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Transcarbamylase activity in fetal liver and in liver of partially hepatectomized parabiotic rats
ARCHIVES
OF
BIOCHEMISTRY
AND
Transcarbamylase
BIOPHYSICS
421428
109,
Activity
in Fetal
Hepatectomized SAXGDUK Department
of Physiological
KIM2
Chemistry,
(1965)
Liver
in Liver
Parabiotic AND
October
of Partially
Rats’
PHILIP
Medical Sciences Madison, Wisconsin
Received
and
I’. WHEY Building,
University
of
Wisconsin,
23, 1964
Carbamyl phosphate-aspartate transcarbamylase (aspartate transcarbamylase) has been measured in livers of embryonic rats and guinea pigs. The levels of enzyme activity were found to be highest in the earliest fetal livers and to decrease during development to low levels of the adult animal. In case of the rat, early fetal liver was found to have 5.7 times the specific activity of aspartate transcarbamylase found in adult liver. Aspartate transcarbamylase activity in intact and regenerating liver of partiall. hepatectomized single, parabiotic pairs, and a parabiotic triplet has been measured. The level of aspartate transcarbamylase activity has been found to rise not on13 during regeneration of the partially hepatectomized livers but also in the intact livers of the parabiotic animals. The level of carbamyl phosphate synthetase was found not to change significantly during liver regeneration in contrast to the level of ornithine transcarbamylase, which was found to decrease as much as 4070 120 hours after partial hepatectomy. The significance of an increase in aspartate transcarbamylase with a concomitant decrease of ornithine transcarbamylase during regeneration of liver is discussed in terms of the regulatory role of these enzymes in growth and differentiation. Attempts to influence the level of aspartate transcarbamylase activity in regenerating liver of rats by injection of different pyrimidine derivatives and precursors revealed no significant effect except, possibly in the case of erotic acid.
It has been shown previously that carbamyl phosphate-aspartate transcarbamylase (aspartat,e kanscarbamylase) act’ivity is increased in hepatoma nodules (1) and during the regeneration of rat liver (2). The increased enzymic activity in t)he case of regenerating liver returns to the normal level when regeneration of the liver is complete. In order to gain more insight int’o the relationship of t,his enzyme to cell growth, we
have studied aspartate transcarbamylase act’ivity in embryonic liver at different, stages and in regenerating liver of individual and parabiotic rats. The use of partially hepatectomized, parabiotic rats has provided information on the question as to the existence of a circulat,ing factor (or factors) which might be involved in the regulation of the level of aspartate tjranscarbamylase in the regenerative or growth process. RIore det,ailed aspects of this study have been recorded in a doctoral dissertation (3). Propert,ies and distribution of aspart,ate transcarbamylase have been recently reviewed (4.
1 This study was supported in part by a grant from the Wisconsin Alumni Research Foundation, and by grant C-3571 from the National Cancer Institute, National Institutes of Health, U. S. Public Health Service. z Present address: Department of Biochemistry, University of Ottawa Faculty of Medicine, Ottawa 2, Canada.
EXPERIMENTAL Preparation of liver rats were anesthetized 421
for enzyfrre assay. with ether, the
After liver
the was
422
KIM
AND
immediately removed from the animal and immersed in ice-cold isotonic KC1 solution. A portion of the liver was blotted with filter paper and then homogenized in 9 volumes of KC1 by the use of a glass hand homogenizer. The homogenized preparation was centrifuged in the Spinco model L centrifuge for 30 minutes at 105,000 9. Previous studies established that the enzyme is present in the supernatant fraction of liver (5). The result,ing supernatant solution was removed by means of a pipette and kept in an ice bath until the incubation was started. Assay for aspartate transcarbamylase. The assay system for aspartate transcarbamylase had the folloning composition: 350 rmoles diethanolamine HCl, pH 9.2; 70 pmoles sodium aspartate, pH 9.2; 70 pmoles of the crystalline diammonium salt of carbamyl phosphate (6); and 0.5-1.0 ml of the enzyme preparation in a total volume of 3.5 ml. The mixture was incubated for 60 minutes unless otherwise indicated. The reaction was stopped by the addition of 0.7 ml of 1.8 M perchloric acid solution containing 0.7 M silver acetate.3 The silver ions serve to remove chloride ions, which interfere with the color reaction. The deproteinized mixture was cent,rifuged, and the excess silver ions, and other interfering cations, were removed by passing 3 ml of supernatant solution through a column (9 mm in diameter) containing 1 gm Dowex-50 (H+). The eluate was collected and made up to 5 ml with distilled Hz0 and then assayed for carbamylaspartic acid by the method of Koritz and Cohen (7). Because the blank may vary, depending on time of incubation, the amount of enzyme used, and the concentration of carbamyl phosphate, each tube had its own blank as a control which contained no aspartate. Specific activity of aspartate transcarbamylase is expressed as micromoles of carbamylaspartic acid synthesized per hour per milligram protein under assay conditions. Optimal conditions of pH and concentrations of substrates were established for the standard assay system. The pH optimum was found to be 9.2. Under the standard assay conditions the amount of carbamyl-aspartate synthesized was found to be linear as a function of time up to 60 minutes and linear as a function of enzyme concentration. Assays were performed at 38°C. Assay for enzyme of urea cycle. The assay methods for the urea cycle enzymes have been described in detail elsewhere (8). Determination of protein. Protein was deter3 This Margaret Chemistry,
modification Marshall, University
was developed by Dr. Department of Physiological of Wisconsin,
COHEN mined by the method of Lowry et al., (9); bovine serum albumin was used as a standard. Operative techniques. The animals (200-250 gm) used in these experiments were obtained from Holtzman Rat Co., Madison, Wisconsin, and were fed a ration of Rockland Mouse Diet and water. After partial hepatectomy, the animals had access to a 10% glucose solution in place of water. Partial hepatectomy was performed according to the method described by Higgins and Anderson (10). Parabiotic pairs and one triplet were prepared by simple subcutaneous joining and a modification of the open celoemic technique (11). Litter mates, weighing between 100 and 150 gm, were subjected to ether anesthesia. A longitudinal incision was made from a point l-2 cm from the base of the tail to just posterior to the ear of each animal. The ventral edges of the skin margins of the two animals were then united with No. 1 black silk or Michel wound clips. The abdominal walls were then incised starting at a point 2-2.5 cm from the region just ventral to the anterior end of the iliac bone and extending to the margin of the ribs. The peritoneal and subcutaneous layers were then sutured separately with medium Chromic 5-O surgical gut, starting from the ventral line. The scapulas on the incision side of each animal were then located and the skin and subcutaneous connective tissue were freed from their dorsal surfaces. The scapulas were sutured by forcing a curved, size-10 needle with medium Chromic No. 1 surgical gut through the superficial muscle and the medial side of the left scapula of one of the animals. The suture was continued through the right scapula of the other animal. The lateral aspects of the scapulas of the two animals were then brought into firm union by tying the two free ends of the suture. Several single sutures were made through the subcutaneous tissue with medium Chromic 3-O surgical gut, and the skin edges were then brought together with Michel wound clips. The clips were removed 1 week after the operation. In order to avoid infection, 6 mg of tetracycline HCl was given intramuscularly to each animal postoperatively. RESULTS
Levels of the urea cycle enzymesduring liver regeneration. The specific activities of carbamyl phosphate synthetase and ornithine transcarbamylase in the regenerating liver are compared with those of control (normal) rat liver in Table I. The specific activity of ornithine transcarbamylase was significantly decreased and reached a minimum value of
TRANSCARBAMYLASES
IN
DIFFERENTIATION TABLE
SPECIFIC
ACTIVITIES
OF CARB~MYL
TRANSCARBAMYLASE
Regeneration
Carbamyl
time (hours)
phosphate
REGENERATION
SYNTHET~SE
AND
OF R.&T
synthetase
sp. act.
423
GROWTH
I
PHOSPH.ITE
DURING
AND
ORNITHINE
LIVER Ornithine
% Change
transcarbamylase
sp. act.
% Change
_____
6.01
zt 0.27
(16)b
12 24
0a
G.90
l
6.61
f
0.94 0.58
(3) (4)
36 48 72
6.14 5.30 6.06
f
0.28
(3)
+
0.10
(3)
f
0.15
(3)
96 120 144
6.54
f
0.21 -
-
234
f
3 (33)
-
+14 +10
247 237
f f
4 9
(3) (4)
+5
197 187 186 170
+ + f *
9 5 8 11
(3) (5) (G) (3)
140
xk
12
(6)
+2 -12 0
(3)
+g -
181 f
17 (3)
a Represents assay value of liver samples removed at time of partial hepatectomy. b Values represent averages f SD. Numbers in parentheses represent number of liver sayed. Assay conditions as previously described (8). Specific activity represents micromoles aspartate per milligram protein per hour.
120 hours after hepatectomy. However, the activity of carbamyl phosphate synthetase remained essentially unchanged during the period of 120 hours of liver regeneration. It was found that the total units of both enzymes are lower in regenerating liver than in normal liver (3). It has been reported that, during regeneration of liver, protein nitrogen is decreased
I
FIG. 1. bamylase rats. Livers pooled for the range livers were represent Experimental
I I 2 4 FET$iAAL;GH
I 6
0
-18 - 20 -20 -20 --IO -23
samples asof carbamyl
to 68 % of normal by 4 days (12), and that the weight is increased to 70-80% of the original liver at the fourth day of the postoperative period (13). ilspartate transcarbamylase activity in fetal liver. Aspartate transcarbamylase activity of liver from rat embryos and young rats has been plotted in Fig. 1 against body weight before birth, and age of rat (days) after birth. It can be seen that the specific act,ivity of aspartate transcarbamylase of early fetal liver was about 5.7 times that of adult rat liver. The activity was highest in the caseof the smallest fetuses which weighed between 0.63 and 0.71 gm; t,hereafter the enzymic ac-
I I I 44 20 40 60 80 DAYS POSTPARTUM
Specific activity of aspartate transcarin livers from fetal, young, and adult of several fetal litter mates were assay. The bars (--) shown represent of body weight of the fetuses whose pooled for assay. The closed circles values for individual liver specimens. conditions as described in the text.
FIG. 2. Specific activity of aspartate transcarbamylase in livers from fetal, young, and adult guinea pigs. Fetuses were obtained from animals of known length of pregnancy. Experimental conditions as described in the text.
424
KIM
AXD
COHEN
tivity decreased rapidly during embryonic TABLE II development, and more gradually aft)er birth, CIRCUL.ITION OF CW-LABELED EHYTHI~OCYTES until a plat.eau was reached. Similar results BETWEEN PAIHS OF PARABIOTIC RATP were obtained in the case of fetal and young guinea pig liver (Fig. 2). The aspartate transcarbamylase activity 1. Whole blood from recipient of Cr513453 of the placenta in rats was also determined labeled erythrocytes 2. Whole blood from second rat of 3920 (3). There appeared to be 110 significant, parabiotic pair relat’ionship between the stage of fetal de3. Erythrocytes from sample 1 2964 velopment and the aspartate transcarbamyl4. Erythrocytes from sample 2 3201 ase activity of placenba. 5. Plasma from sample 1 78 Bspartate tvanscarbamylase in parabiotic 6. Plasma from sample 2 12 ruts. Parabiotic rats were prepared in order to determine whether a circulating factor a Heparinized whole rat blood (2 ml) was incubated at 35” with 3.12 MC of Na&r5Qr for 30 (or factors) might be involved in regulation minutes. Ascorbic acid solution (0.1 ml containing of the levels of aspartate transcarbamylase, was as well as rate of growt)h or cell division of 2.5 mg) was then added and the mixture centrifuged at 4809 for 10 minutes. The plasma liver. l’art,ial hepatectomy of one of t)he was removed and the erythrocytes were washed parabiotic pairs and assay of both the regenand then suspended in 7 ml of 0.859& NaCl. The erating liver and the unoperated liver for suspension of Crsl-labeled erythrocytes was then aspartate transcarbamylase activity would injected intracardially into one of the parabiotic be expected to provide an answer to this animals. Twelve hours after the injection, l-ml question. However, it was necessary to es- samples of blood were removed from the tail veins tablish initially that the parabiotic animals of each of the parabiotic pair. The cpm values have been corrected for background counts. as prepared had a common blood supply. For this purpose, erythrocytes were labeled with Kai’CrOl. Crsl-labeled red blood cells were the viscera were found at the time of partial suspended in 0.85 5%NaCl solut,ion, and 7 ml hepatectomy. of the suspension (about 20,000 cpm/ml) was The right member of each twin pair and injected int’o one nlember of the parabiotic the t)wo end partners of a triplet were hepapair by heart puncture. After 12 hours, 1 ml tectomized. At periods of 12, 48, and 72 of blood, collected from the tail of each mernhours after hepatectomy, the animals were ber of t#he pair, was diluted with 3 ml of SaCl sacrificed and the liver of each member was solution, and the radioact,ivity was det,er- assayed for aspartate t~ranscarbamylase. The mined. The red blood cells were then separesults of the average values and standard are summarized in Table III. rated from the plasma by centrifugation and deviations suspended in isotonic NaCl, and t)he radioTable III also shows the levels of aspartate activity was determined. The counting was transcarbamylase in regenerating liver of done in a well-type scinbillation count.er. The partially hepatectomized single animals asresults are shown in Table II. The slight sayed by t,he same analytical procedure. It radioactivity in the plasma of the donor can can be seen from the Table that an increase be accounted for by the slight hemolysis in aspart)ate transcarbamylase activity ocwhich occurred. curs in both the regenerating liver and the It is apparent from the data of Table II liver from the intact animals. However, it that there was an adequate vascular anastoappears that the increase in aspartate transmosis and circulation between the two partcarbamylase activity of regenerating liver of ners . parabiotic rat’s is less than that seen in the Four pairs of parabiotic rats mere precase of single animals, and that’ the increase pared by simple subcutaneous joining, and in the intact livers of parabiotic rats t,ends to nine pairs were prepared by t,he open celoe- be slightly less than that of the regenerating mic technique. All the pairs of rats were livers. found to be well united and vascularized at Efect of pyrimidines and pyrimidine preaut,opsy. In a few cases, adhesions between cursors. The st,udies of Pardee and co-workSample
Cp/d
TRANSCARBAMYLASES
IN
DIFFERENTIATION TABLE
ASI'rZRT:\TE
TRANSCBRB.~MYL.~SE LIVER
I Liver regener ation (hours)
Animal
Single Single Parabiotic Parabiotic
Single Parabiotic Single Parabiotic Single Parabiotic
0 24 24 24
pairs triplet
pairs
~ 36 36 48
pairs pairs
;
48 72 72
ACTIVITY FROM
SINGLE
AND
III IN
REGENEUTING
AND
PaRABIOTIC Specific
Parabiosis period months)
Controla
l-l.5
-
1.5-5 -
NONKEGENER.STING
T
actkit@
/
Intact
zk 0.13 f 0.27 0.82 (1) 1.32 (1)
1.5
-
AND IhTS
Operated
0.91 0.92
1.5 -
42.5
GROWTH
0.92 0.90 1.02 0.98 0.84
1.5
f f rt r!z
0.09 0.15 0.12 0.09
f
0.10
0.84
(10 (4)
-
1
(2) (2) (2) (4) (2)
(1)
0.82
f 0.17 0.85 (1)
(4)
0.86
zk 0.19 -
(2)
0.91
f
(4)
-
0.15 -
0.91
(1)
,3pe1ated
Intact
-
-
91 G4 64 88
-
93 61 115 75 75 49
-
47 41
54 63 63 of
n Control value for single animals represents mean value of aspartate transcarbamylase activity liver samples removed at time of partial hepatectomy. * Control value for parabiotic pairs represents mean value of aspartate transcarbamylase activity of liver samples removed from one of the parabiotic pairs at time of partial hepatectomy. Numbers in parentheses represent numbers of animals. c Control values for parabiotic triplet represent single values of aspartate transcarbamylase activity of each of the liver samples removed from the 2 outside animals at time of partial hepatectomy. d Specific activity represents micromoles of carbamyl aspartate formed per milligram protein per hour. TABLE IV EFFECT
OF P~RIMIDINE
Uridine Uridine
,ZND DERIVllTIVES OF REGENERATING
acid
175 300 400 425 525 300 500
ON ASP~RT~TE LIVER Specific
Amo~t/ii~~;tion m
Compound
None Carbamyl aspartic Orotic acid Orotic acid Uracil
PRECURSORS ACTIVITY
ControlC
0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
f f f + zt zt f zk
0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
TRANSCARB.~MYLASE
activity6 After
(11) (11) (11) (11) (11) (11) (11) (11)
48 hours
1.01 1.05
regeneration
f 0.10 f 0.10 0.80 (1) 0.77 (1) 1.05 (1) 1.09 (I) 1.15 (1) 1.03 (1)
(3) (2)
a Amounts shown were injected intraperitoneally immediately after partial hepatectomy and at 12-hour intervals thereafter for a total of 5 injections. * Specific activity represents micromoles of carbamyl aspartate formed per milligram protein per hour. c Control values represent determination on sample of liver removed at time of partial hepatectomy. Numbers in parentheses represent numbers of animals.
ers (14, 13) on t,he feedback inhibition of aspart,ate transcarbamylase from Escher&&z coli prompted us to study the effect’ of injections of carbamylaspart,atc, orotate, uracil,
and uridine on t,he levels of aspartate transcarbamylase in regenerating rat, liver. Injections were given intraperitoneally immediat’ely after partial hepatectomy and at 12-hour
426
KIM
AND COHEX
similar type of feedback control in mammalian cells. Thus Hresnick (19-Z) has reported small inhibitions by relatively large amounts of various pyrimidines and pyrimidine nucleotides on rat liver aspartate transcarbamylase. In a more recent st)udy, in which rabbit erythrocyte aspartat)e t,ranscarbamylase preparations were used, Curci and Donachie (22) failed to find any evidence for pyrimidine end-product inhibition. However, very recently, Marchetti et al. (23) have rcportcd that, while erotic acid when fed (1% of the diet) to rats had no effect on the level of aspartate branscarbamylase activity, DISCUSSION it did decreasethe level 50 % when fed along The previously reported relatively high with adenine sulfate (0.25 74). Thus the prelevels of aspartate transcarbamylase in vailing evidence to dat,e is that whatever hepatoma (l), regenerating liver (2), fetal factor (or factors) operates in t)he regulation rat liver and heart (16), and, in the present of the level of liver aspartate transcarbamylstudy, of fetal rat and guinea pig liver aseis not the sameas that which operates on strongly suggest that a relationship exists the enzyme in E. coli. Evidence that, asparbetween the level of activity of this enzyme tate transcarbamylase preparations from and the rate of cell growth or division. Fur- lettuce, Pseudormnas JEuorescens, and Racilther evidence for such a relationship is seen lus subtilis behave differently to pyrimidine from the studies with ascites tumor cells (1) derivatives has been reported by Neumann and intestinal mucosa (5). A striking correla- and Jones (24). tion is demonstrable between the level of Because aspartate transcarbamylasc utiaspartate transcarbamylase and cell growth lizes carbamyl phosphate as a substrate, it in the caseof various parts of growing plants seemedof interest to compare the activity of the enzyme carbamyl phosphat,e synthetase (17). The ret’urn of aspartate transcarbamylase (25) (the enzyme responsible for synthesis of activity to normal levels (2) when regenera- carbamyl phosphate) with ornithine tion is complete further suggest,sthat the transcarbamylase (an enzyme which also activity of this enzyme is somehowrelated to utilizes carbamyl phosphate) in regenerating rate of cell growth or division. liver. Two possible changes might occur The increase in aspartate transcarbamylwhich could explain the increased activity of ase activity in the unoperated liver of para- aspartatc transcarbamylase in regenerating biotic animals with partial hepatectomy of and fetal liver. First, carbamyl phosphate the other one of the pair clearly indicates synthetase activity might increase wit*h the that a circulating factor (or factors) is oper- result that an increase in carbamyl phosating in the regulation of aspartate trans- phate formation would induce an increase in carbamylase activity. aspartatc transcarbamylase. However, it can Rat serum fractions of different kinds have be seen from the present studies that carbeen prepared and tested for their effect on bamyl phosphate synthetase act,ivity does the aspartate transcarbamylase activity of not appear to change significantly during normal and regenerating rat liver, but to regeneration. In the caseof fetal rat liver, the date no single fraction of reproducible and level of carbamyl phosphate synthctase acsignificant activity has been found (3). tivity has been shown to be very low during The studies of Gerhart and Pardee (18) on gestationand to rise rapidly postpart)ulli (26). the feedback inhibition of aspartate trans- It should also be noted that carbamyl phoscarbamylase in E. coli by cytidine triphos- phate synthetase activity of primary hepatophate (the end product of the pyrimidine mas has been reported to be very low (27). pathway) have served to focus interest on a McLean et al. (27) found that carbamyl phosintervals t,hereafter for a total of 5 injections. As can be seenfrom the data of Table IV, no significant effect was observed except in the caseof erotic acid, which appeared to inhibit the usual increase of aspartate transcarbamylase of regenerating liver about 40%. However, a larger number of animals and more systematic studies with a larger series of related compounds will be necessary t’o determine whet’her bhis effect is significant,. In any case, the relat’ively high doses required make it unlikely that this effect is of physiological significance.
TRANSCARBAMYLASES
IN
DIFFERENTIATION
phate synthetase activity showed a transient increase initially during a period of azo dye feeding, followed by a decrease t,o a very low level in the primary hepatoma. On the other hand, aspartate transcarbamylase activity has been shown to be elevated in hepatomas (1). Second, one might cxpcct to see a change in the act,ivit y of the enzyme system ornithine transcarbamylasc, which utilizes a substrate, carbamyl phosphate, in common with aspartat e transcarbamylase (4). It can be see11 from Table I that this enzyme decreases significantly (40 5%at 120 hours) during liver regeneration. However, in regenerating liver the maximum decrease in ornithine t.ranscarbamylase activity appears at 120 hours (Table I) in contrast, to t,he peak of aspartate transcarbamylase activity at 48 hours [Table IV (see also (2)]. Ornithine branscarbamylase activity also begins to return t,o the normal levels later than does aspartate transcarbamylase (Table I). In this connection, McLean et al. (27) have reported that ornithine t.ranscarbamylase decreases significantly during 2 weeks of azo dye feeding, with very low levels present in primary hepatoma nodules. Again, the reverse situation has been observed with respect to aspartate transcarbamylase activity (1). The effect of thyroid hormone on the levels of ornithine and aspartate transcarbamylases is of interest. While the level of aspartate transcarbamylase after hepatectomy in athyroid rats was within the range of values found for normal animals, the expected decrease in ornithine transcarbamylase was not observed unless the at’hyroid animals were given thyroxine (28). While a clear explanation for the apparent inverse relationship of aspartate transcarbamylase and ornithine transcarbamylase in regenerating liver, fetal liver, and hepatomas is not at hand, it is of interest to point out the regulatory significance of these observations. In a cell population undergoing rapid growth, such as exist,s in regenerating liver, fetal liver, and hepatoma, one would anticipate that the pathway for pyrimidine biosynthesis (which involves aspartate transcarbamylase as a key step) would be more active to insure the greater demand for pyrimidine nucleotides. On the other hand, the biosynthesis of urea (which involves ornithine
AND
GROWTH
427
transcarbamylase as a key step) may be considered to be a specialized fun&ion characteristic of a highly differentiated liver cell. During conditions of rapid cell division, it would be expectSed that cells, in the face of a limited substrate supply, would tend t’o become dedifferentiated, and in the present instance, to forego the highly differentiated property of urea biosynthesis. The elements of a self-regulatory process thus exist in liver which operate through regulation of the level of aspart’at,e transcarbamylase on the one hand, to insure pyrimidine nucleotide biosynthesis for rapid cell division, at the expense, on the other hand, of ornithine hranscarbamylase which serves a highly differentiated pathway in the nondividing liver cell. The importance of this relationship in understanding the biochemical basis of differentiation and dedifferentiation has been previously discussed (29, 30) and more recently by McLean et al. (27). REFERENCES 1. CALVA, E., LOWENSTEIN, J. M., AND COHEN, P. P., Cancer Res. 19, 101 (1959). 2. C~LVA, E., AND COHEN, P. P., Cancer Res. 19, 679 (1959). 3. KIM, S., Ph.D., Thesis, University of Wisconsin, Madison (1960). 4. COHEN, P. P., AND MARSHALL, M., in “The Enzymes” (P. D. Boyer, H. A. Lardy, and K. Myrbllck, eds.), 2nd edition, Vol. 6, p. 327. Academic Press, New York (1962). 5. LOWENSTEIN, J. M., AND COHEN, P. P., J. Biol. Chem. 220, 57 (1956). 6. METZENBERG, R. L., MARSHALL, M., AND COHEN, P. P., in “Biochemical Preparations” (H. A. Lardy, eds.), Vol. 7, p. 23. Wiley, New York (1960). 7. KORITZ, S. B., AND COHEN, P. P., J. Biol. Chem. 209, 145 (1954). 8. BROWN, G W., JR., AXD COHEN, P. P., J. Biol. Chem. 234, 1769 (1959). 9. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., BND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. HIGGINS, G. M., AND ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 11. BUNSTER, E., AND MEYER, R. K., Anat. Res. 67, 339 (1933). 12. PRICE, J. M., AND LAIRD, A. K., Cancer Res. 10, 650 (1950). 13. THOMSON, R. V., HEBGY, F. C., HUTCHISON, W. C., AND DAVIDSON, J. N., Biochem. J. 63, 460 (1953).
428
KIM
AND
R. A., END PARDEE, A. B., J. Biol. 14. YATES, Chem. 221, 757 (1956). J. C., BND PARDEE, A. B., J. Biol. 15. GERHBRT, Chem. 237, 891 (1962). Y., HURWITZ, R., \ND KILETCH16. NORDMANN, MER, N., Natwe 201, 617 (1964). L. I., ,\ND COHEN, P. P., 4rch. Bio17. STEIN, them. Biophys. 109, 429 (1965) (following article). J. C., AND PARDEE, A. B., Cold 18. GERH~RT, Spring Harbor Symp. Quant. Biol. 28, 491 (1963). E., 19. BRESNICK, Biochim. Biophys. dcta 61, 598 (1962). 20. BRESNICK, E., Biochim. Biophys. dcta 67, 425 (1963). 21. BRESNICK, E., Cancer Res. 22, 1246 (1962). M. K., ‘\ND DONACHIE, W. D., Biochim. 22. CURCI, Biophys. Acta 85, 338 (1964). M., PUDDU, P., .IND C~LD~RER~, 23. MARCHETTI, C. M., Biochem. J. 92, 46 (1964).
COHEN 24. NET:UNS. J., .\piI) JONEH, X E., :lxh. Binthem. 1~iophy.s. 104, 438 (1964). 25. COHEX, P. P., in “The Enz\mes” (I’. D. Buyer, H. A1. Lard>-, and K. Myrbiick, eds.), \‘ol. 6, 2nd edition, p, 477. Academic Press, New York (1962). 26. KENUN, A. L., .~IVD COHEN, 1’. P., Deaelop. Biol. 1, 511 (1959). 27. McLE.+.v, P., REID, E., .\SD GL-KSET, M. W., Biochem. 1. 91, 464 (1964). 28. NAZ.\HIO, M., AND COHEN, P. P., proc. Sot. Ezptl. Biol. Med. 106, 492 (1961). 29. COHEN, P. P., AND BROV-X, G. W., JK., in “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. II, p. 161. Academic Press, New York (1960). 30. COHEN, P. P., .IND S.ILL.\CH, H. J., in “Metabolic Pathways” (I). M. Greenberg, ed.), Vol. II, 2nd edition, p. 1, Academic Press, New York (1961).
OF
BIOCHEMISTRY
AND
Transcarbamylase
BIOPHYSICS
421428
109,
Activity
in Fetal
Hepatectomized SAXGDUK Department
of Physiological
KIM2
Chemistry,
(1965)
Liver
in Liver
Parabiotic AND
October
of Partially
Rats’
PHILIP
Medical Sciences Madison, Wisconsin
Received
and
I’. WHEY Building,
University
of
Wisconsin,
23, 1964
Carbamyl phosphate-aspartate transcarbamylase (aspartate transcarbamylase) has been measured in livers of embryonic rats and guinea pigs. The levels of enzyme activity were found to be highest in the earliest fetal livers and to decrease during development to low levels of the adult animal. In case of the rat, early fetal liver was found to have 5.7 times the specific activity of aspartate transcarbamylase found in adult liver. Aspartate transcarbamylase activity in intact and regenerating liver of partiall. hepatectomized single, parabiotic pairs, and a parabiotic triplet has been measured. The level of aspartate transcarbamylase activity has been found to rise not on13 during regeneration of the partially hepatectomized livers but also in the intact livers of the parabiotic animals. The level of carbamyl phosphate synthetase was found not to change significantly during liver regeneration in contrast to the level of ornithine transcarbamylase, which was found to decrease as much as 4070 120 hours after partial hepatectomy. The significance of an increase in aspartate transcarbamylase with a concomitant decrease of ornithine transcarbamylase during regeneration of liver is discussed in terms of the regulatory role of these enzymes in growth and differentiation. Attempts to influence the level of aspartate transcarbamylase activity in regenerating liver of rats by injection of different pyrimidine derivatives and precursors revealed no significant effect except, possibly in the case of erotic acid.
It has been shown previously that carbamyl phosphate-aspartate transcarbamylase (aspartat,e kanscarbamylase) act’ivity is increased in hepatoma nodules (1) and during the regeneration of rat liver (2). The increased enzymic activity in t)he case of regenerating liver returns to the normal level when regeneration of the liver is complete. In order to gain more insight int’o the relationship of t,his enzyme to cell growth, we
have studied aspartate transcarbamylase act’ivity in embryonic liver at different, stages and in regenerating liver of individual and parabiotic rats. The use of partially hepatectomized, parabiotic rats has provided information on the question as to the existence of a circulat,ing factor (or factors) which might be involved in the regulation of the level of aspartate tjranscarbamylase in the regenerative or growth process. RIore det,ailed aspects of this study have been recorded in a doctoral dissertation (3). Propert,ies and distribution of aspart,ate transcarbamylase have been recently reviewed (4.
1 This study was supported in part by a grant from the Wisconsin Alumni Research Foundation, and by grant C-3571 from the National Cancer Institute, National Institutes of Health, U. S. Public Health Service. z Present address: Department of Biochemistry, University of Ottawa Faculty of Medicine, Ottawa 2, Canada.
EXPERIMENTAL Preparation of liver rats were anesthetized 421
for enzyfrre assay. with ether, the
After liver
the was
422
KIM
AND
immediately removed from the animal and immersed in ice-cold isotonic KC1 solution. A portion of the liver was blotted with filter paper and then homogenized in 9 volumes of KC1 by the use of a glass hand homogenizer. The homogenized preparation was centrifuged in the Spinco model L centrifuge for 30 minutes at 105,000 9. Previous studies established that the enzyme is present in the supernatant fraction of liver (5). The result,ing supernatant solution was removed by means of a pipette and kept in an ice bath until the incubation was started. Assay for aspartate transcarbamylase. The assay system for aspartate transcarbamylase had the folloning composition: 350 rmoles diethanolamine HCl, pH 9.2; 70 pmoles sodium aspartate, pH 9.2; 70 pmoles of the crystalline diammonium salt of carbamyl phosphate (6); and 0.5-1.0 ml of the enzyme preparation in a total volume of 3.5 ml. The mixture was incubated for 60 minutes unless otherwise indicated. The reaction was stopped by the addition of 0.7 ml of 1.8 M perchloric acid solution containing 0.7 M silver acetate.3 The silver ions serve to remove chloride ions, which interfere with the color reaction. The deproteinized mixture was cent,rifuged, and the excess silver ions, and other interfering cations, were removed by passing 3 ml of supernatant solution through a column (9 mm in diameter) containing 1 gm Dowex-50 (H+). The eluate was collected and made up to 5 ml with distilled Hz0 and then assayed for carbamylaspartic acid by the method of Koritz and Cohen (7). Because the blank may vary, depending on time of incubation, the amount of enzyme used, and the concentration of carbamyl phosphate, each tube had its own blank as a control which contained no aspartate. Specific activity of aspartate transcarbamylase is expressed as micromoles of carbamylaspartic acid synthesized per hour per milligram protein under assay conditions. Optimal conditions of pH and concentrations of substrates were established for the standard assay system. The pH optimum was found to be 9.2. Under the standard assay conditions the amount of carbamyl-aspartate synthesized was found to be linear as a function of time up to 60 minutes and linear as a function of enzyme concentration. Assays were performed at 38°C. Assay for enzyme of urea cycle. The assay methods for the urea cycle enzymes have been described in detail elsewhere (8). Determination of protein. Protein was deter3 This Margaret Chemistry,
modification Marshall, University
was developed by Dr. Department of Physiological of Wisconsin,
COHEN mined by the method of Lowry et al., (9); bovine serum albumin was used as a standard. Operative techniques. The animals (200-250 gm) used in these experiments were obtained from Holtzman Rat Co., Madison, Wisconsin, and were fed a ration of Rockland Mouse Diet and water. After partial hepatectomy, the animals had access to a 10% glucose solution in place of water. Partial hepatectomy was performed according to the method described by Higgins and Anderson (10). Parabiotic pairs and one triplet were prepared by simple subcutaneous joining and a modification of the open celoemic technique (11). Litter mates, weighing between 100 and 150 gm, were subjected to ether anesthesia. A longitudinal incision was made from a point l-2 cm from the base of the tail to just posterior to the ear of each animal. The ventral edges of the skin margins of the two animals were then united with No. 1 black silk or Michel wound clips. The abdominal walls were then incised starting at a point 2-2.5 cm from the region just ventral to the anterior end of the iliac bone and extending to the margin of the ribs. The peritoneal and subcutaneous layers were then sutured separately with medium Chromic 5-O surgical gut, starting from the ventral line. The scapulas on the incision side of each animal were then located and the skin and subcutaneous connective tissue were freed from their dorsal surfaces. The scapulas were sutured by forcing a curved, size-10 needle with medium Chromic No. 1 surgical gut through the superficial muscle and the medial side of the left scapula of one of the animals. The suture was continued through the right scapula of the other animal. The lateral aspects of the scapulas of the two animals were then brought into firm union by tying the two free ends of the suture. Several single sutures were made through the subcutaneous tissue with medium Chromic 3-O surgical gut, and the skin edges were then brought together with Michel wound clips. The clips were removed 1 week after the operation. In order to avoid infection, 6 mg of tetracycline HCl was given intramuscularly to each animal postoperatively. RESULTS
Levels of the urea cycle enzymesduring liver regeneration. The specific activities of carbamyl phosphate synthetase and ornithine transcarbamylase in the regenerating liver are compared with those of control (normal) rat liver in Table I. The specific activity of ornithine transcarbamylase was significantly decreased and reached a minimum value of
TRANSCARBAMYLASES
IN
DIFFERENTIATION TABLE
SPECIFIC
ACTIVITIES
OF CARB~MYL
TRANSCARBAMYLASE
Regeneration
Carbamyl
time (hours)
phosphate
REGENERATION
SYNTHET~SE
AND
OF R.&T
synthetase
sp. act.
423
GROWTH
I
PHOSPH.ITE
DURING
AND
ORNITHINE
LIVER Ornithine
% Change
transcarbamylase
sp. act.
% Change
_____
6.01
zt 0.27
(16)b
12 24
0a
G.90
l
6.61
f
0.94 0.58
(3) (4)
36 48 72
6.14 5.30 6.06
f
0.28
(3)
+
0.10
(3)
f
0.15
(3)
96 120 144
6.54
f
0.21 -
-
234
f
3 (33)
-
+14 +10
247 237
f f
4 9
(3) (4)
+5
197 187 186 170
+ + f *
9 5 8 11
(3) (5) (G) (3)
140
xk
12
(6)
+2 -12 0
(3)
+g -
181 f
17 (3)
a Represents assay value of liver samples removed at time of partial hepatectomy. b Values represent averages f SD. Numbers in parentheses represent number of liver sayed. Assay conditions as previously described (8). Specific activity represents micromoles aspartate per milligram protein per hour.
120 hours after hepatectomy. However, the activity of carbamyl phosphate synthetase remained essentially unchanged during the period of 120 hours of liver regeneration. It was found that the total units of both enzymes are lower in regenerating liver than in normal liver (3). It has been reported that, during regeneration of liver, protein nitrogen is decreased
I
FIG. 1. bamylase rats. Livers pooled for the range livers were represent Experimental
I I 2 4 FET$iAAL;GH
I 6
0
-18 - 20 -20 -20 --IO -23
samples asof carbamyl
to 68 % of normal by 4 days (12), and that the weight is increased to 70-80% of the original liver at the fourth day of the postoperative period (13). ilspartate transcarbamylase activity in fetal liver. Aspartate transcarbamylase activity of liver from rat embryos and young rats has been plotted in Fig. 1 against body weight before birth, and age of rat (days) after birth. It can be seen that the specific act,ivity of aspartate transcarbamylase of early fetal liver was about 5.7 times that of adult rat liver. The activity was highest in the caseof the smallest fetuses which weighed between 0.63 and 0.71 gm; t,hereafter the enzymic ac-
I I I 44 20 40 60 80 DAYS POSTPARTUM
Specific activity of aspartate transcarin livers from fetal, young, and adult of several fetal litter mates were assay. The bars (--) shown represent of body weight of the fetuses whose pooled for assay. The closed circles values for individual liver specimens. conditions as described in the text.
FIG. 2. Specific activity of aspartate transcarbamylase in livers from fetal, young, and adult guinea pigs. Fetuses were obtained from animals of known length of pregnancy. Experimental conditions as described in the text.
424
KIM
AXD
COHEN
tivity decreased rapidly during embryonic TABLE II development, and more gradually aft)er birth, CIRCUL.ITION OF CW-LABELED EHYTHI~OCYTES until a plat.eau was reached. Similar results BETWEEN PAIHS OF PARABIOTIC RATP were obtained in the case of fetal and young guinea pig liver (Fig. 2). The aspartate transcarbamylase activity 1. Whole blood from recipient of Cr513453 of the placenta in rats was also determined labeled erythrocytes 2. Whole blood from second rat of 3920 (3). There appeared to be 110 significant, parabiotic pair relat’ionship between the stage of fetal de3. Erythrocytes from sample 1 2964 velopment and the aspartate transcarbamyl4. Erythrocytes from sample 2 3201 ase activity of placenba. 5. Plasma from sample 1 78 Bspartate tvanscarbamylase in parabiotic 6. Plasma from sample 2 12 ruts. Parabiotic rats were prepared in order to determine whether a circulating factor a Heparinized whole rat blood (2 ml) was incubated at 35” with 3.12 MC of Na&r5Qr for 30 (or factors) might be involved in regulation minutes. Ascorbic acid solution (0.1 ml containing of the levels of aspartate transcarbamylase, was as well as rate of growt)h or cell division of 2.5 mg) was then added and the mixture centrifuged at 4809 for 10 minutes. The plasma liver. l’art,ial hepatectomy of one of t)he was removed and the erythrocytes were washed parabiotic pairs and assay of both the regenand then suspended in 7 ml of 0.859& NaCl. The erating liver and the unoperated liver for suspension of Crsl-labeled erythrocytes was then aspartate transcarbamylase activity would injected intracardially into one of the parabiotic be expected to provide an answer to this animals. Twelve hours after the injection, l-ml question. However, it was necessary to es- samples of blood were removed from the tail veins tablish initially that the parabiotic animals of each of the parabiotic pair. The cpm values have been corrected for background counts. as prepared had a common blood supply. For this purpose, erythrocytes were labeled with Kai’CrOl. Crsl-labeled red blood cells were the viscera were found at the time of partial suspended in 0.85 5%NaCl solut,ion, and 7 ml hepatectomy. of the suspension (about 20,000 cpm/ml) was The right member of each twin pair and injected int’o one nlember of the parabiotic the t)wo end partners of a triplet were hepapair by heart puncture. After 12 hours, 1 ml tectomized. At periods of 12, 48, and 72 of blood, collected from the tail of each mernhours after hepatectomy, the animals were ber of t#he pair, was diluted with 3 ml of SaCl sacrificed and the liver of each member was solution, and the radioact,ivity was det,er- assayed for aspartate t~ranscarbamylase. The mined. The red blood cells were then separesults of the average values and standard are summarized in Table III. rated from the plasma by centrifugation and deviations suspended in isotonic NaCl, and t)he radioTable III also shows the levels of aspartate activity was determined. The counting was transcarbamylase in regenerating liver of done in a well-type scinbillation count.er. The partially hepatectomized single animals asresults are shown in Table II. The slight sayed by t,he same analytical procedure. It radioactivity in the plasma of the donor can can be seen from the Table that an increase be accounted for by the slight hemolysis in aspart)ate transcarbamylase activity ocwhich occurred. curs in both the regenerating liver and the It is apparent from the data of Table II liver from the intact animals. However, it that there was an adequate vascular anastoappears that the increase in aspartate transmosis and circulation between the two partcarbamylase activity of regenerating liver of ners . parabiotic rat’s is less than that seen in the Four pairs of parabiotic rats mere precase of single animals, and that’ the increase pared by simple subcutaneous joining, and in the intact livers of parabiotic rats t,ends to nine pairs were prepared by t,he open celoe- be slightly less than that of the regenerating mic technique. All the pairs of rats were livers. found to be well united and vascularized at Efect of pyrimidines and pyrimidine preaut,opsy. In a few cases, adhesions between cursors. The st,udies of Pardee and co-workSample
Cp/d
TRANSCARBAMYLASES
IN
DIFFERENTIATION TABLE
ASI'rZRT:\TE
TRANSCBRB.~MYL.~SE LIVER
I Liver regener ation (hours)
Animal
Single Single Parabiotic Parabiotic
Single Parabiotic Single Parabiotic Single Parabiotic
0 24 24 24
pairs triplet
pairs
~ 36 36 48
pairs pairs
;
48 72 72
ACTIVITY FROM
SINGLE
AND
III IN
REGENEUTING
AND
PaRABIOTIC Specific
Parabiosis period months)
Controla
l-l.5
-
1.5-5 -
NONKEGENER.STING
T
actkit@
/
Intact
zk 0.13 f 0.27 0.82 (1) 1.32 (1)
1.5
-
AND IhTS
Operated
0.91 0.92
1.5 -
42.5
GROWTH
0.92 0.90 1.02 0.98 0.84
1.5
f f rt r!z
0.09 0.15 0.12 0.09
f
0.10
0.84
(10 (4)
-
1
(2) (2) (2) (4) (2)
(1)
0.82
f 0.17 0.85 (1)
(4)
0.86
zk 0.19 -
(2)
0.91
f
(4)
-
0.15 -
0.91
(1)
,3pe1ated
Intact
-
-
91 G4 64 88
-
93 61 115 75 75 49
-
47 41
54 63 63 of
n Control value for single animals represents mean value of aspartate transcarbamylase activity liver samples removed at time of partial hepatectomy. * Control value for parabiotic pairs represents mean value of aspartate transcarbamylase activity of liver samples removed from one of the parabiotic pairs at time of partial hepatectomy. Numbers in parentheses represent numbers of animals. c Control values for parabiotic triplet represent single values of aspartate transcarbamylase activity of each of the liver samples removed from the 2 outside animals at time of partial hepatectomy. d Specific activity represents micromoles of carbamyl aspartate formed per milligram protein per hour. TABLE IV EFFECT
OF P~RIMIDINE
Uridine Uridine
,ZND DERIVllTIVES OF REGENERATING
acid
175 300 400 425 525 300 500
ON ASP~RT~TE LIVER Specific
Amo~t/ii~~;tion m
Compound
None Carbamyl aspartic Orotic acid Orotic acid Uracil
PRECURSORS ACTIVITY
ControlC
0.55 0.55 0.55 0.55 0.55 0.55 0.55 0.55
f f f + zt zt f zk
0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08
TRANSCARB.~MYLASE
activity6 After
(11) (11) (11) (11) (11) (11) (11) (11)
48 hours
1.01 1.05
regeneration
f 0.10 f 0.10 0.80 (1) 0.77 (1) 1.05 (1) 1.09 (I) 1.15 (1) 1.03 (1)
(3) (2)
a Amounts shown were injected intraperitoneally immediately after partial hepatectomy and at 12-hour intervals thereafter for a total of 5 injections. * Specific activity represents micromoles of carbamyl aspartate formed per milligram protein per hour. c Control values represent determination on sample of liver removed at time of partial hepatectomy. Numbers in parentheses represent numbers of animals.
ers (14, 13) on t,he feedback inhibition of aspart,ate transcarbamylase from Escher&&z coli prompted us to study the effect’ of injections of carbamylaspart,atc, orotate, uracil,
and uridine on t,he levels of aspartate transcarbamylase in regenerating rat, liver. Injections were given intraperitoneally immediat’ely after partial hepatectomy and at 12-hour
426
KIM
AND COHEX
similar type of feedback control in mammalian cells. Thus Hresnick (19-Z) has reported small inhibitions by relatively large amounts of various pyrimidines and pyrimidine nucleotides on rat liver aspartate transcarbamylase. In a more recent st)udy, in which rabbit erythrocyte aspartat)e t,ranscarbamylase preparations were used, Curci and Donachie (22) failed to find any evidence for pyrimidine end-product inhibition. However, very recently, Marchetti et al. (23) have rcportcd that, while erotic acid when fed (1% of the diet) to rats had no effect on the level of aspartate branscarbamylase activity, DISCUSSION it did decreasethe level 50 % when fed along The previously reported relatively high with adenine sulfate (0.25 74). Thus the prelevels of aspartate transcarbamylase in vailing evidence to dat,e is that whatever hepatoma (l), regenerating liver (2), fetal factor (or factors) operates in t)he regulation rat liver and heart (16), and, in the present of the level of liver aspartate transcarbamylstudy, of fetal rat and guinea pig liver aseis not the sameas that which operates on strongly suggest that a relationship exists the enzyme in E. coli. Evidence that, asparbetween the level of activity of this enzyme tate transcarbamylase preparations from and the rate of cell growth or division. Fur- lettuce, Pseudormnas JEuorescens, and Racilther evidence for such a relationship is seen lus subtilis behave differently to pyrimidine from the studies with ascites tumor cells (1) derivatives has been reported by Neumann and intestinal mucosa (5). A striking correla- and Jones (24). tion is demonstrable between the level of Because aspartate transcarbamylasc utiaspartate transcarbamylase and cell growth lizes carbamyl phosphate as a substrate, it in the caseof various parts of growing plants seemedof interest to compare the activity of the enzyme carbamyl phosphat,e synthetase (17). The ret’urn of aspartate transcarbamylase (25) (the enzyme responsible for synthesis of activity to normal levels (2) when regenera- carbamyl phosphate) with ornithine tion is complete further suggest,sthat the transcarbamylase (an enzyme which also activity of this enzyme is somehowrelated to utilizes carbamyl phosphate) in regenerating rate of cell growth or division. liver. Two possible changes might occur The increase in aspartate transcarbamylwhich could explain the increased activity of ase activity in the unoperated liver of para- aspartatc transcarbamylase in regenerating biotic animals with partial hepatectomy of and fetal liver. First, carbamyl phosphate the other one of the pair clearly indicates synthetase activity might increase wit*h the that a circulating factor (or factors) is oper- result that an increase in carbamyl phosating in the regulation of aspartate trans- phate formation would induce an increase in carbamylase activity. aspartatc transcarbamylase. However, it can Rat serum fractions of different kinds have be seen from the present studies that carbeen prepared and tested for their effect on bamyl phosphate synthetase act,ivity does the aspartate transcarbamylase activity of not appear to change significantly during normal and regenerating rat liver, but to regeneration. In the caseof fetal rat liver, the date no single fraction of reproducible and level of carbamyl phosphate synthctase acsignificant activity has been found (3). tivity has been shown to be very low during The studies of Gerhart and Pardee (18) on gestationand to rise rapidly postpart)ulli (26). the feedback inhibition of aspartate trans- It should also be noted that carbamyl phoscarbamylase in E. coli by cytidine triphos- phate synthetase activity of primary hepatophate (the end product of the pyrimidine mas has been reported to be very low (27). pathway) have served to focus interest on a McLean et al. (27) found that carbamyl phosintervals t,hereafter for a total of 5 injections. As can be seenfrom the data of Table IV, no significant effect was observed except in the caseof erotic acid, which appeared to inhibit the usual increase of aspartate transcarbamylase of regenerating liver about 40%. However, a larger number of animals and more systematic studies with a larger series of related compounds will be necessary t’o determine whet’her bhis effect is significant,. In any case, the relat’ively high doses required make it unlikely that this effect is of physiological significance.
TRANSCARBAMYLASES
IN
DIFFERENTIATION
phate synthetase activity showed a transient increase initially during a period of azo dye feeding, followed by a decrease t,o a very low level in the primary hepatoma. On the other hand, aspartate transcarbamylase activity has been shown to be elevated in hepatomas (1). Second, one might cxpcct to see a change in the act,ivit y of the enzyme system ornithine transcarbamylasc, which utilizes a substrate, carbamyl phosphate, in common with aspartat e transcarbamylase (4). It can be see11 from Table I that this enzyme decreases significantly (40 5%at 120 hours) during liver regeneration. However, in regenerating liver the maximum decrease in ornithine t.ranscarbamylase activity appears at 120 hours (Table I) in contrast, to t,he peak of aspartate transcarbamylase activity at 48 hours [Table IV (see also (2)]. Ornithine branscarbamylase activity also begins to return t,o the normal levels later than does aspartate transcarbamylase (Table I). In this connection, McLean et al. (27) have reported that ornithine t.ranscarbamylase decreases significantly during 2 weeks of azo dye feeding, with very low levels present in primary hepatoma nodules. Again, the reverse situation has been observed with respect to aspartate transcarbamylase activity (1). The effect of thyroid hormone on the levels of ornithine and aspartate transcarbamylases is of interest. While the level of aspartate transcarbamylase after hepatectomy in athyroid rats was within the range of values found for normal animals, the expected decrease in ornithine transcarbamylase was not observed unless the at’hyroid animals were given thyroxine (28). While a clear explanation for the apparent inverse relationship of aspartate transcarbamylase and ornithine transcarbamylase in regenerating liver, fetal liver, and hepatomas is not at hand, it is of interest to point out the regulatory significance of these observations. In a cell population undergoing rapid growth, such as exist,s in regenerating liver, fetal liver, and hepatoma, one would anticipate that the pathway for pyrimidine biosynthesis (which involves aspartate transcarbamylase as a key step) would be more active to insure the greater demand for pyrimidine nucleotides. On the other hand, the biosynthesis of urea (which involves ornithine
AND
GROWTH
427
transcarbamylase as a key step) may be considered to be a specialized fun&ion characteristic of a highly differentiated liver cell. During conditions of rapid cell division, it would be expectSed that cells, in the face of a limited substrate supply, would tend t’o become dedifferentiated, and in the present instance, to forego the highly differentiated property of urea biosynthesis. The elements of a self-regulatory process thus exist in liver which operate through regulation of the level of aspart’at,e transcarbamylase on the one hand, to insure pyrimidine nucleotide biosynthesis for rapid cell division, at the expense, on the other hand, of ornithine hranscarbamylase which serves a highly differentiated pathway in the nondividing liver cell. The importance of this relationship in understanding the biochemical basis of differentiation and dedifferentiation has been previously discussed (29, 30) and more recently by McLean et al. (27). REFERENCES 1. CALVA, E., LOWENSTEIN, J. M., AND COHEN, P. P., Cancer Res. 19, 101 (1959). 2. C~LVA, E., AND COHEN, P. P., Cancer Res. 19, 679 (1959). 3. KIM, S., Ph.D., Thesis, University of Wisconsin, Madison (1960). 4. COHEN, P. P., AND MARSHALL, M., in “The Enzymes” (P. D. Boyer, H. A. Lardy, and K. Myrbllck, eds.), 2nd edition, Vol. 6, p. 327. Academic Press, New York (1962). 5. LOWENSTEIN, J. M., AND COHEN, P. P., J. Biol. Chem. 220, 57 (1956). 6. METZENBERG, R. L., MARSHALL, M., AND COHEN, P. P., in “Biochemical Preparations” (H. A. Lardy, eds.), Vol. 7, p. 23. Wiley, New York (1960). 7. KORITZ, S. B., AND COHEN, P. P., J. Biol. Chem. 209, 145 (1954). 8. BROWN, G W., JR., AXD COHEN, P. P., J. Biol. Chem. 234, 1769 (1959). 9. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L., BND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 10. HIGGINS, G. M., AND ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 11. BUNSTER, E., AND MEYER, R. K., Anat. Res. 67, 339 (1933). 12. PRICE, J. M., AND LAIRD, A. K., Cancer Res. 10, 650 (1950). 13. THOMSON, R. V., HEBGY, F. C., HUTCHISON, W. C., AND DAVIDSON, J. N., Biochem. J. 63, 460 (1953).
428
KIM
AND
R. A., END PARDEE, A. B., J. Biol. 14. YATES, Chem. 221, 757 (1956). J. C., BND PARDEE, A. B., J. Biol. 15. GERHBRT, Chem. 237, 891 (1962). Y., HURWITZ, R., \ND KILETCH16. NORDMANN, MER, N., Natwe 201, 617 (1964). L. I., ,\ND COHEN, P. P., 4rch. Bio17. STEIN, them. Biophys. 109, 429 (1965) (following article). J. C., AND PARDEE, A. B., Cold 18. GERH~RT, Spring Harbor Symp. Quant. Biol. 28, 491 (1963). E., 19. BRESNICK, Biochim. Biophys. dcta 61, 598 (1962). 20. BRESNICK, E., Biochim. Biophys. dcta 67, 425 (1963). 21. BRESNICK, E., Cancer Res. 22, 1246 (1962). M. K., ‘\ND DONACHIE, W. D., Biochim. 22. CURCI, Biophys. Acta 85, 338 (1964). M., PUDDU, P., .IND C~LD~RER~, 23. MARCHETTI, C. M., Biochem. J. 92, 46 (1964).
COHEN 24. NET:UNS. J., .\piI) JONEH, X E., :lxh. Binthem. 1~iophy.s. 104, 438 (1964). 25. COHEX, P. P., in “The Enz\mes” (I’. D. Buyer, H. A1. Lard>-, and K. Myrbiick, eds.), \‘ol. 6, 2nd edition, p, 477. Academic Press, New York (1962). 26. KENUN, A. L., .~IVD COHEN, 1’. P., Deaelop. Biol. 1, 511 (1959). 27. McLE.+.v, P., REID, E., .\SD GL-KSET, M. W., Biochem. 1. 91, 464 (1964). 28. NAZ.\HIO, M., AND COHEN, P. P., proc. Sot. Ezptl. Biol. Med. 106, 492 (1961). 29. COHEN, P. P., AND BROV-X, G. W., JK., in “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. II, p. 161. Academic Press, New York (1960). 30. COHEN, P. P., .IND S.ILL.\CH, H. J., in “Metabolic Pathways” (I). M. Greenberg, ed.), Vol. II, 2nd edition, p. 1, Academic Press, New York (1961).