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A View Of The Comparison of The Regulation of Enzymes in mammalian and Microbial Systems
A VIEW
OF
THE
REGULATION MAMMALIAN
AND
COMPARISON OF
ENZYMES
MICROBIAL
OF
THE
IN SYSTEMS
MASAMI SUDA The Division of Protein Metabolism, Institute for Protein Research, Osaka University, Osaka, Japan
INTRODUCTION
VARIOUS individual mechanisms, operating from the molecular level to the highly organized system, must cooperate in maintaining the selfregulating system which we call homeostasis. However, there are many gaps to bridge before biological significance can be assigned to the response of an individual reaction to new circumstances. In a broad sense, homeostasis is the situation in which both internal and external factors are combined harmoniously. The external factors include physical, nutritional and ecological factors, while the internal factors consist of genetic, neural and hormonal control systems, membrane transport and subcellular control systems. Unicellular organisms are in constant and direct contact with the external environment. This causes the internal computor to signal substrate induction or end-product repression. Therefore, it can be said that the regulatory computor of unicellular organisms works in such an absolutely passive way that it is switched on when the cells happen to come into contact with their nutrients by chance. In contrast with unicellular organisms, mammalian organisms have higher and more active mechanisms to permit them to fight for survival, such as neural and hormonal control systems. Through these monitoring systems, they actively and rapidly respond to their external conditions. On the basis of the data obtained in our laboratory, with the exception of autonomic nerve stimulation which was done in the Department of Anatomy, Medical School, Osaka University, I would like to compare the fitness of these regulating mechanisms of mammalian and bacterial metabolism. 181
182
MASAMI SUDA RESULTS
Micrococcus ,,reae was chosen as material for investigation of meta-
bolic regulation in unicellular organisms. Cells of this organism are able to induce the sequential enzyme systems metabolizing tryptophan and mandelic acid, as shown in Fig. 1. OH CH--COOH
=,
•
COOH
V Mandelic Acid
NN~Benz°ic Acid Oxidase)
Benzoic Acid
qL
OH Catechol
NH="=" "-P V H Tryptophan
Succinic Acid CH=--COOH I CH=--COOH CH3--COOH Acetic Acid
NH=
Anthranilie Acid
FIG. I Metabolic pathways of mandelic acid and tryptophan.
Several years ago,o) we found that p-chlorobenzoic acid served as a nonmetabolizable inducer of benzoate oxidase and also found that o-chlorobenzoic acid competitively inhibited the induced formation of benzoate oxidase by p-chlorobenzoic acid. In the present experiments, we attempted to show where and how the substrate is fixed in the cells. Cells were incubated for 2 hr with sufficient aH-p-chlorobenzoic acid (ZH-p-C1BA) (1.33 × 10r c.p.m.//zmole) to induce benzoate oxidase formation. Then they were harvested and washed 4 times with cold phosphate buffer and once with cold 5% HC104 by centrifugation. The residue was dissolved in 0.1 M hyamine (Packard) solution and an aliquot was mixed with scintillator fluid and the radioactivity was determined in a Packard liquid scintillation spectrometer. The enzyme activity of the induced cells was assayed by measuring the rate of decarboxylation of carboxyl-labelled benzoic acid (5300 c.p.m, per/xmole) in 0.05 M phosphate buffer, pH 7.2 in the presence of chloramphenicol (100/zg]ml). As shown in Table 1 and Fig. 2, there is a competitive relationship between p-CIBA and 6-azathymine (Azt) in enzyme formation.(2) Taking advantage of the antagonistic action of Azt on p-CIBA, the fixation of aH-p-CIBA was determined in the presence and absence of
E N Z Y M E S IN M A M M A L I A N A N D M I C R O B I A L S Y S T E M S
183
Azt. As shown in Fig. 3, the amount of 3H-p-CIBA fixed gradually decreased as the concentration of Azt was increased, and finally the slope reached the lowest level at the point where the concentration of Azt exceeded 100/xg/ml, which was enough to inhibit the enzyme formation completely. The radioactivity remaining in the cells was no longer released even when excess Azt was added. TABLE !
Competitive Inhibition of Action of p-Chlorobenzoic Acid by 6 - A z a t h y m i n e
Concentration of pchlorobenzoic acid (M) 1 x 10-4 5 x 10-5 1 x 10-5 5 x 10-e
Concentration of 6-azathymine (/xg/ml) 25 25 25 25
Inhibition (%)
36.6 70.6 99.8 100.0
T h e percent inhibition was calculated from the benzoate oxidase activity in the absence of the inhibitor. 15
m
× -J>
I 2
I 5
I 10
I 15
I / (p-Ohlorobenzoic acid~ X 10T M FIG. 2
Effect of 6 - a z a t h y m i n e on the rate of synthesis of benzoate oxidase. V is the rate of enzyme synthesis, that is, the increase in e n z y m e activity between 60 rain and 120 rain of incubation in the absence (lower line) and presence (upper line) of 6 - a z a t h y m i n e (10/~g/ml).
These results led us to conclude that there are two kinds of fixation of the substrate, that is, specific and non-specific fixation, where the former is defined as the fixation inhibited by Azt. Figure 3 also shows that there is a good correlation between enzyme activity and specific fixation.
184
MASAMI SUDA
T
i,soo
"Specific Fixation"
1 v
_g
1,000
I.t.
"Non-specific Fixation"
500
I---
I
I
25
0
50
i
I00
1
//
1
400
Concentration of 6-Azathymine (pg/ml) FIG. 3a Effect of 6-azathymine on fixation of SH-p-chlorobenzoic acid. Bacteria were grown overnight at 30* with shaking in nutrient broth (Difco) and then washed once with 0.05 M phosphate buffer, pH 7.2. For induction of benzoate oxidase, the cells were suspended (1 mg dry weighffml) in a synthetic citrate medium containing 5 x 10-SM 3H-p-chlorobenzoic acid (1.33 x 107 c.p.m.//zmole) and 6azathymine at the final concentrations indicated above and incubated for 2 hr at 30 °. Cells were harvested, washed 4 times with cold phosphate buffer and once with cold 5% HCIO4 by centrifugation. The residues were dissolved in a 1 M solution of the hydroxide of hyamine 10X (Packard) and the resultant solution was mixed with liquid scintillator. Radioactivity was determined in a Packard scintillation spectrometer. 100 -': "Specific Fixation" uJ
Enzyme Activity
=0= ~r~5o
I n
25
I
50 100 Conn.entration of 6-Azathymine (/4g/ml)
FIG. 3b Relationship between "specific fixation" and enzyme activity.
400
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
185
Table 2 shows the influence of various inhibitors on induced enzyme formation and specific fixation. Acetate was found to be a kind of catabolite repressor in our system. As shown in the Table, specific fixation was markedly diminished by acetate, o-Chlorobenzoic acid, a powerful competitor with p-C1BA, also completely inhibited specific fixation as well as enzyme formation. The fact that both actinomycin S (4) and chloramphenicol caused complete inhibition of specific fixation, suggests that new formation of specific receptor may be involved in the fixation process. TABLE 2 Effects of lnhibitors and Coinducer on "Specific Fixation" "Specific fixation"
Addition
(%)
p-Chlorobenzoic acid + o-Chlorobenzoic acid + 6-Azathymine + Acetate + Chloramphenicol + Actinomycin S* + Catechol
(5 x 10 -5 M) (5 x 10-4 M) (250/zg/ml) (1 × 10 -2 M) (50/zg/mi) ( 10 ttg/ml) (4 × 10 -a M)
100.0 0 0 2.1 0 0 0.6
Enzyme activity
(%)
100.0 0 0 0.8 0 3.2 317.4
*Cells were pretreated with E D T A (1 mg/ml) for 30 rain. (a)
Catechol, the product of the benzoate oxidase reaction, had a very interesting effect on both enzyme induction and specific fixation. Enzyme Catech01 ( + ) ~ ~ " ' ~
20,000
I0,000 W
Catechol (--1
0
10_7
5XlO_ ~
10- 6
5XIO -6 I0 -~
5X i0'_s
Concentration of p-Chlorobenzoie Acid (M)
FIG. 4 Effect of catechol on benzoate oxidase induction by p-chlorobenzoic acid. The upper fine represents the enzyme activity induced in the presence of catechol (4 × 10-3M). The lower line represents the control experiment with p-C1BA alone.
186
MASAMI SUDA
induction was markedly stimulated by catechol3~ As shown in Fig. 4, catechol greatly lowered the effective concentration of p-C1BA for enzyme induction. Catechol was also found to stimulate the induction by the natural substrate, benzoic acid. E~cen at a concentration of 10-~ M, at which p-CIBA alone failed to induce the enzyme, the coexistence of 4 x 10-aM catechol evoked an enzyme level of more than that obtained with a 50 times higher concentration ofp-C1BA. On the other hand, it was found that catechol almost completely abolished the specific fixation of p-CIBA. This apparently paradoxical phenomenon led us to assume that the fixation ofp-C1BA to the receptor is by itself not enough but that another step is required for enzyme induction to occur which is stimulated by catechol and after this p-CIBA is liberated from the receptor. We would like to call effectors like catechol coinducers. It must be noted here that the inhibitory effect of Azt on enzyme formation was abolished by catechol, as shown in Table 3.
TABLE 3 Effect of Catechol on Inhibition of Enzyme Induction by 6-Azathymine Concentration of 6-azathymine (/zg/ml)
Concentration of catechol (M)
Inhibition* (%)
100 100
0 4 × 10-a
98.1 1.4
*The percent inhibition was calculated from the benzoate oxidase activity with inhibitor. The cells were suspended (1.0 mg dry weight/ml) in synthetic citrate medium containing p-CIBA (5 x 10-5 M) for 2 hr at 30 ° in the presence of catechol and 6-azathymine as indicated above.
Some of the properties of the intracellular receptor are shown in Table 4. It seems to be a protein-like substance, although a full understanding of it must await further studies. The number ofp-CIBA molecules fixed in a single cell was calculated to be roughly 1000. This relatively large amount of fixation makes it rather unlikely that the receptor is the repressor itself.~ From the above findings, the process of benzoate oxidase induction is summarized in Fig. 5.
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS TABLE 4 Chemical Nature of the Receptor: Effect of Hydrolyzing Enzymes on Fixation Fixation (c.p.m./mg protein)
Treatment Total
Non-specific
Specific
321 186 225 331 321
97 92 75 93 85
224 (100.0) 114 (50.9) 150 (67.0) 238 (106.3) 236 (105.4)
Control Nagase (500 p.g/ml) Trypsin (5000 g.g/ml) RNase (500 p.g/rnl) DNase (500/~g/ml)
Cells were incubated with 5 x 10-5 M p-CIBA (1.33 × l0 T c.p.m./ /zmole) in the presence and absence of Azt (250 p.g/ml) and harvested by centrifugation after 3 hr incubation. The cell pellets were washed 4 times with cold 0.05 M phosphate buffer, pH 7.2, and once with cold 5% HCIO(. They were suspended in 0.04 M Tris buffer, pH 7.2, neutralized with 0.2 M N a O H and then treated with lysozyme (100/zg/ml) and E D T A (1 mg/ml) to disrupt them. After 15 rain incubation, MgSO4 was added at a final concentration of 10 -2 M to stop the lysozyme reaction and the debris was removed by centrifugation at 10,000 × g for 30 min. The supernatant was treated with HCIO4 and the precipitate formed was dissolved in Tris buffer and neutralized as described above. The total and nonspecific fixations were calculated to be 321 and 97 c.p.m./mg protein, respectively, as shown for the control in the figure. Aiiquots of 2 ml were treated for 2 hr at 30 ° with each of the hydrolyzing enzymes indicated above and the reactions were terminated with cold HCIO4 (5%). After washing once with cold 5%HCIO4, the pellets were dissolved in a 1 M solution of the hydroxide of hyamine 10X (Packard) and radioactivity was determined in a Packard scintillation spectrometer.
/
flop.
- - -
Azt •
(liberated) ~
[~++|
( ~ " ~ " Oerepression) Receptor : Repressor FIG. 5 Schematic diagram of the derepression caused by binding of inducer and coinducer to receptor. Re.
fop.
:
187
188
MASAMI SUDA
Thus, when p-CIBA alone is present, it first combines with a receptor (Rc), and Azt or o-C1BA compete with it in this step. Further fixation of coinducer to the receptor stimulates, or is necessary for, the fixation of the latter to the repressor, resulting in the derepression of enzyme synthesis on the one hand and liberation of p-CIBA on the other. Fixation of the coinducer to the receptor also causes a conformational change in the latter so that its affinity for p-C1BA increases markedly. From these findings, it can be said that the coinducer works as a catabolite activator, accelerating the turnover of the inducer on the receptor through a "push and pull reaction". From this idea on the mode of action of the coinducer in bacterial cells the possibility arises that hormones in higher organisms might act in a similar way by binding to a receptor to cause an allosteric change of it and increase its sensitivity to a metabolic inducer. ._> J
Serine Dehydratase Aotivity
50 J=
E
30 20
n
= H
10
g
"6 120 E =L II0 A ~0 J~
f,
40
Growth Curve
I00 90 ~
Diet Change .
~
,
,
~
~
=G_~u~acid-diet =~ , ~ o,___o.__,.o .,~'°
_ _ ~ ~ .o..~ ~..o-
Serme-Dlet
80 m
Daysafter
Diet
Cha~e
FIG. 6 Effect of dietary serine on serine dehydratase. Male rats of the Wistar strain, weighing 60-80 g, were maintained for 4 days on a complete amino acid-diet containing glutamic acid as the sole source of non-essential amino acids (glutamic acid diet). Then, the glutamic acid in the diet was replaced by serine in the experimental group (serine diet). The growth curve was followed up to 8 days after the diet change as shown in the lower part of the Figure. The pooled livers from three rats were homogenized with 2 vol. of 0.1 M potassium phosphate buffer, pH 7.5, containing 1 x 10-4 M pyridoxal phosphate and 1 x 10-SM E D T A in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 10,000 x g for 15 min and the resulting supernatant was used for enzyme assay. The assay system contained 200/~moles of EDTA, 2/zmoles of dithiothreitol, 200ttmoles of borate buffer, pH 8.3, and enzyme in a final vol. of 2 ml. The reaction was started by addition of serine and terminated by addition of 2.0 ml of 0.1% D N P in 2 N HCI after 5-min incubation at 37 °. The pyruvate formed was determined by the method of Selim and Greenberg.~s)
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
189
In this connection, I will now discuss some characteristics of hormonal and dietary control of metabolism in higher animals. Young rats of the Wistar strain were maintained on a complete amino acid diet containing glutamic acid as the sole source of non-essential amino acids. After 4 days on this diet, glutamic acid was replaced by serine. As shown in Fig. 6, the growth of the animals was restored to the control level after a lag period of 2 days, in contrast to the results in Greenstein's experiments, ts) With the restoration of the growth rate after administration of serine, the activity of liver serine dehydratase was remarkably elevated, as shown in Fig. 6. It is also shown in Fig. 7 that serine dehydratase was induced 4 hr after the intraperitoneai injection of a single dose of serine when rats were maintained for 2 weeks on a synthetic diet not containing non-essential amino acids. Under these dietary conditions, the growth of the animals is restricted, because of the lack of an available nitrogen source. Scrine Oehydratase Activity ,umoles Pyruvate formed/hr/g Liver
Essential Amino Acids +
50
I00
150
i
i
i
Serine
I
I
I
Serne
25
Serine l Essential Amino Acids
50
J
75
J
Glutamic Acid NaCI
FIG. 7 Effect of injection of serine on liver serine dehydratase. Male rats of the Wistar strain, weighing 90-120 g, were maintained for 14 days on a complete synthetic amino acid diet not containing any non-essential amino acids (in the lower Figure) or with serine as the sole source of non-essential amino acids (in the upper Figure). Then, the animals were injected intraperitoneally with 0.5 mmole of serine, 0.5 mmole of glutamic acid or 2.0 ml of 0.95% NaCI. Four hr later the animals were sacrificed by cervical dislocation and their livers were removed for assay of serine dehydratase. The assay system was the same as that described in the legend of Fig. 6. Each group consisted of three rats.
The question arises as to what elevated the serine dehydratase activity. With regard to the increased activity of serine dehydratase, the problem of whether serine serves as an essential nitrogen source or an available
190
MASAMI
SUDA
source of pyruvate for gluconeogenesis must be taken into consideration according to the metabolic requirement of the liver. In the nutritional experiments described above, serine serves as an essential nitrogen source, since there is sufficient carbohydrate for glucose synthesis. It was also observed that there was an elevation of serine dehydratase activity after administration of serine even after adrenalectomy. It would appear, therefore, that serine plays a main role as an inducer to meet the nutritional requirement of a nitrogen source without participation of the adrenal hormone. We reported previously that serine dehydratase increased during gluconeogenesis. (¢) Results with diabetic rats showed that enzyme activity was induced by treatment with hydrocortisone in insulin deficiency. As shown in Fig. 8, there is a reciprocal relationship between serine dehydratase and glucokinase which is under dietary and hormonal regulation. This supports the hypothesis that serine dehydratase activity increases when the supply of glucose decreases, since serine must provide its carbon skeleton for gluconeogenesis. These findings indicate that the activity of serine dehydratase is under the regulation of dietary Glucokinase
Serine dehydratase.
F-
(I)
I c~
(m) ,,5 0
+
2
I ,
I
,
I I 3
I I
I
I
I
(VI)
3
(Vll)
s
(I)
6 ---T--I I
I
2.0
0.5 0.4 0.3 0.2 0.1 /~moleof pyruvateformed/mm./mg.protein
,
61o J.o
12.0 '
m#moles of C-6-P formed/mirL/mg,protein FIG. 8
Reciprocal relationship between serine dehydratase and glucokinas¢ under dietary and hormonal regulation. The conditions of the male Donryu rats w e r e : (I) normal, (II) diabetic + insulin, (III) adrenalectomized, (IV) adrenalectomized + diabetic, (V) fasted for 3 days, (VI) fed on a 91% casein diet for 3 days, (VII) diabetic, (VIII) adrenalectomized + diabetic + hydrocortisone acetate. Glucokinase activity was determined 2 or 3 days after alloxan treatment (70 mg/kg body weight) and 5 to 7 days after adrenalectomy. In (II), the animals were fasted for 2 days and were then injected intravenously with 50 mg of alloxan monohydrate/kg body weight. Protamin¢ zinc insulin (6.5 units/day) was administered intramuscularly 5, 29 and 53 hr before sacrifice. Animals were killed for enzyme assay 3 days after injection of alloxan. ¢7)
ENZYMES
IN MAMMALIAN
AND
MICROBIAL
SYSTEMS
191
serine and of hormones. F u r t h e r investigation of the problem of whether the i n c r e a s e d a c t i v i t y after f e e d i n g or i n j e c t i n g s e r i n e is d u e to s o - c a l l e d "substrate induction" in microorganisms or the "substrate type induction" in h i g h e r a n i m a l s ; d e s c r i b e d b y K n o x , will o n l y b e p o s s i b l e w h e n the p u r e a n t i b o d y for s e r i n e d e h y d r a t a s e is a v a i l a b l e . T h e e n z y m e protein exhibiting serine dehydratase activity appears to h a v e t h e a c t i v i t y o f c y s t a t h i o n i n e s y n t h e t a s e , c a t a l y z i n g the f o r m a t i o n o f c y s t a t h i o n i n e , a n i n t e r m e d i a t e in the s y n t h e s i s o f c y s t e i n e f r o m "g
Oyst~thionine Synthetase Activity
,o
J=
20
~
o
E
~t -~
120
Growth 0urve II0
Glutamic AeidDiet Change
7 ~O"
-~ al
.,..o
~ -o~'~°~Serine-Diet
8O (
70 I -2
I
|
-I
0
I
I
I
I
I 2 3 4 Days after Diet Change
I
I
I
I
5
6
7
8
FIG. 9 Effect of dietary serine on cystatldonine synthetase. The experimental conditions were the same as in Fig. 6. The liver homogenate was fractionated with a solution of saturated ammonium sulfate to remove cystathionase activity. The fraction precipitating from 0.45 saturation of ammonium sulfate, which contained almost all the cystathionine synthetase, was collected and dissolved in 0.1 M potassium phosphate, pH 7.5, containing 1 × 10-4M pyridoxal phosphate and 1 × 10-3 M EDTA. The assay of cystathionine synthetase was performed by a modification of the method of Mudd e t al. (1°) T h e assay system contained 2/~moles of l*C-serine (250,000 c.p.m.), 20/zmoles of L-homocysteine 0.5/zmole of pyridoxal phosphate, 1/zmole of EDTA, 40/~moles of borate buffer, pH 8.3, and enzyme in a final voi. of 0.5 ml. The reaction was started with ~4C-serine and terminated with 0.5 ml of 8% TCA after 15-rain incubation at 37°. The reaction mixture was centrifuged and the residue was washed with 1.0 ml of cold 4% TCA. The supernatant and washing were combined, diluted with 18 ml of cold water and applied to a column (0.9 × 3.0 cm) of Dowex 50W (H ÷, 200-400 meshes). The column was washed with 30 nd of 0.4 N HCI followed by 15 nd of water to remove serine and then eluted with each 2 ml vol. of 1 M pyridine solution. Cystathionine was eluted in the 3rd and 4th fractions. Aliquots of 0.5 mi of the pooled cystathionine fraction were dried on a planehet under an infra-red lamp and their radioactivities were measured in a gas flow counter.
192
MASAMI SUDA
methionine, from serine and homocysteine. (s,9) To determine the identity of the protein molecule which has these two enzyme activities, studies were made of whether cystathionine synthetase activity increases in response to the same stimuli which enhance serine dehydratase activity. Assay of cystathionine synthetase was carded out using homocysteine and C14-serine.O°) The activity was calculated from the radioactivity in the cystathionine fraction eluted from a column of Dowex 50W-X8 (H+). As illustrated in Fig. 9, cystathionine synthetase activity showed little or no response to dietary serine, while serine dehydratase activity showed a marked response, as mentioned previously (Fig. 6). A similar finding was obtained in regard to hormonal regulation (Fig. 10). In this experiment, rats were made diabetic by treatment with alloxan. The activity of serine dehydratase increased to almost 40 times the starting level during the experimental period, e_nd in parallel with elevation of the blood sugar, whereas that of cystathionine synthetase showed little change.
I 4,00o
40" 1.000
.-, .>
"5
<~
= i ¢1
~
3,000
: "
T
30"
E
2o! 500// // /
I,O~i
/11" Cystathionine
f
7
~
Synthetase
.....
/ /
~gu0
_J
~E
Serine Dehydratas ~'- e $/
:
I
24
I
48 72 -Hours after AIIoxan-Treatment
I
96
I
120
FIG. l0 Effect of diabetes on cystathionine synthetase and serine dehydratase. Male rats of the Wistar strain, weighing approximately 100 g, were maintained on a non-protein diet and then injected intravenously with alloxan (70 mg/kg body weight) 2 days after changing the diet. The method used for assay for cystathionine synthetase and serine dehydratase activities are described in Fig. 9 and Fig. 6, respectively.
These facts suggest that the two enzyme reactions are not catalyzed by a single protein. In this connection it is interesting that Brown and Mallady reported at the Federation Meeting this year that the two enzymes could be separated by chromatography on hydroxylapatite, t11) We have previously presumed that the sparing action of L-cystine for the dietary requirement for L-methionine of growing rats was based on
ENZYMES IN M A M M A L I A N A N D MICROBIAL SYSTEMS
193
the d e c r e a s e d activity of serine dehydratase-cystathionine synthetase.O2) W e h a v e s h o w n that serine d e h y d r a t a s e activity is s u p p r e s s e d b y the inhibitor(s) which is formed on incubating L-cystine with a liver extract in vitro which was later identified as cystine desulfurase (cystathionase). It was found that the inhibitor could be replaced b y inorganic sulfur. (13"14) Since serine d e h y d r a t a s e was found to be different f r o m cystathionine synthetase as we discussed above, we again tried to determine w h e t h e r cystathionine synthetase itself is repressed b y L-cystine. Rats were maintained on two kinds of synthetic diet as previously reported312) O n e diet contained 0.5% L-methionine and the other contained 0.16% L-methionine and 0.274% L-cystine315) T h e two diets contained the s a m e a m o u n t of sulfur. T h e r e was no difference b e t w e e n the growth rates of animals on the two diets. A t 10 a.m., 1 p.m. and 4 p.m. on the fifth day of administration of these diets, three rats f r o m each group were sacrificed and their liver cystathionine synthetase activities were determined. A t 10 a.m. on the day of the experiment, the food was r e m o v e d to avoid undesirable effects o f feeding. As shown in Fig. 11, the e n z y m e activity of each group was definitely repressed w h e n a part of the dietary methionine was replaced b y cystine.
Clock Time
Cystathionine Synthetase Activity (pmoles Cystathionine formed/hr/g Liver) 5 10 15
1 0 a . m , ~ / ~ l
' ;0.5 % Methionine 0.274% Cystine + 0.16% Methionine
Ip.m
J 0.5 % Methionlne 0.274% Cystine + 0.16% Methionine
4p.m.
I 0.5 % Methionme 0.274% Cystine + 0.16% Methionine
Fro. 11 Repression of cystathionine synthetase by dietary cystine. Male rats of the Wistar strain, weighing 80-100g, were maintained on a synthetic amino acid-diet containing 0.5% L-methionine (Diet I). On the 6th day, the 0.34% of 0.5% Lmethionine in the diet was replaced by 0.274% L-eystine (Diet II, 0.16% Lmethionine+0.274% L-cystine) in the experimental groups. The animals of both experimental and control groups were maintained for a further 4 days on Diet II and Diet I, respectively. Food was removed at 10 a.m. of the 10th day and the activity of liver cystathionine synthetase was determined at intervals of 3 hr. Each group consisted of three rats.
194
MASAMI SUDA
However, no distinct difference between the serine dehydratase activities in the two groups was found. These results indicate that cystathionine synthetase is controlled by feedback repression by L-cystine (Fig. 12). The mechanism of this repression is now under investigation.
Methionine
1
T
r----- Homocysteine III Serine ) i Dehydratase Serine
I
"-
Oystathionine--" Synthetase| 7
I Cystathionase H=S
- ~
Pyruvate
I
+ NH:,
~r-ketobutyrate+NH:, T~ Cysteine
) Oystathionine
)
1
Pyruvate
I I t
t I I
NH:, <
I
0ystine J
FIG. 12 Diagram of the mechanism of control of synthesis of cysfine from methionine.
N o w I am going to talk about the dual action of hormones on the enzymes in the anabolic and catabolic pathways of serine, which are illustrated in Fig. 13.
o-s,- -® i
OHm0--~
NAD
HC--OH I COOH I
NADH
_~ I
CHf CI__O~
I' | 3- phosphoglyoerate dehydrogenase
ADP
ATP
OOOH
T
~ oI = o
Olu
d~r- KG
..*
carboxy kinase
CH=O--~
I I COOH
]
?H,OH
CH~ )C==O ( COOH
T
H(/'--NH= COOH
methylase |
I OOOH i o..... ,, I
lo o--no pyruvate
OH,O--O
) 0=O I OOOH
carboxyllase
FIG. 13 The catabolic and anabolic pathways of serine.
@ CH=NH= ( COOH
195
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
3-Phosphoglycerate dehydrogenase is a shunt enzyme for the synthesis of serine, branching off from sugar metabolism. (1~-1s) The activity of this enzyme was scarcely detectable in rat liver when animals were maintained on laboratory chow. However, it appeared when they were fed on a diet containing no protein. The effect of casein content in diet on the level of this enzyme is shown in Fig. 14. Change from a diet containing no protein to o n e containing 30% casein caused a decrease in enzyme activity (Fig. 15). i
i
i
i
i
?
1.5
i.s
6
I t *'° 0.5
0.5
_o
0
o. ,J,
0
50
Casein
in
I00 diet,
%
FIG. i 4 Effect of casein concentration in diet on 3-phosphoglycerate dehydrogenase and glycerate dehydrogenase. Male Wistar rats, weighing 100-120 g, were fed on synthetic diets with various casein contents for 4 days. Livers were homogenized with 2 vol. of 0.1 M potassium phosphate buffer, pH 7.2, containing 0.15 M KCI, centrifuged at 105,000 x g for 1 hr and the supernatant was passed through a column of Sephadex G-25. The assay mixture for 3-phosphoglycerate dehydrogenase contained 10/xmoles of sodium 3-phosphoglycerate, 1/zmole of N A D , 300/~moles of NaC1, 100 txmoles of hydrazine, 100/~moles of Tris-HC1 buffer, pH 9.0, and enzyme in a total vol. of 1 ml. The substrate was omitted in the blank. The reduction of N A D at 25 ° was followed at 340 m/~ in a Gitford recording spectrophotometer. Glycerate dehydrogenase was measured by essentially the same method except that 3-phosphoglycerate and N A D were replaced by glycerate and N A D P , respectively.
On the contrary, glycerate dehydrogenase, another enzyme which has been postulated to be the first enzyme in serine biosynthesis in rat liver (19,2o) showed only a slight response to change of diet. When rats were fed on a synthetic diet, in which non-essential amino acids were replaced by ammonium citrate, 3-phosphoglycerate dehydrogenase activity increased to the level of that in animals on a non-protein diet and this increase was suppressed by replacing the ammonium citrate in the diet by serine and glycine (Table 5).
196
MASAMI SUDA i
~
Lab.
i
i
,
l
i
N°npr°tein
i
i
i
9 I 6
30% Casein
chow
(DIET) z.0
2.0 1
~f
%
1.0
1,0
¢~ ¢=
O
g
~IF
n
0
I
i
[
A
I
f
i
I
5
i
I
i
10
Days
FIG. 15 Effect of diet on 3-phosphoglycerate dehydrogenase and glycerate dehydrogenase in rat liver. Male Wistar rats were fed on laboratory chow, then on a non-protein diet for 5 days, and finally on a 30% casein diet for 5 days. The assay systems used for 3-phosphoglycerate and glycerate dehydrogenases are described in the legend of Fig. 14. TABLE 5 Effect of Dietary Serine and Glycine on Rat Liver 3-Phosphoglycerate Dehydrogenase Additions to diet Ammoniumcitrate, dibasic Sodium glutamate Serine Glycine Serine + Glycine
Amount in 100 g diet 3-Phosphoglycerate dehydrogenase (mmoles) (mu/mg protein) 20 40 40 40 20 for each
1.29 (_+0.21) 1.01 (_--*-0.74) 0.75 (_-+-0.38) 0.37 (_-*-0.39) 0.28 (_-*O.18)
Male Wister strain rats were fed for 5 days on synthetic diets containing 5.4% essential amino acid mixture plus the additions listed in the table. Three rats were used for each group and standard deviations were shown in the parentheses. Condition for enzyme assay was described under Fig. 14. T a b l e 6 s h o w s the r e c i p r o c a l effects o f h o r m o n e s a n d the diet o n 3p 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 a n d s e r i n e d e h y d r a t a s e with a d d i t i o n a l data on serine hydroxymethylase, catalyzing glycine formation from s e r i n e i n the p r e s e n c e o f t e t r a h y d r o f o l i c acid a n d p y r i d o x a l p h o s p h a t e . W i t h regard to t h e s e r e g u l a t i o n s , a s h a r p c o n t r a s t c a n b e s e e n b e t w e e n the e n z y m e s c a t a l y z i n g the s y n t h e s i s ( 3 - 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 ) a n d the d e g r a d a t i o n ( s e r i n e d e h y d r a t a s e ) o f s e r i n e (see Fig. 16).
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS !
i
L
,
i
I
i
I
197
I
91% Caseindiet 30% Caseindiet Lab.cho~%Casein~ E 3rowth hormone Nonprotein diet
E i_
",
+ Glucagon
* +
Adrenal hm'mone
I
'~ -t- Diabetes 40~0 3000 ' 2000 ' 10b 0 Serine dehydratase, u/mg. liver
. . . . 0' 5 . . . . 1.0 3-Phosphoglyceratedehydrogenase,mu/mg, prof.
FIG. 16 Reciprocal relationship between 3-phosphoglycerate dehydrogenase and serine dehydratase under various conditions. The hormones were administered intraperitoneally in the following manner. Growth hormone (bovine): 0.2 mg/100g body weight was injected once a day for 4 days for 3-phosphoglycerate dehydrogenase, and 0.1 mg/100g body weight was injected once a day for 3 days for serine dehydratase. Glucagon: 0.1 nag/100 g body weight was injected 4 times a day for 3 days for 3-phosphoglycerate dehydrogenase. 0.1 nag1100 g body weight was injected twice at an interval of 3 hr for serine dehydratase and the rats were sacrificed 3 hr after the second injection. Adrenal hormones: 2 mg triamcinolone/ 100 g body weight were injected once a day for 3 days for 3-phosphoglycerate dehydrogenase and 5 mg hydrocortisone/100 g body weight were injected once a day for 3 days for serine dehydratase.
The 3-phosphoglycerate dehydrogenase is induced, (a) by feeding a low protein diet, (b) when insulin or the growth hormone is in a predominant state and (c) when there is a deficiency of hydrocortisone or glucagon, while serine dehydratase is induced, (a') by feeding a high protein diet, (b') when insulin and the growth hormone are deficient and (c') when hydrocortisone or glucagon is in a predominant state. In other words, the factors which induced 3-phosphoglycerate dehydrogenase caused the reduction of serine dehydratase and vice versa. For example, the administration of the growth hormone after hypophysectomy caused the induction of the dehydrogenase and the reduction of serine dehydratase. The other factors also showed reciprocal effects on the increase and decrease of these two enzymes. Much evidence has suggested that glycine is mainly formed from serine in higher animals. This led us to assume that the induction of serine hydroxymethylase would take place in parallel with the induction of 3phosphoglycerate dehydrogenase. However, as shown in Table 6, it was unexpectedly found that the regulation of serine hydroxymethylase had an exactly reciprocal relationship with that of 3-phosphoglycerate
198
MASAMI SUDA TABLE 6 Reciprocal Effects of Hormones and Diets on the Enzymes Catalyzing the Synthesis and Degradation of Serine 3-Phosphoglycerate dehydrogenase
Diabetes Diabetes + Hormonal control
Insulin Growth hormone Glucagon Hydrocortisone
~High protein Dietary ~Non-protein control [Starvation Feedback repression Substrate induction
~ t
Serine dehydratase
~" ~
Serine hydroxymethylase
t
t ~ t
t t
t
t t t
t
+ +
Assay for serine hydroxymethylase was based on the increase of the absorbance at 340 m/.L due to the reduction of N A D P by methylene-THFA (tetrahydrofolic acid) formed from serine and T H F A . Male rats of Wistar strain, weighing 80-120g, were used. Livers were homogenized with 3 vol. of 25% sucrose containing 10-3 M EDTA, pH 7.4, and the homogenate was centrifuged at 105,000xg for 60 min in a Spinco model L centrifuge. The resultant supernatant was used for the enzyme assay. Reaction system contained enzyme preparation, 10 /.~moles of L-serine, 0.3 mg of D L - T H F A , 0.5 t~mole of N A D P , 1.2 units of purified methylene T H F A dehydrogenase from a bacterial strain, 150 ~moles of potassium phosphate buffer, pH 7.3, and water in a final vol. of 1.0 ml. The reaction was started by adding serine and the change of the absorbance at 340 m/~ was spectrophotometrically followed at 25 °.
dehydrogenase and in fact varied in parallel with that of serine dehydratase. In other words, formation of glycine is decreased under conditions which permit synthesis of serine, while the interconversion between serine and glycine occurs under conditions which permit catabolism of excess serine provided by feeding or accumulation. It should be mentioned again that one hormone showed reciprocal and dual actions on the two enzymes in the same tissue, where the one plays an important role in the anabolic process of a compound, while the other is active in its catabolism. In addition to these facts, the anabolic enzyme is controlled by feedback repression, while the catabolic enzyme is controlled by substrate (type) induction in higher animals as well as microorganisms. As discussed previously, there is no great difference between unicellular and higher organisms with respect to the regulation mechanism, if
OF
THE
REGULATION MAMMALIAN
AND
COMPARISON OF
ENZYMES
MICROBIAL
OF
THE
IN SYSTEMS
MASAMI SUDA The Division of Protein Metabolism, Institute for Protein Research, Osaka University, Osaka, Japan
INTRODUCTION
VARIOUS individual mechanisms, operating from the molecular level to the highly organized system, must cooperate in maintaining the selfregulating system which we call homeostasis. However, there are many gaps to bridge before biological significance can be assigned to the response of an individual reaction to new circumstances. In a broad sense, homeostasis is the situation in which both internal and external factors are combined harmoniously. The external factors include physical, nutritional and ecological factors, while the internal factors consist of genetic, neural and hormonal control systems, membrane transport and subcellular control systems. Unicellular organisms are in constant and direct contact with the external environment. This causes the internal computor to signal substrate induction or end-product repression. Therefore, it can be said that the regulatory computor of unicellular organisms works in such an absolutely passive way that it is switched on when the cells happen to come into contact with their nutrients by chance. In contrast with unicellular organisms, mammalian organisms have higher and more active mechanisms to permit them to fight for survival, such as neural and hormonal control systems. Through these monitoring systems, they actively and rapidly respond to their external conditions. On the basis of the data obtained in our laboratory, with the exception of autonomic nerve stimulation which was done in the Department of Anatomy, Medical School, Osaka University, I would like to compare the fitness of these regulating mechanisms of mammalian and bacterial metabolism. 181
182
MASAMI SUDA RESULTS
Micrococcus ,,reae was chosen as material for investigation of meta-
bolic regulation in unicellular organisms. Cells of this organism are able to induce the sequential enzyme systems metabolizing tryptophan and mandelic acid, as shown in Fig. 1. OH CH--COOH
=,
•
COOH
V Mandelic Acid
NN~Benz°ic Acid Oxidase)
Benzoic Acid
qL
OH Catechol
NH="=" "-P V H Tryptophan
Succinic Acid CH=--COOH I CH=--COOH CH3--COOH Acetic Acid
NH=
Anthranilie Acid
FIG. I Metabolic pathways of mandelic acid and tryptophan.
Several years ago,o) we found that p-chlorobenzoic acid served as a nonmetabolizable inducer of benzoate oxidase and also found that o-chlorobenzoic acid competitively inhibited the induced formation of benzoate oxidase by p-chlorobenzoic acid. In the present experiments, we attempted to show where and how the substrate is fixed in the cells. Cells were incubated for 2 hr with sufficient aH-p-chlorobenzoic acid (ZH-p-C1BA) (1.33 × 10r c.p.m.//zmole) to induce benzoate oxidase formation. Then they were harvested and washed 4 times with cold phosphate buffer and once with cold 5% HC104 by centrifugation. The residue was dissolved in 0.1 M hyamine (Packard) solution and an aliquot was mixed with scintillator fluid and the radioactivity was determined in a Packard liquid scintillation spectrometer. The enzyme activity of the induced cells was assayed by measuring the rate of decarboxylation of carboxyl-labelled benzoic acid (5300 c.p.m, per/xmole) in 0.05 M phosphate buffer, pH 7.2 in the presence of chloramphenicol (100/zg]ml). As shown in Table 1 and Fig. 2, there is a competitive relationship between p-CIBA and 6-azathymine (Azt) in enzyme formation.(2) Taking advantage of the antagonistic action of Azt on p-CIBA, the fixation of aH-p-CIBA was determined in the presence and absence of
E N Z Y M E S IN M A M M A L I A N A N D M I C R O B I A L S Y S T E M S
183
Azt. As shown in Fig. 3, the amount of 3H-p-CIBA fixed gradually decreased as the concentration of Azt was increased, and finally the slope reached the lowest level at the point where the concentration of Azt exceeded 100/xg/ml, which was enough to inhibit the enzyme formation completely. The radioactivity remaining in the cells was no longer released even when excess Azt was added. TABLE !
Competitive Inhibition of Action of p-Chlorobenzoic Acid by 6 - A z a t h y m i n e
Concentration of pchlorobenzoic acid (M) 1 x 10-4 5 x 10-5 1 x 10-5 5 x 10-e
Concentration of 6-azathymine (/xg/ml) 25 25 25 25
Inhibition (%)
36.6 70.6 99.8 100.0
T h e percent inhibition was calculated from the benzoate oxidase activity in the absence of the inhibitor. 15
m
× -J>
I 2
I 5
I 10
I 15
I / (p-Ohlorobenzoic acid~ X 10T M FIG. 2
Effect of 6 - a z a t h y m i n e on the rate of synthesis of benzoate oxidase. V is the rate of enzyme synthesis, that is, the increase in e n z y m e activity between 60 rain and 120 rain of incubation in the absence (lower line) and presence (upper line) of 6 - a z a t h y m i n e (10/~g/ml).
These results led us to conclude that there are two kinds of fixation of the substrate, that is, specific and non-specific fixation, where the former is defined as the fixation inhibited by Azt. Figure 3 also shows that there is a good correlation between enzyme activity and specific fixation.
184
MASAMI SUDA
T
i,soo
"Specific Fixation"
1 v
_g
1,000
I.t.
"Non-specific Fixation"
500
I---
I
I
25
0
50
i
I00
1
//
1
400
Concentration of 6-Azathymine (pg/ml) FIG. 3a Effect of 6-azathymine on fixation of SH-p-chlorobenzoic acid. Bacteria were grown overnight at 30* with shaking in nutrient broth (Difco) and then washed once with 0.05 M phosphate buffer, pH 7.2. For induction of benzoate oxidase, the cells were suspended (1 mg dry weighffml) in a synthetic citrate medium containing 5 x 10-SM 3H-p-chlorobenzoic acid (1.33 x 107 c.p.m.//zmole) and 6azathymine at the final concentrations indicated above and incubated for 2 hr at 30 °. Cells were harvested, washed 4 times with cold phosphate buffer and once with cold 5% HCIO4 by centrifugation. The residues were dissolved in a 1 M solution of the hydroxide of hyamine 10X (Packard) and the resultant solution was mixed with liquid scintillator. Radioactivity was determined in a Packard scintillation spectrometer. 100 -': "Specific Fixation" uJ
Enzyme Activity
=0= ~r~5o
I n
25
I
50 100 Conn.entration of 6-Azathymine (/4g/ml)
FIG. 3b Relationship between "specific fixation" and enzyme activity.
400
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
185
Table 2 shows the influence of various inhibitors on induced enzyme formation and specific fixation. Acetate was found to be a kind of catabolite repressor in our system. As shown in the Table, specific fixation was markedly diminished by acetate, o-Chlorobenzoic acid, a powerful competitor with p-C1BA, also completely inhibited specific fixation as well as enzyme formation. The fact that both actinomycin S (4) and chloramphenicol caused complete inhibition of specific fixation, suggests that new formation of specific receptor may be involved in the fixation process. TABLE 2 Effects of lnhibitors and Coinducer on "Specific Fixation" "Specific fixation"
Addition
(%)
p-Chlorobenzoic acid + o-Chlorobenzoic acid + 6-Azathymine + Acetate + Chloramphenicol + Actinomycin S* + Catechol
(5 x 10 -5 M) (5 x 10-4 M) (250/zg/ml) (1 × 10 -2 M) (50/zg/mi) ( 10 ttg/ml) (4 × 10 -a M)
100.0 0 0 2.1 0 0 0.6
Enzyme activity
(%)
100.0 0 0 0.8 0 3.2 317.4
*Cells were pretreated with E D T A (1 mg/ml) for 30 rain. (a)
Catechol, the product of the benzoate oxidase reaction, had a very interesting effect on both enzyme induction and specific fixation. Enzyme Catech01 ( + ) ~ ~ " ' ~
20,000
I0,000 W
Catechol (--1
0
10_7
5XlO_ ~
10- 6
5XIO -6 I0 -~
5X i0'_s
Concentration of p-Chlorobenzoie Acid (M)
FIG. 4 Effect of catechol on benzoate oxidase induction by p-chlorobenzoic acid. The upper fine represents the enzyme activity induced in the presence of catechol (4 × 10-3M). The lower line represents the control experiment with p-C1BA alone.
186
MASAMI SUDA
induction was markedly stimulated by catechol3~ As shown in Fig. 4, catechol greatly lowered the effective concentration of p-C1BA for enzyme induction. Catechol was also found to stimulate the induction by the natural substrate, benzoic acid. E~cen at a concentration of 10-~ M, at which p-CIBA alone failed to induce the enzyme, the coexistence of 4 x 10-aM catechol evoked an enzyme level of more than that obtained with a 50 times higher concentration ofp-C1BA. On the other hand, it was found that catechol almost completely abolished the specific fixation of p-CIBA. This apparently paradoxical phenomenon led us to assume that the fixation ofp-C1BA to the receptor is by itself not enough but that another step is required for enzyme induction to occur which is stimulated by catechol and after this p-CIBA is liberated from the receptor. We would like to call effectors like catechol coinducers. It must be noted here that the inhibitory effect of Azt on enzyme formation was abolished by catechol, as shown in Table 3.
TABLE 3 Effect of Catechol on Inhibition of Enzyme Induction by 6-Azathymine Concentration of 6-azathymine (/zg/ml)
Concentration of catechol (M)
Inhibition* (%)
100 100
0 4 × 10-a
98.1 1.4
*The percent inhibition was calculated from the benzoate oxidase activity with inhibitor. The cells were suspended (1.0 mg dry weight/ml) in synthetic citrate medium containing p-CIBA (5 x 10-5 M) for 2 hr at 30 ° in the presence of catechol and 6-azathymine as indicated above.
Some of the properties of the intracellular receptor are shown in Table 4. It seems to be a protein-like substance, although a full understanding of it must await further studies. The number ofp-CIBA molecules fixed in a single cell was calculated to be roughly 1000. This relatively large amount of fixation makes it rather unlikely that the receptor is the repressor itself.~ From the above findings, the process of benzoate oxidase induction is summarized in Fig. 5.
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS TABLE 4 Chemical Nature of the Receptor: Effect of Hydrolyzing Enzymes on Fixation Fixation (c.p.m./mg protein)
Treatment Total
Non-specific
Specific
321 186 225 331 321
97 92 75 93 85
224 (100.0) 114 (50.9) 150 (67.0) 238 (106.3) 236 (105.4)
Control Nagase (500 p.g/ml) Trypsin (5000 g.g/ml) RNase (500 p.g/rnl) DNase (500/~g/ml)
Cells were incubated with 5 x 10-5 M p-CIBA (1.33 × l0 T c.p.m./ /zmole) in the presence and absence of Azt (250 p.g/ml) and harvested by centrifugation after 3 hr incubation. The cell pellets were washed 4 times with cold 0.05 M phosphate buffer, pH 7.2, and once with cold 5% HCIO(. They were suspended in 0.04 M Tris buffer, pH 7.2, neutralized with 0.2 M N a O H and then treated with lysozyme (100/zg/ml) and E D T A (1 mg/ml) to disrupt them. After 15 rain incubation, MgSO4 was added at a final concentration of 10 -2 M to stop the lysozyme reaction and the debris was removed by centrifugation at 10,000 × g for 30 min. The supernatant was treated with HCIO4 and the precipitate formed was dissolved in Tris buffer and neutralized as described above. The total and nonspecific fixations were calculated to be 321 and 97 c.p.m./mg protein, respectively, as shown for the control in the figure. Aiiquots of 2 ml were treated for 2 hr at 30 ° with each of the hydrolyzing enzymes indicated above and the reactions were terminated with cold HCIO4 (5%). After washing once with cold 5%HCIO4, the pellets were dissolved in a 1 M solution of the hydroxide of hyamine 10X (Packard) and radioactivity was determined in a Packard scintillation spectrometer.
/
flop.
- - -
Azt •
(liberated) ~
[~++|
( ~ " ~ " Oerepression) Receptor : Repressor FIG. 5 Schematic diagram of the derepression caused by binding of inducer and coinducer to receptor. Re.
fop.
:
187
188
MASAMI SUDA
Thus, when p-CIBA alone is present, it first combines with a receptor (Rc), and Azt or o-C1BA compete with it in this step. Further fixation of coinducer to the receptor stimulates, or is necessary for, the fixation of the latter to the repressor, resulting in the derepression of enzyme synthesis on the one hand and liberation of p-CIBA on the other. Fixation of the coinducer to the receptor also causes a conformational change in the latter so that its affinity for p-C1BA increases markedly. From these findings, it can be said that the coinducer works as a catabolite activator, accelerating the turnover of the inducer on the receptor through a "push and pull reaction". From this idea on the mode of action of the coinducer in bacterial cells the possibility arises that hormones in higher organisms might act in a similar way by binding to a receptor to cause an allosteric change of it and increase its sensitivity to a metabolic inducer. ._> J
Serine Dehydratase Aotivity
50 J=
E
30 20
n
= H
10
g
"6 120 E =L II0 A ~0 J~
f,
40
Growth Curve
I00 90 ~
Diet Change .
~
,
,
~
~
=G_~u~acid-diet =~ , ~ o,___o.__,.o .,~'°
_ _ ~ ~ .o..~ ~..o-
Serme-Dlet
80 m
Daysafter
Diet
Cha~e
FIG. 6 Effect of dietary serine on serine dehydratase. Male rats of the Wistar strain, weighing 60-80 g, were maintained for 4 days on a complete amino acid-diet containing glutamic acid as the sole source of non-essential amino acids (glutamic acid diet). Then, the glutamic acid in the diet was replaced by serine in the experimental group (serine diet). The growth curve was followed up to 8 days after the diet change as shown in the lower part of the Figure. The pooled livers from three rats were homogenized with 2 vol. of 0.1 M potassium phosphate buffer, pH 7.5, containing 1 x 10-4 M pyridoxal phosphate and 1 x 10-SM E D T A in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged at 10,000 x g for 15 min and the resulting supernatant was used for enzyme assay. The assay system contained 200/~moles of EDTA, 2/zmoles of dithiothreitol, 200ttmoles of borate buffer, pH 8.3, and enzyme in a final vol. of 2 ml. The reaction was started by addition of serine and terminated by addition of 2.0 ml of 0.1% D N P in 2 N HCI after 5-min incubation at 37 °. The pyruvate formed was determined by the method of Selim and Greenberg.~s)
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
189
In this connection, I will now discuss some characteristics of hormonal and dietary control of metabolism in higher animals. Young rats of the Wistar strain were maintained on a complete amino acid diet containing glutamic acid as the sole source of non-essential amino acids. After 4 days on this diet, glutamic acid was replaced by serine. As shown in Fig. 6, the growth of the animals was restored to the control level after a lag period of 2 days, in contrast to the results in Greenstein's experiments, ts) With the restoration of the growth rate after administration of serine, the activity of liver serine dehydratase was remarkably elevated, as shown in Fig. 6. It is also shown in Fig. 7 that serine dehydratase was induced 4 hr after the intraperitoneai injection of a single dose of serine when rats were maintained for 2 weeks on a synthetic diet not containing non-essential amino acids. Under these dietary conditions, the growth of the animals is restricted, because of the lack of an available nitrogen source. Scrine Oehydratase Activity ,umoles Pyruvate formed/hr/g Liver
Essential Amino Acids +
50
I00
150
i
i
i
Serine
I
I
I
Serne
25
Serine l Essential Amino Acids
50
J
75
J
Glutamic Acid NaCI
FIG. 7 Effect of injection of serine on liver serine dehydratase. Male rats of the Wistar strain, weighing 90-120 g, were maintained for 14 days on a complete synthetic amino acid diet not containing any non-essential amino acids (in the lower Figure) or with serine as the sole source of non-essential amino acids (in the upper Figure). Then, the animals were injected intraperitoneally with 0.5 mmole of serine, 0.5 mmole of glutamic acid or 2.0 ml of 0.95% NaCI. Four hr later the animals were sacrificed by cervical dislocation and their livers were removed for assay of serine dehydratase. The assay system was the same as that described in the legend of Fig. 6. Each group consisted of three rats.
The question arises as to what elevated the serine dehydratase activity. With regard to the increased activity of serine dehydratase, the problem of whether serine serves as an essential nitrogen source or an available
190
MASAMI
SUDA
source of pyruvate for gluconeogenesis must be taken into consideration according to the metabolic requirement of the liver. In the nutritional experiments described above, serine serves as an essential nitrogen source, since there is sufficient carbohydrate for glucose synthesis. It was also observed that there was an elevation of serine dehydratase activity after administration of serine even after adrenalectomy. It would appear, therefore, that serine plays a main role as an inducer to meet the nutritional requirement of a nitrogen source without participation of the adrenal hormone. We reported previously that serine dehydratase increased during gluconeogenesis. (¢) Results with diabetic rats showed that enzyme activity was induced by treatment with hydrocortisone in insulin deficiency. As shown in Fig. 8, there is a reciprocal relationship between serine dehydratase and glucokinase which is under dietary and hormonal regulation. This supports the hypothesis that serine dehydratase activity increases when the supply of glucose decreases, since serine must provide its carbon skeleton for gluconeogenesis. These findings indicate that the activity of serine dehydratase is under the regulation of dietary Glucokinase
Serine dehydratase.
F-
(I)
I c~
(m) ,,5 0
+
2
I ,
I
,
I I 3
I I
I
I
I
(VI)
3
(Vll)
s
(I)
6 ---T--I I
I
2.0
0.5 0.4 0.3 0.2 0.1 /~moleof pyruvateformed/mm./mg.protein
,
61o J.o
12.0 '
m#moles of C-6-P formed/mirL/mg,protein FIG. 8
Reciprocal relationship between serine dehydratase and glucokinas¢ under dietary and hormonal regulation. The conditions of the male Donryu rats w e r e : (I) normal, (II) diabetic + insulin, (III) adrenalectomized, (IV) adrenalectomized + diabetic, (V) fasted for 3 days, (VI) fed on a 91% casein diet for 3 days, (VII) diabetic, (VIII) adrenalectomized + diabetic + hydrocortisone acetate. Glucokinase activity was determined 2 or 3 days after alloxan treatment (70 mg/kg body weight) and 5 to 7 days after adrenalectomy. In (II), the animals were fasted for 2 days and were then injected intravenously with 50 mg of alloxan monohydrate/kg body weight. Protamin¢ zinc insulin (6.5 units/day) was administered intramuscularly 5, 29 and 53 hr before sacrifice. Animals were killed for enzyme assay 3 days after injection of alloxan. ¢7)
ENZYMES
IN MAMMALIAN
AND
MICROBIAL
SYSTEMS
191
serine and of hormones. F u r t h e r investigation of the problem of whether the i n c r e a s e d a c t i v i t y after f e e d i n g or i n j e c t i n g s e r i n e is d u e to s o - c a l l e d "substrate induction" in microorganisms or the "substrate type induction" in h i g h e r a n i m a l s ; d e s c r i b e d b y K n o x , will o n l y b e p o s s i b l e w h e n the p u r e a n t i b o d y for s e r i n e d e h y d r a t a s e is a v a i l a b l e . T h e e n z y m e protein exhibiting serine dehydratase activity appears to h a v e t h e a c t i v i t y o f c y s t a t h i o n i n e s y n t h e t a s e , c a t a l y z i n g the f o r m a t i o n o f c y s t a t h i o n i n e , a n i n t e r m e d i a t e in the s y n t h e s i s o f c y s t e i n e f r o m "g
Oyst~thionine Synthetase Activity
,o
J=
20
~
o
E
~t -~
120
Growth 0urve II0
Glutamic AeidDiet Change
7 ~O"
-~ al
.,..o
~ -o~'~°~Serine-Diet
8O (
70 I -2
I
|
-I
0
I
I
I
I
I 2 3 4 Days after Diet Change
I
I
I
I
5
6
7
8
FIG. 9 Effect of dietary serine on cystatldonine synthetase. The experimental conditions were the same as in Fig. 6. The liver homogenate was fractionated with a solution of saturated ammonium sulfate to remove cystathionase activity. The fraction precipitating from 0.45 saturation of ammonium sulfate, which contained almost all the cystathionine synthetase, was collected and dissolved in 0.1 M potassium phosphate, pH 7.5, containing 1 × 10-4M pyridoxal phosphate and 1 × 10-3 M EDTA. The assay of cystathionine synthetase was performed by a modification of the method of Mudd e t al. (1°) T h e assay system contained 2/~moles of l*C-serine (250,000 c.p.m.), 20/zmoles of L-homocysteine 0.5/zmole of pyridoxal phosphate, 1/zmole of EDTA, 40/~moles of borate buffer, pH 8.3, and enzyme in a final voi. of 0.5 ml. The reaction was started with ~4C-serine and terminated with 0.5 ml of 8% TCA after 15-rain incubation at 37°. The reaction mixture was centrifuged and the residue was washed with 1.0 ml of cold 4% TCA. The supernatant and washing were combined, diluted with 18 ml of cold water and applied to a column (0.9 × 3.0 cm) of Dowex 50W (H ÷, 200-400 meshes). The column was washed with 30 nd of 0.4 N HCI followed by 15 nd of water to remove serine and then eluted with each 2 ml vol. of 1 M pyridine solution. Cystathionine was eluted in the 3rd and 4th fractions. Aliquots of 0.5 mi of the pooled cystathionine fraction were dried on a planehet under an infra-red lamp and their radioactivities were measured in a gas flow counter.
192
MASAMI SUDA
methionine, from serine and homocysteine. (s,9) To determine the identity of the protein molecule which has these two enzyme activities, studies were made of whether cystathionine synthetase activity increases in response to the same stimuli which enhance serine dehydratase activity. Assay of cystathionine synthetase was carded out using homocysteine and C14-serine.O°) The activity was calculated from the radioactivity in the cystathionine fraction eluted from a column of Dowex 50W-X8 (H+). As illustrated in Fig. 9, cystathionine synthetase activity showed little or no response to dietary serine, while serine dehydratase activity showed a marked response, as mentioned previously (Fig. 6). A similar finding was obtained in regard to hormonal regulation (Fig. 10). In this experiment, rats were made diabetic by treatment with alloxan. The activity of serine dehydratase increased to almost 40 times the starting level during the experimental period, e_nd in parallel with elevation of the blood sugar, whereas that of cystathionine synthetase showed little change.
I 4,00o
40" 1.000
.-, .>
"5
<~
= i ¢1
~
3,000
: "
T
30"
E
2o! 500// // /
I,O~i
/11" Cystathionine
f
7
~
Synthetase
.....
/ /
~gu0
_J
~E
Serine Dehydratas ~'- e $/
:
I
24
I
48 72 -Hours after AIIoxan-Treatment
I
96
I
120
FIG. l0 Effect of diabetes on cystathionine synthetase and serine dehydratase. Male rats of the Wistar strain, weighing approximately 100 g, were maintained on a non-protein diet and then injected intravenously with alloxan (70 mg/kg body weight) 2 days after changing the diet. The method used for assay for cystathionine synthetase and serine dehydratase activities are described in Fig. 9 and Fig. 6, respectively.
These facts suggest that the two enzyme reactions are not catalyzed by a single protein. In this connection it is interesting that Brown and Mallady reported at the Federation Meeting this year that the two enzymes could be separated by chromatography on hydroxylapatite, t11) We have previously presumed that the sparing action of L-cystine for the dietary requirement for L-methionine of growing rats was based on
ENZYMES IN M A M M A L I A N A N D MICROBIAL SYSTEMS
193
the d e c r e a s e d activity of serine dehydratase-cystathionine synthetase.O2) W e h a v e s h o w n that serine d e h y d r a t a s e activity is s u p p r e s s e d b y the inhibitor(s) which is formed on incubating L-cystine with a liver extract in vitro which was later identified as cystine desulfurase (cystathionase). It was found that the inhibitor could be replaced b y inorganic sulfur. (13"14) Since serine d e h y d r a t a s e was found to be different f r o m cystathionine synthetase as we discussed above, we again tried to determine w h e t h e r cystathionine synthetase itself is repressed b y L-cystine. Rats were maintained on two kinds of synthetic diet as previously reported312) O n e diet contained 0.5% L-methionine and the other contained 0.16% L-methionine and 0.274% L-cystine315) T h e two diets contained the s a m e a m o u n t of sulfur. T h e r e was no difference b e t w e e n the growth rates of animals on the two diets. A t 10 a.m., 1 p.m. and 4 p.m. on the fifth day of administration of these diets, three rats f r o m each group were sacrificed and their liver cystathionine synthetase activities were determined. A t 10 a.m. on the day of the experiment, the food was r e m o v e d to avoid undesirable effects o f feeding. As shown in Fig. 11, the e n z y m e activity of each group was definitely repressed w h e n a part of the dietary methionine was replaced b y cystine.
Clock Time
Cystathionine Synthetase Activity (pmoles Cystathionine formed/hr/g Liver) 5 10 15
1 0 a . m , ~ / ~ l
' ;0.5 % Methionine 0.274% Cystine + 0.16% Methionine
Ip.m
J 0.5 % Methionlne 0.274% Cystine + 0.16% Methionine
4p.m.
I 0.5 % Methionme 0.274% Cystine + 0.16% Methionine
Fro. 11 Repression of cystathionine synthetase by dietary cystine. Male rats of the Wistar strain, weighing 80-100g, were maintained on a synthetic amino acid-diet containing 0.5% L-methionine (Diet I). On the 6th day, the 0.34% of 0.5% Lmethionine in the diet was replaced by 0.274% L-eystine (Diet II, 0.16% Lmethionine+0.274% L-cystine) in the experimental groups. The animals of both experimental and control groups were maintained for a further 4 days on Diet II and Diet I, respectively. Food was removed at 10 a.m. of the 10th day and the activity of liver cystathionine synthetase was determined at intervals of 3 hr. Each group consisted of three rats.
194
MASAMI SUDA
However, no distinct difference between the serine dehydratase activities in the two groups was found. These results indicate that cystathionine synthetase is controlled by feedback repression by L-cystine (Fig. 12). The mechanism of this repression is now under investigation.
Methionine
1
T
r----- Homocysteine III Serine ) i Dehydratase Serine
I
"-
Oystathionine--" Synthetase| 7
I Cystathionase H=S
- ~
Pyruvate
I
+ NH:,
~r-ketobutyrate+NH:, T~ Cysteine
) Oystathionine
)
1
Pyruvate
I I t
t I I
NH:, <
I
0ystine J
FIG. 12 Diagram of the mechanism of control of synthesis of cysfine from methionine.
N o w I am going to talk about the dual action of hormones on the enzymes in the anabolic and catabolic pathways of serine, which are illustrated in Fig. 13.
o-s,- -® i
OHm0--~
NAD
HC--OH I COOH I
NADH
_~ I
CHf CI__O~
I' | 3- phosphoglyoerate dehydrogenase
ADP
ATP
OOOH
T
~ oI = o
Olu
d~r- KG
..*
carboxy kinase
CH=O--~
I I COOH
]
?H,OH
CH~ )C==O ( COOH
T
H(/'--NH= COOH
methylase |
I OOOH i o..... ,, I
lo o--no pyruvate
OH,O--O
) 0=O I OOOH
carboxyllase
FIG. 13 The catabolic and anabolic pathways of serine.
@ CH=NH= ( COOH
195
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS
3-Phosphoglycerate dehydrogenase is a shunt enzyme for the synthesis of serine, branching off from sugar metabolism. (1~-1s) The activity of this enzyme was scarcely detectable in rat liver when animals were maintained on laboratory chow. However, it appeared when they were fed on a diet containing no protein. The effect of casein content in diet on the level of this enzyme is shown in Fig. 14. Change from a diet containing no protein to o n e containing 30% casein caused a decrease in enzyme activity (Fig. 15). i
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i
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i
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1.5
i.s
6
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o. ,J,
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Casein
in
I00 diet,
%
FIG. i 4 Effect of casein concentration in diet on 3-phosphoglycerate dehydrogenase and glycerate dehydrogenase. Male Wistar rats, weighing 100-120 g, were fed on synthetic diets with various casein contents for 4 days. Livers were homogenized with 2 vol. of 0.1 M potassium phosphate buffer, pH 7.2, containing 0.15 M KCI, centrifuged at 105,000 x g for 1 hr and the supernatant was passed through a column of Sephadex G-25. The assay mixture for 3-phosphoglycerate dehydrogenase contained 10/xmoles of sodium 3-phosphoglycerate, 1/zmole of N A D , 300/~moles of NaC1, 100 txmoles of hydrazine, 100/~moles of Tris-HC1 buffer, pH 9.0, and enzyme in a total vol. of 1 ml. The substrate was omitted in the blank. The reduction of N A D at 25 ° was followed at 340 m/~ in a Gitford recording spectrophotometer. Glycerate dehydrogenase was measured by essentially the same method except that 3-phosphoglycerate and N A D were replaced by glycerate and N A D P , respectively.
On the contrary, glycerate dehydrogenase, another enzyme which has been postulated to be the first enzyme in serine biosynthesis in rat liver (19,2o) showed only a slight response to change of diet. When rats were fed on a synthetic diet, in which non-essential amino acids were replaced by ammonium citrate, 3-phosphoglycerate dehydrogenase activity increased to the level of that in animals on a non-protein diet and this increase was suppressed by replacing the ammonium citrate in the diet by serine and glycine (Table 5).
196
MASAMI SUDA i
~
Lab.
i
i
,
l
i
N°npr°tein
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9 I 6
30% Casein
chow
(DIET) z.0
2.0 1
~f
%
1.0
1,0
¢~ ¢=
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g
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n
0
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5
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10
Days
FIG. 15 Effect of diet on 3-phosphoglycerate dehydrogenase and glycerate dehydrogenase in rat liver. Male Wistar rats were fed on laboratory chow, then on a non-protein diet for 5 days, and finally on a 30% casein diet for 5 days. The assay systems used for 3-phosphoglycerate and glycerate dehydrogenases are described in the legend of Fig. 14. TABLE 5 Effect of Dietary Serine and Glycine on Rat Liver 3-Phosphoglycerate Dehydrogenase Additions to diet Ammoniumcitrate, dibasic Sodium glutamate Serine Glycine Serine + Glycine
Amount in 100 g diet 3-Phosphoglycerate dehydrogenase (mmoles) (mu/mg protein) 20 40 40 40 20 for each
1.29 (_+0.21) 1.01 (_--*-0.74) 0.75 (_-+-0.38) 0.37 (_-*-0.39) 0.28 (_-*O.18)
Male Wister strain rats were fed for 5 days on synthetic diets containing 5.4% essential amino acid mixture plus the additions listed in the table. Three rats were used for each group and standard deviations were shown in the parentheses. Condition for enzyme assay was described under Fig. 14. T a b l e 6 s h o w s the r e c i p r o c a l effects o f h o r m o n e s a n d the diet o n 3p 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 a n d s e r i n e d e h y d r a t a s e with a d d i t i o n a l data on serine hydroxymethylase, catalyzing glycine formation from s e r i n e i n the p r e s e n c e o f t e t r a h y d r o f o l i c acid a n d p y r i d o x a l p h o s p h a t e . W i t h regard to t h e s e r e g u l a t i o n s , a s h a r p c o n t r a s t c a n b e s e e n b e t w e e n the e n z y m e s c a t a l y z i n g the s y n t h e s i s ( 3 - 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 ) a n d the d e g r a d a t i o n ( s e r i n e d e h y d r a t a s e ) o f s e r i n e (see Fig. 16).
ENZYMES IN MAMMALIAN AND MICROBIAL SYSTEMS !
i
L
,
i
I
i
I
197
I
91% Caseindiet 30% Caseindiet Lab.cho~%Casein~ E 3rowth hormone Nonprotein diet
E i_
",
+ Glucagon
* +
Adrenal hm'mone
I
'~ -t- Diabetes 40~0 3000 ' 2000 ' 10b 0 Serine dehydratase, u/mg. liver
. . . . 0' 5 . . . . 1.0 3-Phosphoglyceratedehydrogenase,mu/mg, prof.
FIG. 16 Reciprocal relationship between 3-phosphoglycerate dehydrogenase and serine dehydratase under various conditions. The hormones were administered intraperitoneally in the following manner. Growth hormone (bovine): 0.2 mg/100g body weight was injected once a day for 4 days for 3-phosphoglycerate dehydrogenase, and 0.1 mg/100g body weight was injected once a day for 3 days for serine dehydratase. Glucagon: 0.1 nag/100 g body weight was injected 4 times a day for 3 days for 3-phosphoglycerate dehydrogenase. 0.1 nag1100 g body weight was injected twice at an interval of 3 hr for serine dehydratase and the rats were sacrificed 3 hr after the second injection. Adrenal hormones: 2 mg triamcinolone/ 100 g body weight were injected once a day for 3 days for 3-phosphoglycerate dehydrogenase and 5 mg hydrocortisone/100 g body weight were injected once a day for 3 days for serine dehydratase.
The 3-phosphoglycerate dehydrogenase is induced, (a) by feeding a low protein diet, (b) when insulin or the growth hormone is in a predominant state and (c) when there is a deficiency of hydrocortisone or glucagon, while serine dehydratase is induced, (a') by feeding a high protein diet, (b') when insulin and the growth hormone are deficient and (c') when hydrocortisone or glucagon is in a predominant state. In other words, the factors which induced 3-phosphoglycerate dehydrogenase caused the reduction of serine dehydratase and vice versa. For example, the administration of the growth hormone after hypophysectomy caused the induction of the dehydrogenase and the reduction of serine dehydratase. The other factors also showed reciprocal effects on the increase and decrease of these two enzymes. Much evidence has suggested that glycine is mainly formed from serine in higher animals. This led us to assume that the induction of serine hydroxymethylase would take place in parallel with the induction of 3phosphoglycerate dehydrogenase. However, as shown in Table 6, it was unexpectedly found that the regulation of serine hydroxymethylase had an exactly reciprocal relationship with that of 3-phosphoglycerate
198
MASAMI SUDA TABLE 6 Reciprocal Effects of Hormones and Diets on the Enzymes Catalyzing the Synthesis and Degradation of Serine 3-Phosphoglycerate dehydrogenase
Diabetes Diabetes + Hormonal control
Insulin Growth hormone Glucagon Hydrocortisone
~High protein Dietary ~Non-protein control [Starvation Feedback repression Substrate induction
~ t
Serine dehydratase
~" ~
Serine hydroxymethylase
t
t ~ t
t t
t
t t t
t
+ +
Assay for serine hydroxymethylase was based on the increase of the absorbance at 340 m/.L due to the reduction of N A D P by methylene-THFA (tetrahydrofolic acid) formed from serine and T H F A . Male rats of Wistar strain, weighing 80-120g, were used. Livers were homogenized with 3 vol. of 25% sucrose containing 10-3 M EDTA, pH 7.4, and the homogenate was centrifuged at 105,000xg for 60 min in a Spinco model L centrifuge. The resultant supernatant was used for the enzyme assay. Reaction system contained enzyme preparation, 10 /.~moles of L-serine, 0.3 mg of D L - T H F A , 0.5 t~mole of N A D P , 1.2 units of purified methylene T H F A dehydrogenase from a bacterial strain, 150 ~moles of potassium phosphate buffer, pH 7.3, and water in a final vol. of 1.0 ml. The reaction was started by adding serine and the change of the absorbance at 340 m/~ was spectrophotometrically followed at 25 °.
dehydrogenase and in fact varied in parallel with that of serine dehydratase. In other words, formation of glycine is decreased under conditions which permit synthesis of serine, while the interconversion between serine and glycine occurs under conditions which permit catabolism of excess serine provided by feeding or accumulation. It should be mentioned again that one hormone showed reciprocal and dual actions on the two enzymes in the same tissue, where the one plays an important role in the anabolic process of a compound, while the other is active in its catabolism. In addition to these facts, the anabolic enzyme is controlled by feedback repression, while the catabolic enzyme is controlled by substrate (type) induction in higher animals as well as microorganisms. As discussed previously, there is no great difference between unicellular and higher organisms with respect to the regulation mechanism, if