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Possible alternative functions of rat liver malic enzyme
ARCHIVES
OF BIOCHEMISTRY
AND
Possible MICHAEL The University
BIOPHYSICS
Alternative J. STARK,’
of Texas
Health
Science
166, 174-180
(1975)
Functions BARRY Center
of Rat Liver Malic
THOMPSON,* at Dalla.s
Received
July
AND
Southwestern
RENE
Medical
Enzyme FRENKEL3
School,
Dallas,
Texas
75235
17, 1974
The induction of rat liver malic enzyme by restriction of protein intake has been studied in conjunction with the biosynthesis of fatty acids, fatty acid synthetase, glutathione reductase, and other “lipogenic” enzymes in the various experimental animals. No correlation has been detected between malic enzyme activity and lipogenesis under these conditions. Conversely, a positive correlation between malic enzyme and glutathione reductase has been noted. Possible functions of malic enzyme which appear consistent with these observations are postulated.
The marked induction of hepatic malic enzyme (EC1.1.1.40) in rats maintained on a low-protein diet for 7-10 days has been described recently (1, 2). This type of induction differs from the induction observed after treatment with high concentrations of dietary carbohydrate (specially sucrose) (2), since only malic enzyme activity is enhanced under these conditions, while glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase remain unaffected. These experimental observations lend credence to the suggestion that malic enzyme may play an important role in cellular metabolic regulation, not necessarily linked to lipogenesis (2,3). In a previous abstract (4) it was suggested that malic enzyme may not be exclusively a “lipogenie” enzyme due to its capability to generate reducing equivalents in the form of NADPH, but no direct measurements of fatty acid synthetase had been made. The following paper describes in detail the lack of correlation between lipogenesis (as measured by biosynthetic capacity or enzyme activity) and the activity of malic enzyme in rat liver. In addition, some possible ‘Submitted by MJS in partial fulfillment for the requirements of the Ph.D. degree to the UTHSC at Dallas. *Recipient of a Chilton Foundation Fellowship. 3Supported by Grants AM13275 from the National Institutes of Health and I-385 from The Robert A. Welch Foundation of Houston, Texas. 174 Copyright All rights
0 1975 by Academic Press, of reprodurtion in any form
Inc. reserved.
alternative roles for malic enzyme in rat liver are presented in light of the enzymatic evidence already obtained. MATERIALS
AND
METHODS
Male albino Holtzman rats, weighing between 80 and 90 g at the initiation of all experiments were used throughout these studies. These animals were approximately 30 days of age at the beginning of the experimental diets. The rats were housed individually in steel cages with wire bottoms. After an initial adaptation period of 3 days on a commercial laboratory chow, the rats were allowed to feed ad lib. on either an 18% or a 0.5% protein diet for the duration of the studies. The composition of the diets is given in .Table I. At appropriate time intervals, rats were killed by decapitation. The liver was immediately removed and chilled in ice. For the enzyme assays, weighed samples of liver were homogenized with 9 vol of a solution containing 10 mM morpholinopropane sulfonic acid (MOPS), 1 mM EDTA, and 0.25 M sucrose at pH 7.4. The homogenates were centrifuged at 20,OOOg for 15 min at 2-4”C, and the supernatant extracts tested for various enzyme activities. The activities of malic enzyme isocitrate dehydrogenase, and glucose-6phosphate dehydrogenase was determined as previously described (1). Citrate cleavage enzyme was determined in a final volume of 1 ml of a solution containing: 0.10 M MOPS (pH 7.5); 10 mM MgCl,; 1 mM dithiothreitol (DTT); 20 mM potassium citrate; 25 &ml malate dehydrogenase (MDH); 5 mM ATP; and 0.025 mM NADH. The reaction was initiated by the addition of 30 nmoles of coenzyme A and followed by measuring the change in absorption at 340 nm. Fatty acid synthetase activity was determined by a modification of the method of Collins et al. (6), in a l-ml
POSSIBLE
FUNCTIONS
OF MALIC
TABLE COMPOSITION
Diet Dextrine Sucrose Lactalbumin Casein Salt mixtureb Salt mixture’ Vitamin mixtureb Brewers yeast? Choline’ Wesson oil Vegetable oil
I
OF EXPERIMENTAL
I”
Diet
2210 1105 900 -
3085 1105 25 -
250 -
250 -
25
25 -
50 ml 500 -
50 ml 500 -
solution containing 0.2 M potassium phosphate (pH 6.5), 3 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM DTT, and 0.10 mM NADPH. The reaction was initiated by the addition of acetyl coenzyme A and malonyl coenzyme A to tinal concentrations of 0.050 mM and 0.10 miti, respectively. The activity of glutamate-oxalacetate transaminase was determined by measuring the change of absorption at 340 nm in a 2-ml solution containing 88 mM potassium phosphate buffer (pH 7.6;‘7); 7.4 mM cY-ketoglutarate; 0.15 mM NADH; and 25 I.rg of malate dehydrogenase. The reaction was initiated by addition of L-asparmte (pH 7.6) to a final concentration of 0.2 M. Glutamatepyruvate transaminase activity was assayed in a similar manner The reaction mixture contained: 93 mM potassium phosphate buffer (pH 7.6); 7.4 mM a-ketoglutarate: 0.12 mM NADH; and 25 fig of lactate dehydrogenase iLDH). The reaction was initiated by addition of L-alanine (pH 7.6) to a final concentration of 33.3 mM. Glutathione reductase was determined in a 2-ml solution containing 58 mM potassium phosphate buffer (pH 6.5) and 0.15 mM NADPH. The reaction was initiated by addition of oxidized glutathione (GSSG) to a final concentration of 0.75 mM. Enzymic activity was measured by following the change in absorption at 340 nm. All activities were determined at :!5”C in a Gilford Model 240 recording spectrophotomcter. A unit of enzyme activity is defined as that amount of enzyme catalyzing the reduction (or oxidation) of 1 Fmole of pyridine nucleotide per minute at 25°C. Fatty acid biosynthetic capacity was determined by either an in clitro or an in uiuo system. The in uitro system measured the incorporation of [“Clacetate into lipids in a microsome-free (105,OOOg) supernatant fraction. The system was prepared as follows: One-
DIETS
II”
-
LIIn grams/5 kilograms. b Rogers and Harper (Ref. 5). c U.S.P. No. 2. d U.S.P. p Choline chloride was added to the diet as an aqueous
175
ENZYME
solution
Diet III”
containing
Diet IV”
3400 900 -
675 3400 -
200 -
200 -
100 -
100 -
400
400
25 -
1 g/5 ml.
milliliter samples of postmicrosomal extract were placed in screw-capped tubes which contained 0.1 ml of 0.12 M ATP, 0.1 ml of 0.15 M MgCl,, 0.1 ml of 0.12 M KHCO,, 0.14 ml of 0.3 M potassium citrate (pH 7.4). 0.15 ml of 0.1 M cysteine, 0.03 ml of 1.0 M malate (pH 7.4), and 0.13 ml of 0.1 M potassium phosphate (pH 7.4). These samples were incubated at 37°C for 30 min. After this initial incubation, 0.15 ml of 0.1 M potassium [P-“Clacetate (0.1 pCi/pmole), 0.05 ml of 0.01 M Coenzyme A, and 0.1 ml of 0.03 M NADP’ were added to each tube and the reactions allowed to proceed for various time intervals. The reactions were stopped by addition of 2 ml of 4 N KOH in 25% ethanol. The samples were loosely capped and placed in an autoclave where they were autoclaved at 20 psi for 30 min. After cooling, 1.5 ml of 10 N H,SO, was added to each sample in order to release the free fatty acids. Each sample was subsequently extracted three times with 5-ml portions of petroleum ether. The organic layers were separated, transferred to glass scintillation vials, evaporated to dryness in an 80°C water bath and then placed in a 100°C oven for 10 min. Ten milliliters of PCS (AmershamiSearle, Arlington Heights, IL) were added to each vial. The vials were shaken and the radioactivity determined in a Beckman Liquid Scintillation Counter. For zero time controls, the alcoholic KOH was added prior to addition of the labeled acetate. The in uiuo system of determining fatty acid biosynthetic capacity employed the incorporation of 3H,0 into fatty acids, essentially as described by others (7-9). The rats were injected with 1 mCi (10 mCi/ml) of 3H,0 intraperitoneally, 1 hr prior to their demise by decapitation. Blood samples were collected and the plasma prepared for subsequent determination of the specific radioactivity of the body water.
176
STARK.
THOMPSON
AND
as sucrose or as dextrinel does not appear to affect the rate of fatty acid synthesis. Simultaneously with these assays the activities of malic enzyme, glucose-6-phosphate dehydrogenase, and NADP+-isooitrate dehydrogenase were determined in the postmicrosomal supernatant fraction. The results obtained under these experimental conditions have been previously reported (2). Briefly stated, no alteration of enzymic activity is observed on diet I. In animals maintained on diet II, malic enzyme activity is induced in a rapid and sustained manner, while neither of the other two NADP+-dehydrogenases measured are affected. Animals maintained on diet III (high sucrose-W% protein) show elevated malic enzyme and glucose-6-phosphate dehydrogenase activity while the activity of isocitrate dehydrogenase remains unaltered. On diet IV, malic enzyme alone is induced. The induction curve differs from that seen on either diet II or diet III, seeming to be influenced by both high levels of dietary sucrose and reduced dietary protein. Therefore, although malic enzyme is readily inducible either by elevated dietary sucrose or by restricted dietary protein, there is no concomitant increase in the in vitro rate of fatty acid biosynthesis from acetate under the conditions of our study. Hence, it appears that malic enzyme need not necessarily be confined to a role of providing reducing equivalents for fatty acid biosynthesis as suggested in previous studies (11, 12). In another series of studies, summarized
The liver was removed and immediately chilled in ice. Weighed samples were homogenized in 3 vol of chloroform-methanol (2: 1). The phases were separated by centrifugation, the organic layer removed, and subsequently dried in a boiling water bath. Saponification and extraction of the fatty acids was carried out as described above. Plasma samples were diluted in 0.9% NaCl and aliquots were counted in PCS. Discussions about the advantages of emphasizing ‘H,O incorporation for estimates of the in uiuo synthesis of fatty acids are given in Refs. 8 and 9. Diets and dietary components were purchased from Nutritional Biochemicals Corporation, Cleveland, OH. L-Cysteine was purchased from Eastman Kodak Corp. MOPS, DTT, NADH, NADPH, NADP+, GSSG, a-ketoglutarate, L-alanine, L-aspartic acid, malic acid, glucose-6-phosphate, m-isocitric acid, coenzyme A, acetyl coenzyme A, and malonyl coenzyme A were purchased from Sigma. Sucrose, MgCl,, potassium citrate, potassium phosphate, EDTA, and KHCO, were purchased from J. T. Baker Chemical Company, Phillipsburg, NJ. ATP, MDH, and LDH were from Boehringer Mannheim. [2-“C]Acetate and “H,O were purchased from Amersham-Searle. Protein concentrations were determined by the Lowry technique (11) with bovine serum albumin as the standard protein. RESULTS
A summary of the fatty acid biosynthetic rate from [Z-“Clacetate in the postmicrosomal supernatant extracts is given in Table II. There is no apparent difference in the synthetic rates observed with this method under these conditions and neither dietary protein quality nor quantity appear to alter the in vitro capacity to synthesize fatty acids from acetate. Likewise, the form of the dietary carbohydrate (whether TABLE In vitro
SYNTHEW
Days on diet
OF FATIK
a The biosynthetic supernatant extract. from three animals.
II
ACIDS FROM ACETATE IN MICROSOME-FREE SUPERNATANT LIVERS OF RATS RECEIVING DIFFERENT DIETS~
Laboratory chow
Acetate Diet I (18% lactalbumin)
0 4 6 15 19
FRENKEL
2.6 f 0.5 2.3 i 0.6 2.9 * 0.4
incorporation (0.5%
(pmoleslg Diet II lactalbumin)
2.4 zt 0.4 1.9 l 0.4 -
rates of fatty acids from [2-“Clacetate Conditions for the assay are described
SOLUTIONS
2.2 2.2 2.2 2.6 was measured under Methods.
PREPARED FROM
protein/hr) Diet IV (0.5% casein) -
* + f zt
0.8 0.6 0.3 0.8
in duplicate Each point
3.3 f 0.9 2.5 + 0.7 2.4 zt 0.2 in the postmicrosomal represents the average
POSSIBLE
FUNCTIONS
OF MALIC
In viva fatty acid biosynthesis was measured by determining the incorporation of 3H,0 into fatty acids. The data obtained from this study are given in Table IV. Essentially identical results were obtained in three separate experiments in which no readily observable induction of fatty acid biosynthesis could be detected concomitant with the increase in malic enzyme activity. Throughout these studies, the activity of NADP+-isocitrate dehydrogenase was not appreciably altered. In another series of experiments, the activity of other enzymes which might act upon primary or secondary products of the malic enzyme-catalyzed reaction was determined. Glutamate-oxalacetate transaminase activity appears to be insensitive to low dietary protein concentration. Citrate-cleavage enzyme and glutamatepyruvate transaminase activities are drastically reduced in the livers of animals maintained on 0.5% protein. In each of the above cases, the source of protein does not seem to make a difference, as identical results are obtained whether the protein source is lactalbumin or casein. However, glutathione reductase activity is appreciably higher in the liver of animals maintained on a 0.5% protein diet than in control animals maintained on an 18% protein diet, after about 1 wk from the initiation of the experimental diets. Figure 1 is an example of activities of malic
in Table III, liver enzyme activities were determined in animals maintained on a low-protein diet (diet II). As is noted from the table, although malic enzyme activity is induced by restricted dietary protein, fatty acid synthetase activity continues to decrease. GlucoseS-phosphate dehydrogenase activity is essentially unaffected by the diet. Citrate cleavage enzyme activity remains at a constant level during the 8 days shown in the table, but it eventually diminishes under conditions of continuous protein restriction (unpublished observations) . TABLE ACTWITIES LIP~CENIC
4 6 8
III
OF MALIC ENZYME AND OTHER HEPATIC ENZYMES DURING THE INITIAL STAGES OF PROTEIN RFSTRICTIOP MEb
G6PDb
CCEb
FASb
23.99 18.36 32.92
9.45 11.25 7.97
3.83 2.89 3.76
2.27 1.76 0.75
Dayson diet
a Malic enzyme (ME), glucose-6-phosphate dehydrogenase (GGPD), citrate cleavage enzyme (CCE), and fatty acid synthetase (FAS) activities were measured in aliquots of postmitochondrial supernatant fractions prepared from the liver of rats maintained for the indicated number of days on a 0.5% lactalbumin diet (diet II). For details, see text. b Activities given as units per gram of soluble
TABLE MALIC Days on diet
ENZYME
9.2, 9.7, 10.0, 19.7, 16.8, 16.8, 27.1, 18.4,
10.9,8.6 22.4 10.5,7.1 20.3 20.0, 17.7 13.2, 17.3 18.3, 24.6 21.8,12.7
AND FA~Y
T
Diet I Malic enzyme activityb
4 5 7 9 11 13 17 21
ACTIVITY
I
177
ENZYME
IV
ACID SYNTHESIS in Diet
Viuo
IN RAT LIVERY
T
II
Fatty acid synthesis’
Malic enzyme activityb
Fatty acid synthesis’
3.5,4.5,6.8 18.8, 13.4 6.0,4.7,3.4 6.0,4.7, 3.5 13.6,4.3,4.8 6.0,3.2,3.4 7.3,6.0,5.7 14.3, 12.1, 5.4
36.1, 39.4, 32.3 35.3, 54.3 39.3, 21.0,42.0 94.0.58.0 107.0,71.0,63.2 74.7,83.8,85.0 87.9, 78.3,75.3 90.9, 53.8,67.8
6.7,6.3,8.1 3.5, 3.0 5.0,4.3,4.0 17.8, 17.0 4.9.3.7, 7.9 4.5, 3.0, 4.4 10.5, 7.5,5.8 4.4
Laboratory Malic enzyme activityb 7.1,7.9 8.0, 5.8 14.3,9.2,6.5 13.6, 16.1,21.3 7.6, 10.1,3.3 13.5,12.0 9.2
chow Fatty acid synthesis’ 5.4,5.8 6.4,4.1 11.8,9.7,8.2 7.9,7.2, 5.1 9.0,9.2, 5.8 4.7, 3.8 6.9
“In uiuo incorporation of 3H,0 into fatty acids and malic enzyme activity were measured in the liver of rats receiving diets I and II or standard laboratory chow for the number of days indicated. Details are given under Methods. *Values shown are units per gram of soluble protein. ’ Micromoles of ‘H,O incorporated into fatty acids per hour per gram liver.
178
STARK,
THOMPSON
enzyme and glutathione reductase in chow-fed animals, animals receiving an 18% diet, and animals receiving a 0.5% diet. The correlation is clearly seen in all cases (correlation coefficient = 0.8). DISCUSSION
In previous studies which have documented induction of lipogenic enzymes, either low-fat or fat-free diets were employed (13-19). In every case, the source of dietary carbohydrate was either sucrose, glucose, or fructose in amounts greater than 58% of the diet. The diets employed throughout the studies reported in this paper have a higher fat content and have replaced the major portion of the carbohydrate content with dextrine. The net effect of this dietary treatment has been the observation of significantly different results from other studies. It is clearly evident that high-sucrose diets cause a rapid increase in malic enzyme as previously reported (2). Furthermore, there can be little doubt that increased fatty acid synthesis can result (11, 12, 15) under similar conditions, although it need not necessarily increase, as demonstrated in the initial experiments of this paper. Although high carbohydrate diets do cause an increase of fatty acid synthetase activity (13, 18), the predominant factor required for such in-
I 6
1 8
I IO
1 I2
I 14
DAYS
FIG. 1. Effects of dietary protein restriction on the activities of malic enzyme and glutathione reductase in rat liver. Enzyme activities were measured as described in Methods: Malic enzyme: laboratory chow (0); diet I (@); diet II (A). Glutathione Reductase: laboratory chow (0); Diet I (0); Diet II (W).
AND
FRENKEL
duction would appear to be restricted dietary fats. The level of dietary fat employed in the current studies was higher than in the cited studies. Although increased activities of malic enzyme and glucose6-phosphate dehydrogenase in the liver of animals on a high-carbohydrate diet are observed (2), the capacity for fatty acid biosynthesis does not appear to be altered. It is recognized that starvation drastically affects the activity of fatty acid synthetase. Likewise, restricted dietary protein is now known to decrease the levels of liver fatty acid synthetase. The experimental data shown above indicate that the biosynthetic rate (as measured in uiuo) of fatty acids is certainly not reduced in the livers of animals maintained on low protein diets. Furthermore, the inclusion of higher levels of dietary lipids than has been employed in other studies argues against an increased requirement for fatty acid synthesis. In one study (17), the administration of exogenous fatty acids reduced the activities of the lipogenic enzymes studied. This suggests that the primary regulatory mechanism in studies employing high-carbohydrate and low-fat diets is a combination of two factors: the inclusion of high carbohydrate in the diet and the restriction of dietary fat. Since we have included higher levels of fat in our diet, the second variable has been eliminated. The activity of glucose-6-phosphate dehydrogenase is not induced by high carbohydrate diets, unless protein is also supplied (2, 20). While the potential role of malic enzyme in lipogenesis observed under some conditions is not affected by these experiments, under the conditions of the studies shown here, there is no correlution between malic enzyme activity and lipogenesis. In rabbit mammary tissue, both fatty acid synthesis and the related enzymes are induced during lactation (21), with no increase in malic enzyme activity. This represents the converse situation to that reported here, again arguing that malic enzyme need not be intimately involved in lipogenesis under all circumstances. A similar lack of correlation was also noted by Tepperman and Tepperman (22) in studies in which corn oil or
POSSIBLE
FUNCTIONS
OF MALIC
hydrogenated coconut oil were employed to supply either saturated or unsaturated fatty acids to the experimental animals. It must be noted that, contrary to the protein deficiency studies (1, 2), a positive correlation between malic enzyme activity and the activity of the dehydrogenases associated with the hexose monophosphate shunt was observed by these investigators. On the other hand, a positive correlation between malic enzyme activity and glutathione reductase activity was observed, regardless of the levels of dietary protein employed. These results suggest several possible metabolic correlations in uiuo. Malic enzyme may be involved indirectly in a transport mechanism at the level of the cell membrane by participating in the “y-glutamyl cycle” demonstrated by Meister (23), since this cycle requires glutathione for transport of amino acids in kidney and other tissues. Glutathione, the most abundant cellular thiol, is maintained predominantly in the reduced form, despite its tendency to oxidize (24), turns over rapidly in liver [the half-life is less than 3 hr (25) ] and is extremely sensitive to protein deprivation (24, 26, 27). In view of a conceivable “high priority” for glutathione to facilitate amino acid transport, a mechanism such as suggested in Fig. 2 is quite attractive. Malic enzyme in this scheme furnishes the reducing equivalents which are required for the formation of reduced glutathione. Under conditions of restricted dietary proADPt
179
ENZYME
tein, this mechanism could be of utmost importance to the animal in view of the requirement for maximum nitrogen conservation. Alternatively, malic enzyme could supply reducing equivalents used in a peroxidase cycle (Fig. 3). Over the age range of rats employed, glutathione peroxidase activity is normally about three times higher than glutathione reductase (27). Even if peroxidase cycles have been postulated previously (28-30), this is the first positive correlation between an enzyme which could serve to supply the required reducing equivalents and the glutathione reductase, in liver. Support for this proposal may be found in the work of Sies et al. (29). In Ehrlich tumor cells, a similar system has
92
PYRUVATE
MALATE
-+ NAHp GSSG
GSH
ROe +
ROOH
Hz0 FIG. 3. Glutathione peroxidase to malic enzyme activity.
Pi
cycle and its coupling
ADPtPi
PYR
CARBOXYLIC
Y-GLUTAMYL AMINO ACID
FIG. 2. Possible
coupling
of malic
enzyme
to the y-glutamyl
“KF
system
of amino
acid
transport.
180
STARK,
THOMPSON
been suggested, but in this case the proposed source of NADPH was thought to reside in the hexose-monophosphate shunt dehydrogenases (31). There is no evidence obtained from the current studies to indicate that either glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase activities are high enough to be involved in such a system. However, the activity of malic enzyme is sufficiently elevated in the system described above to warrant further investigation of the proposed cycle. Whether the glutathione peroxidase activity is altered simultaneously with malic enzyme and glutathione reductase has not been determined at this time. Sies et al. (29) have suggested that the rate-limiting step in such a peroxidasereductase system is probably at the level of generation of NADPH, and an elevation of malic enzyme activity would circumvent this limitation. A peroxidase cycle may be important during protein deprivation, since the P-450-mediated drug-metabolizing system loses activity (32) under these conditions and it is conceivable that some of its functions could be replaced by the proposed peroxidase system. In any case, it appears that malic enzyme activity is of sufficient importance to cellular metabolism as to be considerably increased under conditions of strict nitrogen deprivation and that the role of this enzyme is not relegated solely to its function as a lipogenic enzyme. REFERENCES 1. FRENKEL, R., STARK, M. J., AND STAFFORD, J. III (1972) Biochem. Biophys. Res. Commun. 49, 1684-1689. 2. STARK, M. J., ANDFRENKEL, R. (1974) Life Sci. 14, 1x3-1575. 3. LOCKWOOD, E. A., BAILEY, E., AND TAYLOR, C. B. (1970) Biochem. J. 118, 155-162. 4. FRENKEL, R., AND STARK, M. J. (1973) 9th Int. Cong. Biochem. Abstr., p. 377. 5. ROGERS, Q. R., AND HARPER, A. E. (1965) J. Nutr. 87, 267-273. 6. COLLINS, J. M., CRAIG, M. C., NEPOKROEFF, C. M.,
AND
7. 8. 9. 10.
11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28. 29.
30. 31. 32.
FRENKEL
KENNAN, A. L., AND PORTER, J. W. (1971) Arch. Biochem. Biophys. 143, 343-353. WINDMUELLER, H. G., AND SPAETH, A. E. (1966) J. Biol. Chem. 241, 2891-2899. JUNGAS, R. L. (1968) Biochemistry 7, 3708-3717. LOWENSTEIN, J. M. (1971) J. Biol. Chem. 246, 629-632. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. WISE, E. M., AND BALL, E. G. (1964) Proc. Nat. Acad. Sci. USA 52, 1255-1263. YOUNG, J. W., SHRAGO, E., AND LARDY, H. A. (1964) Biochemistry 3, 1687-1692. BURTON, D. N., COLLINS, J. M., KENNAN, A. L., AND PORTER, J. W. (1969) J. Biol. Chem. 244, 4510-4516. MAJORS, P. W., AND KILBURN, E. (1969) J. Biol. Chem. 244, 6254-6262. ALLMAN, D. M., HUBBARD, D. D., AND GIBSON, D. M. (1965) J. Lipid Res. 6, 63-74. KORNACKER, M. S., AND LOWENSTEIN, J. M. (1964) Biochim. Biophys. Acta 84,490-492. KORNACKER, M. S., AND LOWENSTEIN, J. M. (1965) Biochem. J. 94, 209-215. MUTO, Y., AND GIBSON, D. M. (1970) Biochem. Biophys. Res. Commun. 38, 9-15. FITCH, W. M., AND CHAIKOFF, I. L. (1960) J. Biol. Chem. 235, 554-557. SASSON, H. F., DROR, WATSON, J. J., AND JOHNSON, B. C. (1973) J. Nutr. 103, 321-335. MELLENBERGER, R. W., AND BAUMAN, D. E. (1974) Biochem. J. 138, 373-379. TEPPERMAN, H. M., AND TEPPERMAN, J. (1965) Amer. J. Physiol. 209, 773-780. MEISTER, A. (1973) Science 180, 33-39. JOCELYN, P. C. (1972) “Biochemistry of the SH Group”, pp. 175-181, Academic Press, New York. ANDERSON, E. I., AND MOSHER, W. A. (1951) J. Biol. Chem. 188, 717-722. EDWARDS, S., AND WESTERFIELD, W. W. (1952) Proc. Sot. Exp. Biol. Med. 79, 57-59. LINDAN, O., AND WORK, E. (1953) Biochem. J. 55, 554-562. PINTO, R. E., AND BARTLEY, W. (1969) Biochem. J. 112, 109-115. &ES, H., GERSTENECKER, C., MENZEL, H., AND FLOHE, L. (1972) Fed. Eur Biochim. Sot. Lett. 27, 171-175. HOCHSTEIN, P., AND UTLEY, H. (1988). Mol. Pharmacol. 4, 574-579. HOSODA, S., AND NAKAMURA, W. (1970) Biochim. Biophys. Acta 222, 53-64. ANTHONY, L. (1973) J. Nutr. 103, 811-820.
OF BIOCHEMISTRY
AND
Possible MICHAEL The University
BIOPHYSICS
Alternative J. STARK,’
of Texas
Health
Science
166, 174-180
(1975)
Functions BARRY Center
of Rat Liver Malic
THOMPSON,* at Dalla.s
Received
July
AND
Southwestern
RENE
Medical
Enzyme FRENKEL3
School,
Dallas,
Texas
75235
17, 1974
The induction of rat liver malic enzyme by restriction of protein intake has been studied in conjunction with the biosynthesis of fatty acids, fatty acid synthetase, glutathione reductase, and other “lipogenic” enzymes in the various experimental animals. No correlation has been detected between malic enzyme activity and lipogenesis under these conditions. Conversely, a positive correlation between malic enzyme and glutathione reductase has been noted. Possible functions of malic enzyme which appear consistent with these observations are postulated.
The marked induction of hepatic malic enzyme (EC1.1.1.40) in rats maintained on a low-protein diet for 7-10 days has been described recently (1, 2). This type of induction differs from the induction observed after treatment with high concentrations of dietary carbohydrate (specially sucrose) (2), since only malic enzyme activity is enhanced under these conditions, while glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase remain unaffected. These experimental observations lend credence to the suggestion that malic enzyme may play an important role in cellular metabolic regulation, not necessarily linked to lipogenesis (2,3). In a previous abstract (4) it was suggested that malic enzyme may not be exclusively a “lipogenie” enzyme due to its capability to generate reducing equivalents in the form of NADPH, but no direct measurements of fatty acid synthetase had been made. The following paper describes in detail the lack of correlation between lipogenesis (as measured by biosynthetic capacity or enzyme activity) and the activity of malic enzyme in rat liver. In addition, some possible ‘Submitted by MJS in partial fulfillment for the requirements of the Ph.D. degree to the UTHSC at Dallas. *Recipient of a Chilton Foundation Fellowship. 3Supported by Grants AM13275 from the National Institutes of Health and I-385 from The Robert A. Welch Foundation of Houston, Texas. 174 Copyright All rights
0 1975 by Academic Press, of reprodurtion in any form
Inc. reserved.
alternative roles for malic enzyme in rat liver are presented in light of the enzymatic evidence already obtained. MATERIALS
AND
METHODS
Male albino Holtzman rats, weighing between 80 and 90 g at the initiation of all experiments were used throughout these studies. These animals were approximately 30 days of age at the beginning of the experimental diets. The rats were housed individually in steel cages with wire bottoms. After an initial adaptation period of 3 days on a commercial laboratory chow, the rats were allowed to feed ad lib. on either an 18% or a 0.5% protein diet for the duration of the studies. The composition of the diets is given in .Table I. At appropriate time intervals, rats were killed by decapitation. The liver was immediately removed and chilled in ice. For the enzyme assays, weighed samples of liver were homogenized with 9 vol of a solution containing 10 mM morpholinopropane sulfonic acid (MOPS), 1 mM EDTA, and 0.25 M sucrose at pH 7.4. The homogenates were centrifuged at 20,OOOg for 15 min at 2-4”C, and the supernatant extracts tested for various enzyme activities. The activities of malic enzyme isocitrate dehydrogenase, and glucose-6phosphate dehydrogenase was determined as previously described (1). Citrate cleavage enzyme was determined in a final volume of 1 ml of a solution containing: 0.10 M MOPS (pH 7.5); 10 mM MgCl,; 1 mM dithiothreitol (DTT); 20 mM potassium citrate; 25 &ml malate dehydrogenase (MDH); 5 mM ATP; and 0.025 mM NADH. The reaction was initiated by the addition of 30 nmoles of coenzyme A and followed by measuring the change in absorption at 340 nm. Fatty acid synthetase activity was determined by a modification of the method of Collins et al. (6), in a l-ml
POSSIBLE
FUNCTIONS
OF MALIC
TABLE COMPOSITION
Diet Dextrine Sucrose Lactalbumin Casein Salt mixtureb Salt mixture’ Vitamin mixtureb Brewers yeast? Choline’ Wesson oil Vegetable oil
I
OF EXPERIMENTAL
I”
Diet
2210 1105 900 -
3085 1105 25 -
250 -
250 -
25
25 -
50 ml 500 -
50 ml 500 -
solution containing 0.2 M potassium phosphate (pH 6.5), 3 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM DTT, and 0.10 mM NADPH. The reaction was initiated by the addition of acetyl coenzyme A and malonyl coenzyme A to tinal concentrations of 0.050 mM and 0.10 miti, respectively. The activity of glutamate-oxalacetate transaminase was determined by measuring the change of absorption at 340 nm in a 2-ml solution containing 88 mM potassium phosphate buffer (pH 7.6;‘7); 7.4 mM cY-ketoglutarate; 0.15 mM NADH; and 25 I.rg of malate dehydrogenase. The reaction was initiated by addition of L-asparmte (pH 7.6) to a final concentration of 0.2 M. Glutamatepyruvate transaminase activity was assayed in a similar manner The reaction mixture contained: 93 mM potassium phosphate buffer (pH 7.6); 7.4 mM a-ketoglutarate: 0.12 mM NADH; and 25 fig of lactate dehydrogenase iLDH). The reaction was initiated by addition of L-alanine (pH 7.6) to a final concentration of 33.3 mM. Glutathione reductase was determined in a 2-ml solution containing 58 mM potassium phosphate buffer (pH 6.5) and 0.15 mM NADPH. The reaction was initiated by addition of oxidized glutathione (GSSG) to a final concentration of 0.75 mM. Enzymic activity was measured by following the change in absorption at 340 nm. All activities were determined at :!5”C in a Gilford Model 240 recording spectrophotomcter. A unit of enzyme activity is defined as that amount of enzyme catalyzing the reduction (or oxidation) of 1 Fmole of pyridine nucleotide per minute at 25°C. Fatty acid biosynthetic capacity was determined by either an in clitro or an in uiuo system. The in uitro system measured the incorporation of [“Clacetate into lipids in a microsome-free (105,OOOg) supernatant fraction. The system was prepared as follows: One-
DIETS
II”
-
LIIn grams/5 kilograms. b Rogers and Harper (Ref. 5). c U.S.P. No. 2. d U.S.P. p Choline chloride was added to the diet as an aqueous
175
ENZYME
solution
Diet III”
containing
Diet IV”
3400 900 -
675 3400 -
200 -
200 -
100 -
100 -
400
400
25 -
1 g/5 ml.
milliliter samples of postmicrosomal extract were placed in screw-capped tubes which contained 0.1 ml of 0.12 M ATP, 0.1 ml of 0.15 M MgCl,, 0.1 ml of 0.12 M KHCO,, 0.14 ml of 0.3 M potassium citrate (pH 7.4). 0.15 ml of 0.1 M cysteine, 0.03 ml of 1.0 M malate (pH 7.4), and 0.13 ml of 0.1 M potassium phosphate (pH 7.4). These samples were incubated at 37°C for 30 min. After this initial incubation, 0.15 ml of 0.1 M potassium [P-“Clacetate (0.1 pCi/pmole), 0.05 ml of 0.01 M Coenzyme A, and 0.1 ml of 0.03 M NADP’ were added to each tube and the reactions allowed to proceed for various time intervals. The reactions were stopped by addition of 2 ml of 4 N KOH in 25% ethanol. The samples were loosely capped and placed in an autoclave where they were autoclaved at 20 psi for 30 min. After cooling, 1.5 ml of 10 N H,SO, was added to each sample in order to release the free fatty acids. Each sample was subsequently extracted three times with 5-ml portions of petroleum ether. The organic layers were separated, transferred to glass scintillation vials, evaporated to dryness in an 80°C water bath and then placed in a 100°C oven for 10 min. Ten milliliters of PCS (AmershamiSearle, Arlington Heights, IL) were added to each vial. The vials were shaken and the radioactivity determined in a Beckman Liquid Scintillation Counter. For zero time controls, the alcoholic KOH was added prior to addition of the labeled acetate. The in uiuo system of determining fatty acid biosynthetic capacity employed the incorporation of 3H,0 into fatty acids, essentially as described by others (7-9). The rats were injected with 1 mCi (10 mCi/ml) of 3H,0 intraperitoneally, 1 hr prior to their demise by decapitation. Blood samples were collected and the plasma prepared for subsequent determination of the specific radioactivity of the body water.
176
STARK.
THOMPSON
AND
as sucrose or as dextrinel does not appear to affect the rate of fatty acid synthesis. Simultaneously with these assays the activities of malic enzyme, glucose-6-phosphate dehydrogenase, and NADP+-isooitrate dehydrogenase were determined in the postmicrosomal supernatant fraction. The results obtained under these experimental conditions have been previously reported (2). Briefly stated, no alteration of enzymic activity is observed on diet I. In animals maintained on diet II, malic enzyme activity is induced in a rapid and sustained manner, while neither of the other two NADP+-dehydrogenases measured are affected. Animals maintained on diet III (high sucrose-W% protein) show elevated malic enzyme and glucose-6-phosphate dehydrogenase activity while the activity of isocitrate dehydrogenase remains unaltered. On diet IV, malic enzyme alone is induced. The induction curve differs from that seen on either diet II or diet III, seeming to be influenced by both high levels of dietary sucrose and reduced dietary protein. Therefore, although malic enzyme is readily inducible either by elevated dietary sucrose or by restricted dietary protein, there is no concomitant increase in the in vitro rate of fatty acid biosynthesis from acetate under the conditions of our study. Hence, it appears that malic enzyme need not necessarily be confined to a role of providing reducing equivalents for fatty acid biosynthesis as suggested in previous studies (11, 12). In another series of studies, summarized
The liver was removed and immediately chilled in ice. Weighed samples were homogenized in 3 vol of chloroform-methanol (2: 1). The phases were separated by centrifugation, the organic layer removed, and subsequently dried in a boiling water bath. Saponification and extraction of the fatty acids was carried out as described above. Plasma samples were diluted in 0.9% NaCl and aliquots were counted in PCS. Discussions about the advantages of emphasizing ‘H,O incorporation for estimates of the in uiuo synthesis of fatty acids are given in Refs. 8 and 9. Diets and dietary components were purchased from Nutritional Biochemicals Corporation, Cleveland, OH. L-Cysteine was purchased from Eastman Kodak Corp. MOPS, DTT, NADH, NADPH, NADP+, GSSG, a-ketoglutarate, L-alanine, L-aspartic acid, malic acid, glucose-6-phosphate, m-isocitric acid, coenzyme A, acetyl coenzyme A, and malonyl coenzyme A were purchased from Sigma. Sucrose, MgCl,, potassium citrate, potassium phosphate, EDTA, and KHCO, were purchased from J. T. Baker Chemical Company, Phillipsburg, NJ. ATP, MDH, and LDH were from Boehringer Mannheim. [2-“C]Acetate and “H,O were purchased from Amersham-Searle. Protein concentrations were determined by the Lowry technique (11) with bovine serum albumin as the standard protein. RESULTS
A summary of the fatty acid biosynthetic rate from [Z-“Clacetate in the postmicrosomal supernatant extracts is given in Table II. There is no apparent difference in the synthetic rates observed with this method under these conditions and neither dietary protein quality nor quantity appear to alter the in vitro capacity to synthesize fatty acids from acetate. Likewise, the form of the dietary carbohydrate (whether TABLE In vitro
SYNTHEW
Days on diet
OF FATIK
a The biosynthetic supernatant extract. from three animals.
II
ACIDS FROM ACETATE IN MICROSOME-FREE SUPERNATANT LIVERS OF RATS RECEIVING DIFFERENT DIETS~
Laboratory chow
Acetate Diet I (18% lactalbumin)
0 4 6 15 19
FRENKEL
2.6 f 0.5 2.3 i 0.6 2.9 * 0.4
incorporation (0.5%
(pmoleslg Diet II lactalbumin)
2.4 zt 0.4 1.9 l 0.4 -
rates of fatty acids from [2-“Clacetate Conditions for the assay are described
SOLUTIONS
2.2 2.2 2.2 2.6 was measured under Methods.
PREPARED FROM
protein/hr) Diet IV (0.5% casein) -
* + f zt
0.8 0.6 0.3 0.8
in duplicate Each point
3.3 f 0.9 2.5 + 0.7 2.4 zt 0.2 in the postmicrosomal represents the average
POSSIBLE
FUNCTIONS
OF MALIC
In viva fatty acid biosynthesis was measured by determining the incorporation of 3H,0 into fatty acids. The data obtained from this study are given in Table IV. Essentially identical results were obtained in three separate experiments in which no readily observable induction of fatty acid biosynthesis could be detected concomitant with the increase in malic enzyme activity. Throughout these studies, the activity of NADP+-isocitrate dehydrogenase was not appreciably altered. In another series of experiments, the activity of other enzymes which might act upon primary or secondary products of the malic enzyme-catalyzed reaction was determined. Glutamate-oxalacetate transaminase activity appears to be insensitive to low dietary protein concentration. Citrate-cleavage enzyme and glutamatepyruvate transaminase activities are drastically reduced in the livers of animals maintained on 0.5% protein. In each of the above cases, the source of protein does not seem to make a difference, as identical results are obtained whether the protein source is lactalbumin or casein. However, glutathione reductase activity is appreciably higher in the liver of animals maintained on a 0.5% protein diet than in control animals maintained on an 18% protein diet, after about 1 wk from the initiation of the experimental diets. Figure 1 is an example of activities of malic
in Table III, liver enzyme activities were determined in animals maintained on a low-protein diet (diet II). As is noted from the table, although malic enzyme activity is induced by restricted dietary protein, fatty acid synthetase activity continues to decrease. GlucoseS-phosphate dehydrogenase activity is essentially unaffected by the diet. Citrate cleavage enzyme activity remains at a constant level during the 8 days shown in the table, but it eventually diminishes under conditions of continuous protein restriction (unpublished observations) . TABLE ACTWITIES LIP~CENIC
4 6 8
III
OF MALIC ENZYME AND OTHER HEPATIC ENZYMES DURING THE INITIAL STAGES OF PROTEIN RFSTRICTIOP MEb
G6PDb
CCEb
FASb
23.99 18.36 32.92
9.45 11.25 7.97
3.83 2.89 3.76
2.27 1.76 0.75
Dayson diet
a Malic enzyme (ME), glucose-6-phosphate dehydrogenase (GGPD), citrate cleavage enzyme (CCE), and fatty acid synthetase (FAS) activities were measured in aliquots of postmitochondrial supernatant fractions prepared from the liver of rats maintained for the indicated number of days on a 0.5% lactalbumin diet (diet II). For details, see text. b Activities given as units per gram of soluble
TABLE MALIC Days on diet
ENZYME
9.2, 9.7, 10.0, 19.7, 16.8, 16.8, 27.1, 18.4,
10.9,8.6 22.4 10.5,7.1 20.3 20.0, 17.7 13.2, 17.3 18.3, 24.6 21.8,12.7
AND FA~Y
T
Diet I Malic enzyme activityb
4 5 7 9 11 13 17 21
ACTIVITY
I
177
ENZYME
IV
ACID SYNTHESIS in Diet
Viuo
IN RAT LIVERY
T
II
Fatty acid synthesis’
Malic enzyme activityb
Fatty acid synthesis’
3.5,4.5,6.8 18.8, 13.4 6.0,4.7,3.4 6.0,4.7, 3.5 13.6,4.3,4.8 6.0,3.2,3.4 7.3,6.0,5.7 14.3, 12.1, 5.4
36.1, 39.4, 32.3 35.3, 54.3 39.3, 21.0,42.0 94.0.58.0 107.0,71.0,63.2 74.7,83.8,85.0 87.9, 78.3,75.3 90.9, 53.8,67.8
6.7,6.3,8.1 3.5, 3.0 5.0,4.3,4.0 17.8, 17.0 4.9.3.7, 7.9 4.5, 3.0, 4.4 10.5, 7.5,5.8 4.4
Laboratory Malic enzyme activityb 7.1,7.9 8.0, 5.8 14.3,9.2,6.5 13.6, 16.1,21.3 7.6, 10.1,3.3 13.5,12.0 9.2
chow Fatty acid synthesis’ 5.4,5.8 6.4,4.1 11.8,9.7,8.2 7.9,7.2, 5.1 9.0,9.2, 5.8 4.7, 3.8 6.9
“In uiuo incorporation of 3H,0 into fatty acids and malic enzyme activity were measured in the liver of rats receiving diets I and II or standard laboratory chow for the number of days indicated. Details are given under Methods. *Values shown are units per gram of soluble protein. ’ Micromoles of ‘H,O incorporated into fatty acids per hour per gram liver.
178
STARK,
THOMPSON
enzyme and glutathione reductase in chow-fed animals, animals receiving an 18% diet, and animals receiving a 0.5% diet. The correlation is clearly seen in all cases (correlation coefficient = 0.8). DISCUSSION
In previous studies which have documented induction of lipogenic enzymes, either low-fat or fat-free diets were employed (13-19). In every case, the source of dietary carbohydrate was either sucrose, glucose, or fructose in amounts greater than 58% of the diet. The diets employed throughout the studies reported in this paper have a higher fat content and have replaced the major portion of the carbohydrate content with dextrine. The net effect of this dietary treatment has been the observation of significantly different results from other studies. It is clearly evident that high-sucrose diets cause a rapid increase in malic enzyme as previously reported (2). Furthermore, there can be little doubt that increased fatty acid synthesis can result (11, 12, 15) under similar conditions, although it need not necessarily increase, as demonstrated in the initial experiments of this paper. Although high carbohydrate diets do cause an increase of fatty acid synthetase activity (13, 18), the predominant factor required for such in-
I 6
1 8
I IO
1 I2
I 14
DAYS
FIG. 1. Effects of dietary protein restriction on the activities of malic enzyme and glutathione reductase in rat liver. Enzyme activities were measured as described in Methods: Malic enzyme: laboratory chow (0); diet I (@); diet II (A). Glutathione Reductase: laboratory chow (0); Diet I (0); Diet II (W).
AND
FRENKEL
duction would appear to be restricted dietary fats. The level of dietary fat employed in the current studies was higher than in the cited studies. Although increased activities of malic enzyme and glucose6-phosphate dehydrogenase in the liver of animals on a high-carbohydrate diet are observed (2), the capacity for fatty acid biosynthesis does not appear to be altered. It is recognized that starvation drastically affects the activity of fatty acid synthetase. Likewise, restricted dietary protein is now known to decrease the levels of liver fatty acid synthetase. The experimental data shown above indicate that the biosynthetic rate (as measured in uiuo) of fatty acids is certainly not reduced in the livers of animals maintained on low protein diets. Furthermore, the inclusion of higher levels of dietary lipids than has been employed in other studies argues against an increased requirement for fatty acid synthesis. In one study (17), the administration of exogenous fatty acids reduced the activities of the lipogenic enzymes studied. This suggests that the primary regulatory mechanism in studies employing high-carbohydrate and low-fat diets is a combination of two factors: the inclusion of high carbohydrate in the diet and the restriction of dietary fat. Since we have included higher levels of fat in our diet, the second variable has been eliminated. The activity of glucose-6-phosphate dehydrogenase is not induced by high carbohydrate diets, unless protein is also supplied (2, 20). While the potential role of malic enzyme in lipogenesis observed under some conditions is not affected by these experiments, under the conditions of the studies shown here, there is no correlution between malic enzyme activity and lipogenesis. In rabbit mammary tissue, both fatty acid synthesis and the related enzymes are induced during lactation (21), with no increase in malic enzyme activity. This represents the converse situation to that reported here, again arguing that malic enzyme need not be intimately involved in lipogenesis under all circumstances. A similar lack of correlation was also noted by Tepperman and Tepperman (22) in studies in which corn oil or
POSSIBLE
FUNCTIONS
OF MALIC
hydrogenated coconut oil were employed to supply either saturated or unsaturated fatty acids to the experimental animals. It must be noted that, contrary to the protein deficiency studies (1, 2), a positive correlation between malic enzyme activity and the activity of the dehydrogenases associated with the hexose monophosphate shunt was observed by these investigators. On the other hand, a positive correlation between malic enzyme activity and glutathione reductase activity was observed, regardless of the levels of dietary protein employed. These results suggest several possible metabolic correlations in uiuo. Malic enzyme may be involved indirectly in a transport mechanism at the level of the cell membrane by participating in the “y-glutamyl cycle” demonstrated by Meister (23), since this cycle requires glutathione for transport of amino acids in kidney and other tissues. Glutathione, the most abundant cellular thiol, is maintained predominantly in the reduced form, despite its tendency to oxidize (24), turns over rapidly in liver [the half-life is less than 3 hr (25) ] and is extremely sensitive to protein deprivation (24, 26, 27). In view of a conceivable “high priority” for glutathione to facilitate amino acid transport, a mechanism such as suggested in Fig. 2 is quite attractive. Malic enzyme in this scheme furnishes the reducing equivalents which are required for the formation of reduced glutathione. Under conditions of restricted dietary proADPt
179
ENZYME
tein, this mechanism could be of utmost importance to the animal in view of the requirement for maximum nitrogen conservation. Alternatively, malic enzyme could supply reducing equivalents used in a peroxidase cycle (Fig. 3). Over the age range of rats employed, glutathione peroxidase activity is normally about three times higher than glutathione reductase (27). Even if peroxidase cycles have been postulated previously (28-30), this is the first positive correlation between an enzyme which could serve to supply the required reducing equivalents and the glutathione reductase, in liver. Support for this proposal may be found in the work of Sies et al. (29). In Ehrlich tumor cells, a similar system has
92
PYRUVATE
MALATE
-+ NAHp GSSG
GSH
ROe +
ROOH
Hz0 FIG. 3. Glutathione peroxidase to malic enzyme activity.
Pi
cycle and its coupling
ADPtPi
PYR
CARBOXYLIC
Y-GLUTAMYL AMINO ACID
FIG. 2. Possible
coupling
of malic
enzyme
to the y-glutamyl
“KF
system
of amino
acid
transport.
180
STARK,
THOMPSON
been suggested, but in this case the proposed source of NADPH was thought to reside in the hexose-monophosphate shunt dehydrogenases (31). There is no evidence obtained from the current studies to indicate that either glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase activities are high enough to be involved in such a system. However, the activity of malic enzyme is sufficiently elevated in the system described above to warrant further investigation of the proposed cycle. Whether the glutathione peroxidase activity is altered simultaneously with malic enzyme and glutathione reductase has not been determined at this time. Sies et al. (29) have suggested that the rate-limiting step in such a peroxidasereductase system is probably at the level of generation of NADPH, and an elevation of malic enzyme activity would circumvent this limitation. A peroxidase cycle may be important during protein deprivation, since the P-450-mediated drug-metabolizing system loses activity (32) under these conditions and it is conceivable that some of its functions could be replaced by the proposed peroxidase system. In any case, it appears that malic enzyme activity is of sufficient importance to cellular metabolism as to be considerably increased under conditions of strict nitrogen deprivation and that the role of this enzyme is not relegated solely to its function as a lipogenic enzyme. REFERENCES 1. FRENKEL, R., STARK, M. J., AND STAFFORD, J. III (1972) Biochem. Biophys. Res. Commun. 49, 1684-1689. 2. STARK, M. J., ANDFRENKEL, R. (1974) Life Sci. 14, 1x3-1575. 3. LOCKWOOD, E. A., BAILEY, E., AND TAYLOR, C. B. (1970) Biochem. J. 118, 155-162. 4. FRENKEL, R., AND STARK, M. J. (1973) 9th Int. Cong. Biochem. Abstr., p. 377. 5. ROGERS, Q. R., AND HARPER, A. E. (1965) J. Nutr. 87, 267-273. 6. COLLINS, J. M., CRAIG, M. C., NEPOKROEFF, C. M.,
AND
7. 8. 9. 10.
11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28. 29.
30. 31. 32.
FRENKEL
KENNAN, A. L., AND PORTER, J. W. (1971) Arch. Biochem. Biophys. 143, 343-353. WINDMUELLER, H. G., AND SPAETH, A. E. (1966) J. Biol. Chem. 241, 2891-2899. JUNGAS, R. L. (1968) Biochemistry 7, 3708-3717. LOWENSTEIN, J. M. (1971) J. Biol. Chem. 246, 629-632. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. WISE, E. M., AND BALL, E. G. (1964) Proc. Nat. Acad. Sci. USA 52, 1255-1263. YOUNG, J. W., SHRAGO, E., AND LARDY, H. A. (1964) Biochemistry 3, 1687-1692. BURTON, D. N., COLLINS, J. M., KENNAN, A. L., AND PORTER, J. W. (1969) J. Biol. Chem. 244, 4510-4516. MAJORS, P. W., AND KILBURN, E. (1969) J. Biol. Chem. 244, 6254-6262. ALLMAN, D. M., HUBBARD, D. D., AND GIBSON, D. M. (1965) J. Lipid Res. 6, 63-74. KORNACKER, M. S., AND LOWENSTEIN, J. M. (1964) Biochim. Biophys. Acta 84,490-492. KORNACKER, M. S., AND LOWENSTEIN, J. M. (1965) Biochem. J. 94, 209-215. MUTO, Y., AND GIBSON, D. M. (1970) Biochem. Biophys. Res. Commun. 38, 9-15. FITCH, W. M., AND CHAIKOFF, I. L. (1960) J. Biol. Chem. 235, 554-557. SASSON, H. F., DROR, WATSON, J. J., AND JOHNSON, B. C. (1973) J. Nutr. 103, 321-335. MELLENBERGER, R. W., AND BAUMAN, D. E. (1974) Biochem. J. 138, 373-379. TEPPERMAN, H. M., AND TEPPERMAN, J. (1965) Amer. J. Physiol. 209, 773-780. MEISTER, A. (1973) Science 180, 33-39. JOCELYN, P. C. (1972) “Biochemistry of the SH Group”, pp. 175-181, Academic Press, New York. ANDERSON, E. I., AND MOSHER, W. A. (1951) J. Biol. Chem. 188, 717-722. EDWARDS, S., AND WESTERFIELD, W. W. (1952) Proc. Sot. Exp. Biol. Med. 79, 57-59. LINDAN, O., AND WORK, E. (1953) Biochem. J. 55, 554-562. PINTO, R. E., AND BARTLEY, W. (1969) Biochem. J. 112, 109-115. &ES, H., GERSTENECKER, C., MENZEL, H., AND FLOHE, L. (1972) Fed. Eur Biochim. Sot. Lett. 27, 171-175. HOCHSTEIN, P., AND UTLEY, H. (1988). Mol. Pharmacol. 4, 574-579. HOSODA, S., AND NAKAMURA, W. (1970) Biochim. Biophys. Acta 222, 53-64. ANTHONY, L. (1973) J. Nutr. 103, 811-820.