, response of

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enzymes ofserine biosynthesis and of alternative enzymes ofserine utilization. A major role for serine metabolism in cellular proliferation was emphasized by the coordination of serine synthesis from carbohydrate precursors with the biosynthesis of purine and pyrimidine nucleotides through a metabolic coupling via serine hydroxymethyltransferase.

ACKNOWLEDGEMENTS Various aspects of the author's research work described in this paper were supported by grants from the Wellcome Trust, the United States Public Health Service Grant AM 00567 from the National Institute of Arthritis, Metabolism and Digestive Diseases, and from the International Union against Cancer (U.I.C.C.). I am grateful for Travel Fellowships provided by the Biochemical Society (Boehringer Mannheim Travelling Fellowship) and by the international Union against Cancer (International Cancer Research Technology Transfer Fellowship) to enable me to carry out research work at the Cancer Research Institute of the New England Deaconess Hospital and the Department of Biological Chemistry, Harvard Medical School, Boston, and to Professor W. Eugene Knox .for offering me the hospitality of his laboratory. Although the concepts, and their possible limitations, expressed in this review are the responsibility o f the author, I am appreciative of valuable discussions concerning particular phases of the work with: Dr. Edward V. Rowsell, Professor Deryck G. Walker, Professor W. Eugene Knox (now deceased), Professor George Weber and Dr. Monisha Chaudhuri. I am grateful also to Janet Cole for deciphering seemingly impenetrable calligraphic minutiae.

REFERENCES 1.

E.A. NEWSHOLME,B. CRABTREEand V. A. ZAMMIT,Use of enzymeactivitiesas indices of maximum rates of fuel utilization, pp. 245-258 in Trends in Enzyme Histochemistry and Cytochemistry (Ciba Foundation Symp. 73), Excerpta Medica, Amsterdam (1980). 2. D. PETTE and H. W. HOFER, The constant proportion enzymegroup concept in the selection of reference enzymes in metabolism, pp. 231-244 in Trends in Enzyme Histochemistry and Cytochemistry (Ciba Foundation Symp. 73), Excerpta Medica, Amsterdam (1980). 3. G, WEBER, Enzymologyof cancercells,Part 1,NewEnglandJ. Med. 296,486-493 (1977). 4. G. WEBER, Enzymology of cancer cells, Part2,NewEnglandJ. Med. 296,541-551 (1977). 4a, G. WEBER, Biochemicalstrategy of cancer cells and the design of chemotherapy:G. H. A. Clowes memorial lecture, Cancer Res. 43, 3466-3492 (1983). 5. W.E. KNOX, Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues (2nd edition), S. Karger, Basel (1976). 6, O. GREENGARD, Enzymicdifferentiationin mammaliantissues,pp. 159-205 in Essays in Biochemistry, Volume 7 (P. N. CAMPBELL and F. DICKENS, eds.), AcademicPress, London (1971). 7. H.R.V. ARNSTEIN and A. NEUBERGER,The effectof cobalamin on the quantitative

392

8. 9. 10.

II. 12. 13. 14. 15.

16. 17. 18. 19. 20. 20a. 21. 21a. 22. 22a. 23. 24. 25. 26. 27.

KEITH SNELL utilization of serine, glycine and formate for the synthesis of choline and methyl groups of methionine, Biochem. J. 55, 259-280 (1953). A.L. BLACK, M. KLEIBER and C. F. BAXTER, Glucose as a precursor of amino acids in the intact dairy cow, Biochim. Biophys. Acta 17, 346-353 (1955). H . R . V, ARNSTEIN and D. KEGLEVIC, A comparison of alanine and glucose as precursors of serine and glycine, Biochem. J. 62, 199-205 (1956). H . J . SALLACH, Evidence for a specific alanine-hydroxypyruvate transaminase, pp. 782-787 in Symposium on Amino Acid Metabolism (W. D. McELROY and B. GLASS, eds.), Johns Hopkins Press, Baltimore (1955). H, J. SALLACH, Formation of serine from hydroxypyruvate and L-alanine, Z Biol. Chem. 223, 1101-1108 (1956). J.F. NYCandl. ZABIN, The formation of serine from pyruvate, J. Biol. Chem. 215,35--40 (1955). D.A. WALSH and H. J. SALLACH, Comparative studies on the pathways for serine biosynthesis in animal tissues, J. Biol. Chem. 241, 4068--4076 (1966). A. ICHIHARA and D. M. GREENBERG, Further studies on the pathway of serine formation from carbohydrate, J. Biol. Chem. 224, 331-340 (1957). G.P. CHEUNG, J. P. COTROPIA and H. J. SALLACH, The effects of dietary protein on the hepatic enzymes of serine metabolism in the rabbit, Arch. Biochem. Biophys. 129, 672--682 (1969). E.V. ROWSELL, K. SNELL, J. A. CARNIE and A. H. AL-TAI, Liver L-alanineglyoxylate and L-serine-pyruvate aminotransferase activities: an apparent association with gluconeogenesis, Biochem. J. 115, 1071-1073 (1969). H. LARDY, C. VENEZIALE and F. GABRIELLI, Paths of carbon in gluconeogenesis, FEBS Syrup. 19, 55-62 (1969). P. COHEN, The role of protein phosphorylation in neural and hormonal control of cellular activity, Nature 296, 613-620 (1982). B.E. JOHNSON, D. A. WALSH and H. J. SALLACH, Changes in the activities of glycerate and o-3-phosphoglycerate dehydrogenases in the developing rat liver, Biochim. Biophys. Acta 85, 202-205 (1964). K. SNELL, Liver enzymes of serine metabolism during neonatal development of the rat, Biochem J. 190, 451--455 (1980). Y. NAKANO, M. FUJIOKA and H. WADA, Studies on serine hydroxymethylase isoenzymes from rat liver, Biochim. Biophys. Acta 159, 19-26 (1968). W. E. KNOX, A. HERZFELD and J. HUDSON, Phosphoserine phosphatase distribution in normal and neoplastic rat tissues, Arch. Biochem. Biophys. 132, 397-403 (1969). T. NOGUCHI, Y. TAKADA and R. KIDO, Characteristics of hepatic serine~pyruvate aminotransferase in different mammalian species, Biochem. J. 161,609-614 (1977). K. SNELL and D. G. WALKER, Regulation of hepatic L-serine dehydratase and L-serinepyruvate aminotransferase in the developing neonatal rat, Biochem. J. 144, 519-531 (1974). H. INOUE and H. C. PITOT, Regulation of the synthesis of serine dehydratase isozymes, Advances in Enzyme Regulation 8, 289-296 (1970). A. ICHIHARA and D. M. GREENBERG, Studies on the purification and properties of o-glyceric acid kinase of liver, J. Biol. Chem. 225, 949-958 (1957). D . H . WILLIAMSON, O. LOPES-VIEIRA and B. WALKER, Concentrations of free glucogenic amino acids in livers of rats subjected to various metabolic stresses, Biochem. J. 104, 497-502 (1967). S.A. ADIBI, Interrelationships between level of amino acids in plasma and tissues during starvation, Am. J. Physiol. 221, 829-838 (1971). E. L. TOLMAN, C. M. SCHWORER and L. S. JEFFERSON, Effects of hypophysectomy on amino acid metabolism and gluconeogenesis in the perfused rat liver, J. BioL Chem. 248, 4552--4560 (1973). C. A. CAPERELLI, P. A. BENKOVIC, G. CHETTUR and S. J. BENKOVIC, Purification of a complex catalyzing folate co-factor synthesis and transformylation in de novo purine biosynthesis, J. Biol. Chem, 255, 1885-1890 (1980),

REGULATION OF SERINE METABOLISM 28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40.

41.

42. 43. 44. 45.

46. 47. 48. 49. 50. 51.

393

A. HERZFELD and O. GREENGARD, The effect oflymphoma and other neoplasms on hepatic and plasma enzymes of the host rat, Cancer Res. 37, 231-238 (1977). R. MACHOVICH and O. GREENGARD, Thymidine kinase in rat tissues during growth and differentiation, Biochim. Biophys. A cta 286, 375-381 (1972). W.E. KNOX and L. M. LISTER-ROSENOER, Timing of gestation in rats by fetal and maternal weights, Growth 42, 43-53 (1978). H . G . EICHLER, R, HUBBARD and K. SNELL, The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes, Biosci. Rep. 1, 101-106 (1981). L. SCHIRCH and T. GROSS, Serine transhydroxymethylase: identification as the threonine and allothreonine aldolases, J. Biol. Chem. 243, 5651-5655 (1968). H.E. UMBARGER and M. A. UMBARGER, The biosynthetic pathway of serine in Salmonella typhimurium, Biochim. Biophys. Acta 62, 193-195 (1962). L.I. PIZER, The pathway and control of serine biosynthesis in Escherichia coil, J. Biol. Chem. 238, 3934-3944 (1963). J. C. SLAUGHTER and D. D. DAVIES, Inhibition of 3-phosphoglycerate dehydrogenase by L-serine, Biochem. J, 109, 749-755 (1968). H.J. FALLON, Regulatory phenomena in mammalian serine metabolism, Advances in Enzyme Regulation 5, 107-120 (1967). D.A. WALSH and H. J. SALLACH, Purification and properties of chicken liver D-3phosphoglycerate dehydrogenase, Biochemistry 4, 1076-1085 (1965). W.F. BRIDGERS, The biosynthesis ofserine in mouse brain extracts. J. Biol. Chem. 240, 4591--4597 (1965). L.I. PIZER, Enzymology and regulation of serine biosynthesis in cultured human cells,J. Biol. Chem., 239, 4219-4226 (1964). M. L. UHR and M. K. SNEDDON, Glycine and serine inhibition of D-glycerate dehydrogenase and 3-phosphoglycerate dehydrogenase of rat brain, FEBS Lett. 17, 137-140 (1971). H.J. FALLON, E. J. HACKNEY and W. L. BYRNE, Serine biosynthesis in rat liver: regulation of enzyme concentration by dietary factors, J. Biol. Chem. 241, 4157-4167 (1966). M . O . NEMER, E. M. WISE, F. M. WASHINGTON and D. ELWYN, The rate of turnover of sedne and phosphoserine in rat liver, J. Biol. Chem. 235, 2063-2069 (1960). L.F. BORKENHAGEN and E. P. KENNEDY, The enzymatic exchange of L-serinewith O-phospho-L-serine catalyzed by a specific phosphatase, J. Biol. Chem. 234, 849-853 (1959). F.C. NEUHAUS and W. L. BYRNE, Metabolism of phosphoserine. III. Mechanism of O-phosphoserine phosphatase, Z Biol. Chem. 235, 2019-2024 (1960). W. L. BYRNE, Regulation of serine biosynthesis, pp. 89-100 in Fructose-l,6diphosphatase and its Role in Gluconeogenesis (R. W. McGILVERY and B. M. POGELL, eds.), American Institute of Biological Sciences, Washington (1964). H.J. FALLON, J. L. DAVIS and R. A. GOYER, Effect of protein intake on tissue amino acid levels and the enzymes of serine biosynthesis in the rat, J. Nutr. 96, 220-226 (1968). J. L. DAVIS and H. J. FALLON, Studies on the role of 3-phosphoglycerate dehydrogenase in the regulation of serine biosynthesis in rat liver, J. Biol. Chem. 245, 5838-5846 (1970). H.J. SALLACH, T. A. SANBORN and W. J. BRUIN, Dietary and hormonal regulation of hepatic biosynthetic and catabolic enzymes of serine metabolism in rats, Endocrinology 91, 1054-1063 (1972). J. MAURON, F, MOTTU and G. SPOHR, Reciprocal induction and repression of serine dehydratase and phosphoglycerate dehydrogenase by proteins and dietary-essential amino acids in rat liver, Europ. J. Biochem. 32, 331-342 (1973). S. HAYASHI, T. T A N A K A , J. NAITO and M. SUDA, Dietary and hormonal regulation of serine synthesis in the rat, J. Biochem, 77, 207-219 (1975). S.C. JAMDAR and O. GREENGARD, Phosphoserine phosphatase: developmental formation and hormonal regulation in rat tissues, Arch. Biochem. Biophys, 134, 228-232 (1969).

394 52, 53. 54. 55.

56.

57. 58. 59. 60.

61. 62. 63.

64.

65. 66. 67. 68. 69. 70.

71. 72. 73. 74.

KEITH SNELL R, F. PITTS, A. C. DAMIAN and M. B. MacLEOD, Synthesis of serine by rat kidney in vivo and in vitro, Am. J. PhysioL 219, 584-589 (1970). E.V. ROWSELL, M, M. AL-NAAMA and K, V. ROWSELL, Glycine metabolism in rat kidney cortex slices, Biochem. J. 204, 313-321 (1982). H . A . KREBS, Some aspects of the regulation of fuel supply in omnivorous animals, Advances in Enzyme Regulation 10, 397-420 (1972). K. SNELL, Protein, amino acid and urea metabolism in the neonate, pp. 651-695 in Biochemical Development of the Fetus and Neonate (C. T. JONES, ed.), Elsevier-North Holland, Amsterdam (1982). D . G . WALKER, Developmental aspects of carbohydrate metabolism, pp. 465-496 in Carbohydrate Metabolism and its Disorders, Vol. 1 (F, DICKENS, P. J. RANDLE and W. J. WHELAN, eds.), Academic Press, London (1968). R. MACHOVICH and O. GREENGARD, Thymidine kinase in rat tissues during growth and differentiation, Biochim. Biophys. Acta 286, 375-381 (1972). J.L. DAVIS, H. J. FALLON and H. P. MORRIS, Two enzymes of serine metabolism in rat liver and hepatomas, Cancer Res. 30, 2917-2920 (1970). I.V. SANDOVAL and A. SOLS, Gluconeogenesis from serine by the serine dehydratasedependent pathway in rat liver, Europ. J. Biochem. 43, 609-616 (1974), A. NAGABHUSHAMAN and D. M. GREENBERG, Isolation and properties of a homogenous preparation of cystathionine synthetase, L-serine and L-threonine dehydratases, J. Biol. Chem. 240, 3002-3008 (1965). H. NAKAGAWA, H. KIMURA and S. MIURA, Crystallisation and characteristics of serine dehydratase from rat liver, Biochem. Biophys. Res. Commun. 28, 359-364 (1967). H. NAKAGAWA and H. KIMURA, The properties of crystalline serine dehydratase of rat liver, J. Biochem. 66, 669-683 (1969). J. HOSHINO and H. KROGER, Properties of L-serine dehydratase purified from rat liver after induction by fasting or feeding casein hydrolysate, Hoppe-Seyler's Z. Physiol. Chem, 350, 595-602 (1969). L. GOLDSTEIN, W. E. KNOX and E. J. BEHRMAN,Studies on the nature, inducibility, and assay of the threonine and serine dehydrase activities of rat liver, 3". Biol. Chem. 237, 2855-2860 (1962). J. HOSHINO, D. SIMON and H. KROGER, Identification of one of the L-serine dehydratase isoenzymes from rat liver as L-homoserine dehydratase, Biochem. Biophys. Res. Commun. 44, 872-878 (1971). K. SNELL, The neonatal development of rat liver L-homoserine dehydratase activity, Enzyme 14, 193-200 (1973). H. INOUE, C. B. KASPER and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver: VI. some properties and the metabolic regulation of two isoenzymic forms of serine dehydratase, J. Biol. Chem. 246, 2626-2632 (1971). D. SIMON, J. HOSHINO and H. KROGER, L-Serine dehydratase from rat liver: purification and some properties, Biochim. Biophys. Acta 321, 361-368 (1973). A. PESTANA and A. SOLS, Reversible inactivation of rat liver serine dehydratase by its substrates, FEBS Lett. 7, 29-31 (1970). J.-P. JOST, E. A. KHAIRALLAH and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver, V. Regulation of the rate of synthesis and degradation of serine dehydratase by dietary amino acids and glucose, J. Biol. Chem. 243, 305%3066 (1968). R.A. FREEDLAND and E. H. AVERY, Studies on threonine and serine dehydrase, J. Biol. Chem. 239, 3357-3360 (1964). C. PERAINO, Interactions of diet and cortisone in the regulation of adaptive enzymes in rat liver, J. Biol. Chem. 242, 3860-3867 (1967). A. I. HURWITZ and R. A. FREEDLAND, Influence of dietary protein on hydrocortisone-mediated adaptive enzymatic changes in rat liver, Arch. Biochem, Biophys. 127, 548-555 (1968). A. PESTANA, Dietary and hormonal control of enzymes of amino acid catabolism in liver, Europ. J. Biochem. 11,400-404 (1969).

REGULATION OF SERINE METABOLISM 75. 76.

77,

78. 79. 80.

81.

82.

83. 84. 85.

86. 87.

88. 89. 90. 91. 92.

93. 94. 95.

395

M, SUDA, A view of the comparison of the regulation of enzymes in mammalian and microbial systems, Advances in Enzyme Regulation 5, 181-209 (1967). R . D . REYNOLDS, V. R. POTTER and H. C. PITOT, Response of several hepatic adaptive enzymes to a shift from low to high protein diet in intact and adrenalectomized rats, J. Nutr. I01, 797-802 (1971). A. CIHAK, H. INOUE and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver: Characteristics of the L-tryptophan and cortisone-mediated induction of serine dehydratase in the livers of intact and adrenalectomized rats, Arch. Biochem. Biophys. 166, 237-244 (1975). A. B. EISENSTEIN and I. STRACK, Plasma glucagon, liver cyclic AMP and gluconeogenesis in high protein fed rats, Federation Proc. 31, 709 (1972). E. ISHIKAWA and K. NAKAJIMA, Hormonal and dietary control of enzymes in the rat. I. Roles of the pancreas and adrenals in regulation of liver serine dehydratase activity, J. Biochem. 67, 685-692 (1970). H.C. PITOT and C. PERAINO, Studies on the induction and repression of enzymes in rat liver. 1. Induction of threonine dehydrase and ornithine transaminase by oral intubation of casein hydrolysate, J. Biol. Chem. 239, 1783-1788 (1964). C. PERAINO and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver. II. Carbohydrate repression of dietary and hormonal induction of threonine dehydrase and ornithine transaminase, J. Biol. Chem. 239, 4308-4313 (1964). C. PERAINO, R. L. BLAKE and H. C. PITOT, Studies on the induction and repression of enzymes in rat liver. III. Induction of ornithine transaminase and threonine dehydrase by oral intubation of free amino acids, J. Biol. Chem. 240, 3039-3043 (1965). C. PERA1NO, C. LAMAR and H. C. PITOT, Studies on the mechanism of carbohydrate repression in rat liver, Advances in Enzyme Regulation 4, 199-217 (1966). H. NAKAGAWA, S. MIURA, H. KIMURA and T. KANATSUNA, Studies on substrate induction of serine dehydratase of rat liver, J. Biochem, 66, 549-564 (1969). H. MATSUTAKA, K. NAKAJIMA and E. ISHIKAWA, Hormonal and dietary control of enzymes in the rat. IV. Dietary control of some liver enzymes unique to amino acid metabolism and gluconeogenesis and its dependence on pancreatic hormones, J. Biochem. 70, 395-405 (1971). C. REMESEY, P. FAFOURNOUX and C. DEMIGNE, Control of hepatic utilization of serine, glycine and threonine in fed and starved rats, J. Nutr. 113, 28-39 (1983). E.V. R O W S E L L , J.A. CARNIE, S.D. W A H B I , A.H. A L - T A I a n d K . V. ROWSELL, LSerine dehydratase and L-serine-pyruvate aminotransferase activities in different animal species, Comp. Biochem. Physiol. 63B, 543-555 (1979). E.V. ROWSELL, A. H. AL-TAI, J. A. CARNIE and E. V. ROWSELL, Increased liver Lserine-pyruvate aminotransferase activity under gluconeogenic conditions, Biochem. J. 134, 349-351 (1973). E. ISHIKAWA, T. NINAGAWA and M. SUDA, Hormonaland dietary control ofserine dehydratase in rat, J. Biochem. 57, 506-513 (1965). W. W. MAK and H. C. PITOT, Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh, Biochem. J. 198, 499-504 (1981). J.M. MEIER, J. D. McGARRY, G. R, FALOONA, R. H. UNGER and D. W. FOSTER, Studies of the development of diabetic ketosis in the rat, Z LipidRes. 13, 228-233 (1972) J.-P. JOST, A. HSIE, S. D. HUGHES and L. RYAN, Role of cyclic adenosine 3',5'monophosphate in the induction of hepatic enzymes, I. Kinetics of the induction of rat liver serine dehydratase by cyclic adenosine 3',5'-monophosphate, J. Biol. Chem. 245, 351-357 (1970). W.D. WICKS, F, T. KENNEY and K.-L. LEE, Induction of hepatic enzyme synthesis in vivo by adenosine 3',5'-monophosphate, J, Biol. Chem. 244, 6008-6013 (1969), H.W. MOHRENWEISER and H. C. PITOT, Studies on the mechanism of 5-fluoroorotic acid inhibition of serine dehydratase induction, Arch, Bioehem. Biophys. 164, 663-668 (1974). O, GREENGARD and H. K. DEWEY, Initiation by glucagon of the premature

396

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101, 102.

103. 104.

105. 106, 107. 108. 109. 110. 111. 112. 113. 114. 115.

t16.

KEITH SNELL development of tyrosine aminotransferase, serine dehydratase, and glucose-6phosphatase in fetal rat liver, J. Biol. Chem. 242, 2986-2991 (1967). H . C . PITOT, H. INOUE and J. E. KAPLAN, Mechanisms in cellular metabolic regulation, Biochem. Pharmacol. 20, 1035-1039 (1971). W.D. WICKS, Regulation of protein synthesis by cyclic AMP, Advan. Cyclic Nuc. Res. 4, 335-438 (1974). C. NODA, T. NAKAMURA and A. ICHIHARA, Hormonal regulation of serine dehydratase activity in primary cultures of adult rat hepatocytes, Biochem. Biophys. Res. Commun. 100, 65-72 (1981). C. NODA, T. NAKAMURA and A. ICI-IIHARA, a-Adrenergic regulation of enzymes of amino acid metabolism in primary cultures of adult rat hepatocytes, J. Biol. Chem. 258, 1520-1525 (1983). I. T. OLIVER, R. L. MARTIN, C. 3. FISHER and G. C. T. YEOH, Enzymic differentiation in cultured fetal hepatocytes of the rat. Induction of serine dehydratase activity by dexamethasone and dibutyryl cyclic AMP, Differentiation, 234-238 (1983). M.S. EDWARDS, M. D. HITCHEN and K. SNELL, Regulation of multiple forms of Lserine dehydratase in mammalian liver, Biochem. Sac. Trans. 12, in press (1984) R . W . JOHNSON, L. E. ROBERSON and F, T~ KENNEY, Regulation of tyrosine aminotransferase in rat liver. X. Characterization and interconversion of the multiple enzyme forms, J. Biol. Chem. 248, 4521-4527 (1973). J.L. HARGROVE and D. K. GRANNER, Physical properties, limited proteolysis, and acetylation of tyrosine aminotransferase from rat liver, J. Biol. Chem. 256, 8012-8017 (1981). H. NAKAGAWA and H. KIMURA, Purification and properties of cystathionine synthetase from rat liver: separation of cystathionine synthetase from serine dehydratase, Biochem. Biophys. Res. Commun. 32, 208-214 (1968). R.D. FELD and H. J. SALLACH, Purification and properties of hydroxypyruvate: ralanine transaminase from rabbit liver, Arch. Biochem. Biophys. 159, 757-766 (1973). M. FUKASHIMA, Y. AIHARA and A. ICHIYAMA, Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon, J. Biol. Chem. 253, 1187-1194 (1978). T. NOGUCHt, E. OKUNO and R. KIDO, Identity ofisoenzyme 1 of histidine-pyruvate aminotransferase with serine-pyruvate aminotransferase, Biochem. J. 159,607-613 (1976). T. NOGUCHI and Y. TAKADA, Purification and properties of peroxisomal pyruvate (glyoxylate) aminotransferase from rat liver, Biochem. J. 175, 765-768 (1978). T. ODA, M. YANAGISAWA and A. ICHIYAMA, Induction of serine: pyruvate aminotransferase in rat liver organeiles by glucagon and a high-protein diet, ,1".Biochem. 91,219-232 (1982). T. NOGUCI-II, E. OKUNO, Y. TAKADA, Y. MINATOGAWA, K. OKAI and R. KIDO, Characteristics of hepatic alanine-glyoxylate aminotransferase in different mammalian species, Biochem. J. 169, 113-122 (1978). K. SNELL, Studies on L-alanine:glyoxylate aminotransferase in animal tissues, Ph.D. Thesis, University of Manchester, Manchester, U.K. (1971). E. V, ROWSELL, A. H. AL-TAI, J. A. CARNIE and K. V. ROWSELL, Liver L-serinepyruvate aminotransferase activity in different animal species, Biochem. 3. 127, 27P (1972). K. SNELL, Mitochondrial-cytosolic interrelationships involved in gluconeogenesis from serine in rat liver, FEBS Lett. 55, 202-205 (1975). K.V. ROWSELL, L. M. R. AL-NAAMA and P. BENETT, The subcellular distribution of rat liver serine-pyruvate aminotransferase, Biochem J. 202, 483--490 (1982). K. SNELL, Mitochondrial-cytosolic interrelationships in gluconeogenesis from serine, pp. 118-121 in Use of Isolated Liver Cells and Kidney Tubules in Metabolic Studies (J. M. TAGER, H. D. SOLING and J, R. WILLIAMSON, eds.), North-Holland Pub. Co., Amsterdam (1976). T. NOGUCHI, Y. MINATOGAWA, Y. TAKADA, E, OKUNO and R. KIDO, Subcellular distribution of pyruvate (glyoxylate) aminotransferases in rat liver, Biochem. Z 170, 173-175 (1978).

REGULATION OF SERINE METABOLISM 117. 118.

119. 120. 121. 122.

123.

124. 125.

126. 127.

128.

129.

130. 131. 132. 133. 134. 135. 136. 137. 138. 139.

397

P, D. DAWKINS and F. DICKENS, The oxidation of o- and L-glycerate by rat liver, Biochem. J, 94, 353-367 (1965). I.Y. ROSENBLUM, D, H. ANTKOWIAK, H. J. SALLACH, L. E. FLANDERS and L. A. FAHIEN, Purification and regulatory properties of beef liver D-glycerate dehydrogenase, Arch. Biochem. Biophys, 144, 375-383 (1971). E. SUGIMOTO, Y. KITAGAWA, K. NAKANISHI and H. CHIBA, Purification and properties of beef liver o-glycerate dehydrogenase, J. Biochem. 77, 1307- I315 (1972). A. ICHIHARA and D. M, GREENBERG, Studies on the purification and properties of I~glyceric acid kinase of liver, J. Biol. Chem. 225, 949-958 (1957). S . L . VANDER and N. E. TOLBERT, Glyoxylate metabolism by isolated rat liver peroxisomes, Biochim. Biophys. Acta 215, 449-453 (1970). Y, KITAGAWA and E, SUGIMOTO, Possibilityof mitochondrial-cytosoliccooperation in gluconeogenesis from serine via hydroxypyruvate, Biochim. Biophys. Acta582, 276-282 (1979). W. LAMPRECHT, F. HEINZ and T. DIAMANTSTEIN, Phosphorylierung yon ~glycerinsaiire zu 2-phospho-o-glycerinsatire mit gIyceratkinase in Leber. II. Identifizierung des reaktionproduktes durch papier-chromatographie, Hoppe-Seyler's Z. Physiol. Chem. 328, 204-206 (1%2). Y. KITAGAWA, H. KATAYAMA and E. SUGIMOTO, Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein, Biochim. Biophys. Acta 582, 260-275 (1979). J. HOSHINO, D. SIMON, B. ROBERT and H. KROGER, Dietary and cortisone regulation of cytosoi serine and phosphoserine aminotransferase in rat liver, HoppeSeyler's Z. Physiol. Chem. 335, 4-10 (1974) S.C. BHATIA, S. BHATIA and S. ROUS, Gluconeogenesis from L-serine in rat liver, Life Sci. 17, 267-274 (1975). J. HOSHINO, B. ROBERT and H. KROGER, Induction in vivo of rat liver L-serinepyruvate aminotransferase by N6,O2"-dibutyryl cyclic AMP and its inhibition by cortisone, Biochim. Biophys. Acta 338, 418-427 (1974). J. HOSHINO, U. KUHNE, B. FILJAK and H. KROGER, Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol t-serine-pyruvate aminotransferase by dibutyryl adenosine 3',5'-monophosphate, Biochim. Biophys. Acta 399, 42-49 (1975). T. ODA, A. ICHIYAMA, S. MIURA, M. MORI and M. TATIBANA, In vitro synthesis of a putative p~'?cursor of serine-pyruvate aminotransferase of rat liver mitochondria, Biochem. Biophys. Res. Commun. 102, 568-573 (I981). B.D. ROSS, R. HEMS and H. A. KREBS, The rate of gluconeogenesis from various precursors in the perfused rat liver, Biochem, J.. I02, 942-951 (1967). J.H. EXTON and C. R. PARK, Control ofgluconeogenesis in liver. I. General features of gluconeogenesis in the peffused livers of rats, J, Biol. Chem. 242, 2622-2636 (1967). E. ISHIKAWA, T. AIKAWA and H. MATSUTAKA, The role ofalanine and serine in hepatic gluconeogenesis, J. Biochem. 71, 1093-1095 (1972), P.S. BELO, D. R. ROMSOS and G. A. LEVEILLE, Influence of diet on lactate, alanine and serine turnover and incorporation into glucose in the dog, J. Nutr. 107, 397-403 (1977). C. REMES¥, P. FAFOURNOUX and C. DEMIGNE, Control of hepatic utilization of serine, glycine and threonine in fed and starved rat, J. Nutr. 113, 28-39 (1983). H, A. KREBS, D. A. H. BENNETT, P. DE GASQUET, T. GASCOYNE and T, YOSHIDA, Renal gluconeogenesis. The effect of diet on the gluconeogenic capacity of rat-kidney-cortex slices, Biochem. J. 86, 21-27 (1963). D, FRIEDRICHS, On the stimulation of gluconeogenesis by L-lysine in isolated rat kidney cortex tubules, Biochim. Biophys. Acta 392, 255-270 (1975). F, DICKENS and D. H. W1LLIAMSON, Studies on the metabolism ofhydroxypyruvate in animal tissues, Biochem, J. 72,496-508 (1959). F. DICKENS and D. H. WILLIAMSON, Metabolism of D- and L-[3-J4C]glycerate by liver tissue of the rat, Biochem. J. 77, 356-363 (1960). D . H . WILLIAMSON and E. V. ELLINGTON, Hydroxypyruvate as a gluconeogenic substrate in rat hepatocytes, Biochem. J. 146, 277-279 (1975).

398 140. 141. 142. 143. 144.

145. 146. 147. 148. 149.

150. 151. 152. 153. 154. 155, 156, 157. 158. 159. 160. 161. 162,

163.

KEITH SNELL A.B. EISENSTEIN and I. STRACK, Effect of high protein feeding on gluconeogenesis in rat liver, Diabetes 20, 577-585 (1971). A, B. EISENSTEIN, I, STRACK and A. STEINER, Increased hepatic gluconeogenesis without a rise of glucagon secretion in rats fed a high fat diet, Diabetes 23,869-875 (1974). H . D . SOLING, B. WlLLMS, 3. KLEINEKE and M. GEHLHOFF, Regulation of gluconeogenesis in the guinea pig liver, Europ. J. Biochem. 16, 289-302 (1970). K. SNELL, Pathways of gluconeogenesis from t-serine in the neonatal rat, Biochem. J. 142, 433--436 (1974). T. METZ, U. NOGAJ and W. STAIB, Comparative studies on the influence of quinolinic acid on the metabolism of L-serine and L-alanine in the isolated perfused rat liver, HoppeSeyler's Z. PhysioL Chem. 353, 1496-1499 (1972). T . M . CHAN and R. A. FREEDLAND, The role of L-serine dehydratase in the metabolism of L-serine in the perfused rat liver, Biochim. Biophys. Acta 237, 99-106 (1971 ). G.P. BELIVEAU and R. A. FREEDLAND, Effect of starvation and diet composition on two pathways of L-serine metabolism in isolated rat hepatocytes, J. Nutr. 112,686-696 (1982). K. SNELL, Hypoglycaemia caused by indole and quinoline derivatives Biochem. Soc. Trans. 7, 745-749 (1979). N. W. CORNELL, P. LUND, R. HEMS and H. A. KREBS, Acceleration of gluconeogenesis from lactate by lysine, Biochem. J. 134, 671-672 (1973). J. MENDES-MOURAO, A. P. HALESTRAP, D. M. CRISP and C. I. POGSON, The involvement of mitochondrial pyruvate transport in the pathways of gluconeogenesis from serine and alanine in isolated rat and mouse liver cells, FEBS Lett. 53, 29-32 (1975). G. KIKUCHI, The glycine cleavage system: composition, reaction mechanism, and physiological significance, Mol. Cell. Biochem. 1, 169-187 (1973). G.P. RELIVEAU and R. A, FREEDLAND, Metabolism of serine, glycine and threonine in isolated cat hepatocytes Fells Domestica, Comp. Biochem. PhysioL 71B, 13-18 (1982). K. SNELL and D. G. WALKER, Gluconeogenesis in the newborn rat: the substrates and their quantitative significance, Enzyme 15, 40-81 (1973). J . A . CARNIE, E. V. ROWSELL, M. K. DABBAGHIAN, D. R. HOBBS and K. V. ROWSELL, Comparative and developmental studies on 4-hydroxy-2-oxoglutarate aldolase and hydroxyproline oxidase, Comp. Biochem. PhysioL 71B, 681-687 (1982). E. ADAMS, Metabolism of proline and hydroxyproline, Int. Rev. Connect. Tissue Res. 5, 1-91 (1970). U. MAITRA and E, E. DEKKER, Purification and properties of rat liver 2-keto-4hydroxyglutarate aldolase, J. Biol. Chem. 239, 1458-1491 (1964). E.V. ROWSELL, K. SNELL, J. A. CARNIE and K. V. ROWSELL, The subcellular distribution of rat liver t-alanine-glyoxylateaminotransferase in relation to a pathway for glucose formation involving glyoxylate, Biochem. J. 127, 155-165 (1972). K. SNELL, The regulation of rat liver L-alanine-glyoxylate aminotransferase by glucagon in vivo, Biochem. J. 123, 657-659 (1971). K. SNELL and D. G. WALKER, The adaptive behaviour of the isoenzyme forms of rat liver alanine aminotransferases during development, Biochem. J. 128, 403-413 (1972). K. SNELL, Discussion on perinatal mammalian liver, pp. 234-237 in Inborn Errors of Metabolism (F, A. HOMMES and C. J. VAN DEN BERG, eds.), Academic Press, London (1973). K. SNELL, Regulation of protein metabolism during postnatal development, Biochem. Soc. Trans. 9, 367-368 (1981). K~ SNELL, Glucose turnover in the newborn rat, pp. 81-109 in Metabolic Adaptation to Extrauterine Life (R. DE MEYER, ed,), Martinus Nijhoff Pub., The Hague (1981). A. NEUBERGER and F. F. RICHARDS, Protein biosynthesis in mammalian tissues. II. Studies on protein turnover in the whole animal, pp. 243-296 in Mammalian Protein Metabolism, Vol.2 (H. N. MUNRO and J. B. ALLISON, eds.), Academic Press, New York (1964) C.S. GARRETT and K. SNELL, The glyoxylate cycle in developing neonatal rat liver, Biochem. Soc. Trans. 11, 289 (1983).

REGULATION OF SERINE METABOLISM 164. 165.

166. 167. 168.

169. 170. 171. 172.

173. 174. 175. 176.

177.

178. 179.

180. 181. 182.

183. 184. 185.

399

D. YEUNG and I. T. OLIVER, The postnatal induction of serine dehydratase in rat liver, Comp Biochem. PhysioL 40A, 135-144 (1971). H. PHILIPPIDIS, R. W. HANSON, L. RESHEF, M. F. HOPGOOD and F. J. BALLARD, The initial synthesis of proteins during development: Phosphoenolpyruvate carboxylase in rat liver at birth, Biachem. J. 126, 1127-1134 (1972). F.R. BUTCHER and V. R. POTTER, Control of the adenosine 3',5'-monophosphateadenyl cyclase system in the livers of developing rats, Cancer Res. 32, 2141-2147 (1972). S. MIURA and H. NAKAGAWA, Studies on the molecular basis of development of serine dehydratase in rat liver, J. Biochem. 68, 543-548 (1970). V . R . POTTER, M. WATANABE, H. C. PITOT and H. P. MORRIS, Systematic oscillations in metabolic activity in rat liver and hepatomas. Survey of normal diploid and other hepatoma lines, Cancer Res. 29, 55-78 (1969). D. E. KIZER and D. E. LACY, Deletion of serine dehydratase and eystathionine synthetase activities during azo-dye carcinogenesis, Proc. Soc. ExptL BioL Med. 106, 790-794 (1961). N.J. CURTIN and K. SNELL, Enzymic retrodifferentiation during hepatocarcinogenesis and liver regeneration in rats in vivo, Brit. £ Cancer 48, 495-505 (1983). E. REID, Specificity of certain biochemical derangements in hepatic carcinogenesis, Brit. J, Cancer 24, 127-137 (1970). F.R. BUTCHER, D. F. SCOTT, V. R. POTTER and H. P. MORRIS, Endocrine control of cyclic adenosine 3',5'-monophosphate levels in several Morris hepatomas, CancerRes, 32, 2135-2140 (1972). F.R. BUTCHER, D. F. SCOTT, V. R. POTTER and H. P. MORRIS, Naturally occurring and induced levels of amino acid transport and tyrosine aminotransferase in rats bearing Morris hepatomas, Cancer Res. 32, 2127-2134 (1972). G. WEBER and M. A. LEA, The molecular correlation concept of neoplasia, Advances& Enzyme Regulation 4, 115-145 (1966). L. SCHIRCH, Serine hydroxymethyltransferase, Advances in Enzymology 53, 83-112 (1982) H. OGAWA and M. FUJIOKA, Purification and characterization of cytosolic and mitochondrial serine hydroxymethyltransferases from rat liver, J. Biochem. 90, 381-390 (1981). A. G. PALEKAR, S. S. TATE and A. MEISTER, Rat liver aminomalonate decarboxylase. Identity with cytoplasmic serine hydroxymethyltransferase and allothreonine aldolase, J. Biol. Chem. 248, 1158-1167 (1973). M. FUJIOKA, Purification and properties of serine hydroxymethylase from soluble and mitochondrial fractions of rabbit liver, Biochim. Biophys. Acta 1/15, 338-349 (1969). M. AKHTAR, H. A. EL-OBEID and P. M. JORDAN, Mechanistic, inhibitory and stereochemical studies on cytoplasmic and mitocbondrial serine transhydroxymethylases, Biochem. J. 145, 159-169 (1975). L. SCHIRCH and D. PETERSON, Purification and properties of mitochondrial serine hydroxymethyltransferase, J. Biol. Chem, 255, 7801-7806 (1980). K. S, RAMESH and N. A. RAO, Purification and physicochemical, kinetic and immunological properties of allosteric serine hydroxymethyltransferase from monkey liver, Biochem. J. 187, 623-636 (1980). L. SCHIRCH and J. QUASHNOCK, Evidence that tetrahydrofolate does not bind to serine hydroxymethyltransferase with positive homotropic cooperativity, J. Biol. Chem. 256, 6245-6249 (1981). D KEPPLER and A. HOLSTEGE, Pyrimidine nucleotide metabolism and its compartmentation, pp. 147-203 in Metabolic Compartmentation (H. SIESS, ed.), Academic Press, New York (1981). Y. MOTOKAWA and G. KIKUCHI, Glycine metabolism in rat liver mitochondria V. Intramitochondrial localization of the reversible glycine cleavage system and serine hydroxymethyltransferase, Arch. Biochem. Biophys. 146, 461-466 (1971). R.K. HAMPSON, L. L. BARRON and M. S. OLSON, Regulation of the glycine cleavage system in isolated rat liver mitochondrta, J. Biol. Chem. 258, 2993-2999 (1983).

400 186. 187. 188.

189.

190. 191. 192.

193. 194. 195. 196. 197.

198. 199. 200.

201.

KEITH SNELL T. YOSHIDA and G. KIKUCHI, Major pathways of serine and glycine catabolism in various organs of the rat and cock, J. Biochem. 73, 1013-1022 (1973). J.E. KAY, Early effects of phytohaemagglutinin on lymphocyte RNA synthesis, Europ ,L Biochem. 4, 225-232 (1968). N.R. LING, The structure and ultrastructure of lymphocytes and transformed cells, pp. 81-91 in Lymphocyte Stimulation (N. R. LING, ed.), North-Holland Pub. Co., Amsterdam (1968). J. THORNDIKE, T.-T, PELLINIEMI and W. S. BECK, Serine hydroxymethyltransferase activity and serine incorporation in leukocytes, Cancer Res. 39, 3435-3440 (1979). D.A. HUME, J. L. RADIK, E. FERBER and M. J. WEIDEMANN, Aerobic glycolysis and lymphocyte transformation, Biochem. J. 174, 703-709 (1978). D.A. HUME and M. J. WEIDEMANN, Role and regulation of glucose metabolism in proliferating ceils, J. NatL Cancer Inst. 62, 3-8 (1979). T.A. SANBORN, R. L. KNOWLE and H. J. SALLACH, Regulation of enzymes ofserine and one-carbon metabolism by testosterone in rat prostate, liver and kidney, Endocrinology 97, 1000-1007 (1975). A. HERRANEN and G. C, MUELLER, The effect ofestradiol pretreatment on the serine aldolase activity of rat uteri, Bioehim. Biophys. Acta 24, 223-224 (1957). A. HERRANEN and G. C. MUELLER, Effect ofestradiol on the metabolism ofserine-3C ~4 in surviving uterine segments, J. BioL Chem. 223, 369-375 (1956). J.A. STURMAN, G. E. GAULL and N. C. R. RAIHA, DNA synthesis from the B-carbon of serine by fetal and mature human liver, Biol. Neonate, 27, 17-22 (1975). M.E. BROSNAN, G. W. SYMONDS, D. E. HALL and D. L. SYMONDS, Polyamine metabolism in liver of young rats, Biochem. J. 174, 727-732 (1978). J . R . BERTINO, R. SILBER, M. FREEMAN, A. ALENTY, M. ALBRECHT, B. W. GABRIO and F. M. HUENNEKENS, Studies on normal and leukemic leukocytes. IV. Tetrahydrofolate-dependent enzyme systems and dihydrofolic reductase, J. Clin, lnvestig. 42, 1899-1907 (1963). J.D. REGAN, H. VODOPICK, S. TAKEDA, W. H. LEE and F. M. FAULCON, Serine requirement in leukemic and normal blood cells, Science 163, 1452-1453 (1969). J.W. PORTEOUS, Catalysis and modulation in metabolic systems, Biochem, Soc. Trans. 11, 29-43 (1983). J. MOWBRAY, The autoregulation of multienzymesequences in the intact cell,pp. 19-41 in Short-term Regulation of Liver Metabolism (L, HUE and G. VAN DE WERVE, eds.), Elsevier North-Holland, Amsterdam (1981). L.A. SAUER, J. W. STAYMAN and P. T. DAUCHY, Amino acid, glucose, and lactic acid utilization in vivo by rat tumors, Cancer Res. 42, 4090-4097 (1982).