Differential hormonal regulation of l -glycerol 3-phosphate dehydrogenase in rat brain and skeletal muscle

ARCHIVES

OF BIOCHEMISTRY

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BIOPHYSICS

179,

682-689

(1977)

Differential Hormonal Regulation of L-Glycerol Dehydrogenase in Rat Brain and Skeletal JAMES *Mental

Retardation +Department

F. MCGINNIS”

JEAN

AND

DE

VELLIS*,

3-Phosphate Muscle’ t*’

Research Center, tLaboratoty of Nuclear Medicine and Radiation Biology, of Anatomy, University of California, Los Angeles, California 90024 Received

September

and

20, 1976

The level of L-glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) is regulated in the rat brain by glucocorticoids. Following hypophysectomy, the concentration of brain glycerol-3-P dehydrogenase decreases to about 40% of the control. By immunotitration, we have demonstrated that this decrease in glycerol-3-P dehydrogenase activity is due to fewer enzyme molecules rather than less efficient ones. We also demonstrated that the enzyme remaining in the brain after hypophysectomy is identical to that found in the brains of control littermates, as determined by gel permeation chromatography, pH optimum, heat lability, electrophoretic mobility, and Ouchterlony double-diffusion analysis. Since the concentration of glycerol-3-P dehydrogenase in skeletal muscle is not regulated by glucocorticoids, we also compared the brain enzyme to the muscle enzyme. By the above criteria, skeletal muscle glycerol phosphate dehydrogenase is identical to the brain enzyme. This suggests that the same structural gene codes for glycerol-3-P dehydrogenase in brain and muscle and that the difference in response to glucocorticoids is due to the presence of a specific regulatory mechanism in brain that is absent in muscle.

In the brain of adult rats, the level of glycerol-3-P2 dehydrogenase has been shown to be regulated directly by adrenal and pituitary functions. Following hypophysectomy or adrenalectomy, the specific activity of glycerol-3-P dehydrogenase in the brain decreased to a basal level of about 40% of the level found in control littermates. Injection of adrenocorticotrophic hormone or cortisol in hypophysectomized rats, or cortisol in adrenalectomized rats, restored the adult level of brain glycerol-3-P dehydrogenase. In addition, adrenalectomy had little or no effect on the level of glycerol-3-P dehydrogenase in skeletal muscle (1). Both of these observations could be explained if glycerol-3-P dehydrogenase ex-

isted in two isozymic forms coded for by different structural genes. In that case, one could postulate that the brain has two forms of enzymes and that the muscle has only one form. The concentration of the isozymic form specific to the brain would be regulated by glucocorticoids, whereas the form common to muscle and brain would be synthesized constitutively or regulated by a different mechanism. The existence of multiple molecular forms of a given enzyme within an organism has been well established. Isozymes, the products of different structural genes, need not be expressed equally during different stages of development nor to the same extent in different tissues. The systems best characterized include the H- and M-type lactic dehydrogenases (21, the A, B, and C aldolases (31, and the hexokinases (4). The physiological and evolutionary iniportance of multiple molecular forms of enzymes, including glycerol-3-P dehydrogenase, has been studied in a number of

1 This work was supported by USPHS Grants HD05615 and HD-04612 and by ERDA Contract E(04-1) GEN-12. 2 Abbreviations used: glycerol-3-P, glycerol 3phosphate; GPDH, glycerol 3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis; C6, glioma cells; LDH, lactatedehydrogenase. 682 Ccpyright All rights

0 1977 by Academic press, Inc. of reproduction in any form reserved.

ISSN 9903-9961

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organisms and organs. In birds, the glycerol-3-P dehydrogenase isozymes of liver and breast muscle are believed to have different metabolic functions (5). It has been postulated that the breast muscle isozyme functions in maintaining the NAD redox potential during anaerobic glycolysis, whereas the liver glycerol-3-P dehydrogenase acts primarily in the synthesis of triglycerides. In addition to the isozymes of cytoplasmic glycerol-3-P dehydrogenase found in the chick, different molecular forms of glycerol-3-P dehydrogenase have been purified from rat liver and muscle (61, two electrophoretically different forms were purified from rat brain (7), two immunologically different forms were found in skeletal and cardiac muscle of rats and of rabbits (81, and fetal and adult forms of glycerol-3-P dehydrogenase were demonstrated in mouse brain tissue (9). Similarly, the glycerol-3-P dehydrogenase in brain and in muscle of the rat might have different metabolic roles and fulfill them with isoenzymes. The purpose of this investigation was to examine the mechanism(s) regulating the concentration of glycerol-3-P dehydrogenase in rat brain and skeletal muscle tissue. More specifically, we set out to answer the following questions. Is the basal level of glycerol-3-P dehydrogenase in the brain of a hypophysectomized rat due to the existence of two isozymes, one of which is regulated by the pituitary and one which is not? Is the decrease in enzymic activity in the brain due to a decrease in the number of enzyme molecules or to catalytically less efficient molecules? Similarly, since the muscle glycerol-3-P dehydrogenase is not regulated by glucocorticoids, we were interested in whether this difference was due to a different glycerol-3-P dehydrogenase isozyme in muscle or to different regulatory mechanisms operating in brain and muscle tissue. By immunotitration, Ouchterlony double diffusion, gel permeation chromatography, pH optimum, heat lability, and polyacrylamide gel electrophoresis, we have now shown that the enzyme in the brains of control and hypophysectomized rats is identical and that the decrease in brain

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glycerol-3-P dehydrogenase activity is due to fewer molecules, not less efficient ones. On the basis of similar experiments, our data also indicate that the muscle glycerol3-P dehydrogenase is the same as that present in the brain. Therefore, the reason(s) for the unique regulation of brain glycerol-3-P dehydrogenase by cortisol appears to reside in the brain tissue itself rather than the enzyme molecule. MATERIALS

AND

METHODS

Materials. All chemicals were reagent grade unless indicated otherwise and were obtained from commercial sources. Grade-X a-m-glycerol phosphate (used for staining gels for enzyme activity) was from Sigma Chemical Co., St. Louis, Missouri. All reagents for polyacrylamide gels were from BioRad, Richmond, California. Animals. Sprague-Dawley rats were surgically hypophysectomized by Hormone Assay Laboratories (Chicago, Illinois) and were maintained on Purina Rat Chow ad libitum. The rats were 25 days old at the time of surgery and were killed by decapitation at 47 days of age. The rats used for comparison of glycerol3-P dehydrogenase in muscle with glycerol-3-P dehydrogenase in brain were also killed at 47 days of age and, in each experiment, the brain and muscle tissues were from the same animal. The tissue was rapidly excised, rinsed with cold saline, and homogenized in 7 vol of 10 mM Na phosphate buffer, pH 7.5, containing 1 mM EDTA and 5 mM mercaptoethanol at 4°C. The muscle tissue obtained from the hindlegs was cut into small pieces with scissors and then homogenized in a Sorvall Omnimixer. The brain tissue was homogenized in Teflon-glass Potter-Elvehjem homogenizers at 4°C. The crude homogenates were then centrifuged in a 40 rotor at 40,000 rpm for 1 h in a Beckman L2 65B ultracentrifuge and the supernatants were poured through gauze to remove the lipids which had separated. Enzyme assay. Glycerol-3-P dehydrogenase was assayed as described previously (71. One unit of activity is defined as that amount of enzyme which catalyzes the oxidation of one nanomole of NADH per minute under the conditions of the assay. Protein was assayed by the method of Lowry et al. (10) using crystallized bovine serum albumin as a standard. Immunological titration of GPDH with rabbit antiserum. Rabbit antiserum against purified rat brain GPDH was obtained as described previously (7). The serum was centrifuged at 105,OOOg for 1 h in an ultracentrifuge and removed with a pipet. The lipid layer which formed was left in the tube. The serum was then put through a G-25 (fine) Sephadex column equilibrated with 10 mM Na phosphate buffer con-

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taining 1 mM EDTA and 5 mM mercaptoethanol. For each experiment, the serum was then diluted 50-fold in the same buffer containing 1 mg of bovine serum albumin/ml and various concentrations were added to 200 ~1 of 105,OOOg supernatants from brain and muscle homogenates as indicated. Following incubation at 37°C for 30 min, an aliquot was removed and the amount of glycerol-3-P dehydrogenase activity remaining was determined. Incubation overnight at 4°C did not result in further inactivation of glycerol-3-P dehydrogenase and, since the antibodyglycerol-3-P dehydrogenase complex was inactive, centrifugation was not necessary. Poljacrylamide gel electrophoresis (PAGE). Polya&amide gel electrophoresis was performed essentially as described by Davis (11). The separating gel was 75 mm in length and 7% acrylamide. The gels were cast in 100 x 5-mm (i.d.1 tubes and run at 4”C, 4.0 mAttube, for 2.5 h. Bromophenol blue was included in the upper buffer as a visual marker. The gels were removed and rinsed with 4°C buffer prior to treatment. Staining for glycerol-3-P dehydrogenase activity was performed in foil-wrapped, screwcap tubes containing a-m-glycerol phosphate (0.156 M), nitro blue tetrazolium salt (0.234 mglml), NAD (1.56 mg/ml), and phenazine methosulfate (0.625 mglml) in 0.078 M glycine-NaOH, pH 9.0. The tubes were incubated at 37°C in a circulating water bath for 15 min and the reaction was terminated by replacing the staining solution with 10% acetic acid. Gels were photographed with a Polaroid camera using a Wratten No. 22 gelatin filter to accentuate the bands. Determination of thermal stability. Sets of tubes (in duplicate) containing 200 ~1 of the 105,OOOg supernatants of the enzyme solutions were incubated in a circulating water bath at 50.0 ? O.l”C for the indicated times. The tubes were sealed with Parafilm to prevent evaporation. Immediately upon removal from the bath, the samples were quickly cooled by swirling in an ice-salt slurry (- 15°C) for 5 s and were then maintained at 0°C until completion of the experiment. Aliquots from each tube were assayed for glycerol-3-P dehydrogenase activity and the log of the percentage activity remaining was plotted versus the time at 50°C. rmmunodiffusion. Purified agar (1%) in isotonic saline was used to coat the surface of microscope slides, and wells were cut as indicated with a punch apparatus. To increase the intensity of the immunodiffusion bands, the enzyme from brain was partially purified and concentrated using the affinity column previously described (12). A volume of 20 ~1 of the indicated samples was put into each of the wells and the slide was incubated in a humidified atmosphere at 37°C for 16-24 h. Gel filtration. The column used for comparing the elution profile of glycerol-3-P dehydrogenase was 2.5 x 100 cm. It was fitted with flow adaptors and run in

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an ascending manner using a peristaltic pump to maintain a constant flow rate. The bed (Sephadex G-200) occupied a volume of 490 ml and was equilibrated with 10 mM sodium phosphate, pH 7.5, 5 mM /3-mercaptoethanol, and 1 mM EDTA. The flow rate was 0.34 ml/min and lo-min fractions were collected. The column was calibrated with the following molecular weight standards: ribonuclease A (13,700), chymotrypsinogen (25,0001, ovalbumin (43,500), bovine serum albumin (68,000), lactate dehydrogenase (142,000), aldolase (158,000), catalase (240, OOO), and ferritin (460,000). RESULTS

To investigate the possibility that more than one structural gene exists for glycerol-3-P dehydrogenase, we compared the properties of (a) brain glycerol-3-P dehydrogenase in control and hypophysectomized rats and 0~) compared brain glycerol-3-P dehydrogenase with muscle glycerol-3-P dehydrogenase. It has been previously demonstrated that hypophysectomy resulted in a 60% decrease in the level of brain glycerol-3-P dehydrogenase (1). To determine whether this residual or basal amount of activity might be due to a different isoenzyme of glycerol-3-P dehydrogenase, we compared the glycerol-3-P dehydrogenase activity remaining in the brain 23 days after hypophysectomy with that in the brain of control littermates. The control brains had 50.2 -+-4.7 U/mg of protein, whereas the hypophysectomized brains had 24.0 k 2.0 U/mg of protein, confirming the earlier observation. Since a number of forms of glycerol-3-P dehydrogenase have been resolved by isoelectric focusing (13) and since two electrophoretically distinct forms of glycerol-3-P dehydrogenase have been purified from rat brain tissue (7), we first examined the electrophoretic mobility of glycerol-3-P dehydrogenase from these animals. Aliquots from the high-speed supernatant of brain homogenates of control and experimental animals were subjected to polyacrylamide gel electrophoresis and subsequently stained for glycerol-3-P dehydrogenase activity. The results (Fig. 1) indicate that, in each case, there is only one electrophoretic form of glycerol-3-P dehydrogenase and that this form is the same for both. Similarly, when brain and muscle glycerol-3-P dehydrogenases were electrophoresed (data not shown), only one

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without spurs (data not shown), suggesting identity of the antigens. Similarly, muscle and brain glycerol-3-P dehydrogenases also proved to be identical by this criterion (Fig. 2). The second comparison was by immunotitration in which a constant amount of antibrain glycerol-3-P dehydrogenase serum was incubated with increasing amounts of brain glycerol-3-P dehydrogenase from control or hypophysectomized rats (Fig. 3). The single intercept (equivalence point) on the abscissa suggests that the glycerol-3-P dehydrogenase from each source is immunologically identical and that these molecules are catalytically equal. These data also demonstrate that the brain glycerol-3-P dehydrogenase activity remaining following hypophysectomy actually represents fewer glycerol-3P dehydrogenase molecules rather than catalytically less active molecules. Therefore, either the rate of synthesis of brain glycerol-3-P dehydrogenase has decreased or its rate of degradation has increased in hypophysectomized rats. The addition of increasing amounts of

FIG. 1. Electrophoretic mobility of brain glycerol-3-P dehydrogenase from control and hypophysectomized rats. Each gel contained a total of 15 U of glycerol-3-P dehydrogenase, and, following polyacrylamide gel electrophoresis, they were stained for enzyme activity as described under Materials and Methods. The dark band at the bottom of each gel is due to the tracking dye diffusing out from the front during the enzymatic staining procedure. (A) Control brain glycerol-3-P dehydrogenase. (B) Coelectrophoresis of equal amounts of brain glycerol-3-P dehydrogenase from control and hypophysectomized animals. (C) Hypophysectomized brain glycerol-3-P dehydrogenase.

band of enzyme activity was observed and coelectrophoreses indicated that the glycerol-3-P dehydrogenase of these tissues was electrophoretically identical. The immunological identity of brain glycerol-3-P dehydrogenase from control and hypophysectomized animals was examined by two different methods. In the first case, Ouchterlony immunodiffusion plates gave a continuous precipitation line

FIG. 2. Ouchterlony immunodiffusion against rabbit anti-rat brain glycerol-3-P dehydrogenase serum. The center well contains 20 ~1 of antiserum and the outside wells contain 20 ~1 of the high-speed supernatants (see Materials and Methods) from either rat muscle (M) or rat brain (B). The specific activity of brain GPDH was 0.227 U/mg of protein, whereas the muscle was 1.020 U/mg of protein, and the concentrations were 4.50 and 8.71 U/ml, respectively.

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FIG. 3. Immunotitration of glycerol-3-P dehydrogenase from the brain of control and hypophysectomized rats. Increasing amounts of brain glycerol-3-P dehydrogenase from control and hypophysectomized rats were added to a constant amount of anti-glycerol-3-P dehydrogenase serum and, following incubation, the amount of activity remaining was determined. Control, 0; hypophysectomized, 0.

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the brain glycerol-3-P dehydrogenase is immunologically identical to the muscle glycerol-3-P dehydrogenase as determined by both immunological tests. The molecular weight of brain glycerol3-P dehydrogenase is identical to muscle glycerol-3-P dehydrogenase as determined by gel permeation chromatography on Sephadex G-200 (Fig. 5). The enzymes from brain and from muscle were run separately on the same column and then plotted as the percentage maximum activity vs the elution volume (fraction number). As can be seen, the elution profiles are essentially identical. Brain glycerol-3-P dehydrogenase has an elution volume (V,) of 326.0 ml, whereas the V, of the muscle glycerol-3-P dehydrogenase was 324.6 ml. We calibrated the column with a number of standard proteins of known molecular weights and plotted the relative elution volume (VJV,) vs the log of the molecular weight (Fig. 6). Each point represents the average of three separate runs. The results

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FIG. 4. Immunotitration of glycerol-3-P dehydrogenase from brain and skeletal muscle. Increasing amounts of antiserum were incubated with a constant amount of glycerol-3-P dehydrogenase from brain (0) and from muscle (0) and the amount of activity remaining was determined as described under Materials and Methods.

antiserum to a constant amount of glycerol-3-P dehydrogenase from brain and from muscle yields the results seen in Fig. 4. The single straight line suggests that an equal number of molecules are inactivated by the antiserum irrespective of whether they are from brain or muscle. Therefore,

FIG. 5. Sephadex G-200 chromatography of glycerol-3-P dehydrogenase from rat skeletal muscle (0) and brain (01. Each of the samples (3.6 ml) was run under identical conditions and the enzyme was assayed as described. The flow rate was 0.34 ml/min, and fractions were collected every 10 min. The elution volumes CV,) for muscle and brain glycerol-3-P dehydrogenase were 326.0 and 324.6 ml, respectively.

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indicate that rat brain and muscle glycerol-3-P dehydrogenases have a molecular weight of 70,000. As a further test of the identity of brain glycerol-3-P dehydrogenase in hypophysectomized and control animals, we measured the ability of the enzyme to survive heating at 50°C. These results (Fig. 7) indicate that, irrespective of the source of enzyme, the decay curves are identical.

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FIG. 6. Determination of the molecular weight of brain and muscle glycerol-3-P dehydrogenase. Column conditions and the molecular weights of the standard proteins are decribed under Materials and Methods. The relative elution volumes (VJVJ of standard proteins (0) and of glycerol-3-P dehydrogenase from brain and muscle (0) are plotted vs the logarithm of the molecular weight.

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FIG. 7. Inactivation of glycerol-3-P dehydrogenase by heating at 50°C. The amount of brain glycerol-3-P dehydrogenase from control (0) and hypophysectomized (0) rata was determined after incubation, as described under Materials and Methods.

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FIG. 8. Heat inactivation of glycerol-3-P dehydrogenase. Equal amounts of glycerol-3-P dehydrogenase from brain (0) and from muscle (0) were heated at 50°C for the indicated times and the amount of activity remaining was determined as described under Materials and Methods.

However, unlike any of the previous techniques, the measurement of this parameter indicates that there are two forms of glycerol-3-P dehydrogenase, one of which decays at approximately twice the rate of the other. Extrapolation of the line representing the more stable form suggests that there is about the same amount (60-65%) of each form in the control and hypophysectomized animals. Therefore, the decrease in specific activity of glycerol-3-P dehydrogenase in the brain of hypophysectomized animals does not represent the preferential loss of one of the two heatlabile forms. A comparison of heat lability of muscle and brain glycerol-3-P dehydrogenase is seen in Fig. 8. These results indicate that the muscle enzyme is identical to the brain enzyme and that it too has more than one heat-labile form. The brain and muscle

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also have similar proportions of each form (about 60% of the glycerol-3-P dehydrogenase is the more stable form in muscle). We also determined the pH optima of glycerol-3-P dehydrogenase from brain and muscle (data not shown) and found that they were both very similar, having a pH optimum between pH 7.3 and 7.6. DISCUSSION

The regulation of the concentration of glycerol-3-P dehydrogenase by glucocorticoids appears to be specific to brain cells (1). We were interested in determining whether the mechanism and specificity of the regulation was due to the enzyme itself or to a unique regulatory system in brain cells. Toward this end, we have compared glycerol-3-P dehydrogenase in the brains of control rats with the glycerol-3-P dehydrogenase remaining following hypophysectomy. Similarly, we have compared the properties of brain glycerol-3-P dehydrogenase with glycerol-3-P dehydrogenase in skeletal muscle - a tissue in which its concentration is not regulated by glucocorticoids (1). The simplest explanation for the existence of an “induced” and “uninduced” level of glycerol-3-P dehydrogenase in rat brain and the lack of glucocorticoid regulation of muscle glycerol-3-P dehydrogenase would be the existence of two structural genes for glycerol-3-P dehydrogenase which code for different isoenzymes and which are regulated by two distinct mechanisms. Brain would have both isoenzymes, one of which would be regulated by glucocorticoids and the other (identical to muscle glycerol-3-P dehydrogenase) would not be subject to hormonal control. In this case, adrenalectomy would result in a decline of one of the brain isoenzymes, but would have no effect on the concentration of the other (“basal level”) or on the muscle glycerol-3-P dehydrogenase. Alternatively, there may simply be one structural gene for glycerol-3-P dehydrogenase, and its transcription is differentially regulated in brain cells and in muscle cells. Our data suggest that the former hypothesis is less likely since we have demonstrated, by a number of criteria, that rat brain glycerol3-P dehydrogenase is identical to rat skele-

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tal muscle glycerol-3-P dehydrogenase. We have similarly demonstrated that the brain glycerol-3-P dehydrogenase remaining after hypophysectomy is the same molecular form as is found in the brain of control animals. The identity of glycerol-3P dehydrogenase from either source, however, is only suggestive, since they may differ with respect to untested properties. By immunotitration, we have also shown that the hormonal control of brain glycerol-3-P dehydrogenase is exerted on the level of enzyme concentration rather than enzyme activity. The decrease in specific activity following hypophysectomy is actually due to fewer glycerol-3-P dehydrogenase molecules, not less active ones. This decrease could be accomplished by either a decrease in the rate of synthesis of glycerol-3-P dehydrogenase or an increase in the rate of degradation. This situation is similar to that found in rat brain glioma cells ((26) in culture, where glucocorticoids regulate the number of glycerol-3-P dehydrogenase molecules, not their catalytic efficiency (14). Further studies have shown (15) that the removal of hydrocortisone from induced C6 cells results in a decrease in the rate of synthesis of glycerol-3-P dehydrogenase. It, therefore, seems likely that the decline in the concentration of glycerol-3-P dehydrogenase in vivo is also due to a decreased rate of synthesis. Experiments are currently underway to determine the rates of turnover of glycerol-3-P dehydrogenase in vivo. The molecular weight of brain and muscle glycerol-3-P dehydrogenase is 70,000, a value which is essentially identical to that found for purified rat brain glycerol-3-P dehydrogenase (7) and slightly higher than the values found for rat muscle (6), liver (131, and adipose tissue (15). The different glycerol-3-P dehydrogenase forms which have been isolated from rat brain (7) and demonstrated in rat muscle and liver (6) have not been shown to be the products of different structural genes and probably represent post-translational modifications of the same primary sequence. However, the embryonic and adult forms of glycerol-3-P dehydrogenase found in brain and muscle of the mouse are apparently coded for by separate struc-

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tural genes (9). Unfortunately, the mouse cannot be used to study the hormonal control of brain glycerol-3-P dehydrogenase because glycerol-3-P dehydrogenase is not regulated in mice by glucocorticoids (McGinnis and de Vellis, unpublished data). One of the most sensitive methods presently available for detecting enzyme variants, without purification of the enzyme, is heat inactivation (16). Our results demonstrate that there are at least two heatlabile forms of glycerol-3-P dehydrogenase present in the brain of control and hypophysectomized rats and that these same two forms are also present in muscle. Since the data fit two straight lines much better than a continuous curve, it is most probable that there are only two rather than three or more molecular species of the enzyme present. This suggests that there are no hybrid molecules and, unlike LDH (2) or aldolase (31, the subunits of the two forms of the L-glycerol 3-phosphate dehydrogenase dimer do not dissociate and reassociate independently. It is possible that these two distinct forms of glycerol-3-P dehydrogenase represent the products of different structural genes, but it seems more probable that the difference arises from a post-translational modification of glycerol-3-P dehydrogenase. For example, one form of rat liver glycerol-3-P dehydrogenase can be generated from the more anionic form simply by incubating the homogenate at 4°C for a few days (13). The most direct evidence supporting the existence of two different structural genes for glycerol-3-P dehydrogenase would be the finding of a variant (electrophoretic, heat stability, etc.) of glycerol-3-P dehydrogenase which affected the enzyme in only one of the tissues. Irrespective of their origin, these forms do not explain the differences in regulation, since the two heat-labile forms are represented in muscle and in the brains of control and experimental animals to the same extent. Our data suggest that differential regulation of the concentration of glycerol-3-P dehydrogenase in brain and muscle tissue is due to different control mechanisms, rather than to different isoenzymes. Since

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brain glycerol-3-P dehydrogenase is specifically regulated by hydrocortisone and since the response of “target cells” to glucocorticoids is dependent on the presence of cytoplasmic hormone receptors, it is possible that skeletal muscle does not have any glucocorticoid receptors, has different receptors which do not affect the transcription of glycerol-3-P dehydrogenase mRNA, has an additional control mechanism superimposed, or is entirely regulated by other factors. Studies designed to examine these possibilities are currently underway. ACKNOWLEDGMENTS We thank assistance.

Deborah

Farson

for

expert

technical

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5. WHITE, H. B., AND KAPLAN, N. 0. (1969) J. Biol. Chem. 244, 6031-6039. 6. FONDY, T. P., SOLOMON, J., AND Ross, C. R. (1971) Arch. Biochem. Biophys. 145, 604-611. 7. MCGINNIS, J. F., AND DE VELLIS, J. (1974) Biochim. Biophys. Acta 364, 17-27. 8. LEE, Y. P., AND CHOY, P. C. C. (1974) J. Biol. Chem. 249, 476-481. 9. KOZAK, L. P., AND JENSEN, J. T. (1974) J. Biol. Chem. 249, 7775-7761. 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 11. DAVIS, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404-427. 12. MCGINNIS, J. F., AND DE VELLIS, J. (1974) Biothem. Biophys. Res. Commun. 60, 186-195. 13. Ross, C. R., CURRY, S., SCHWARTZ, A. W., AND FONDY, T. P. (1971) Arch. Biochem. Biophys. 145, 591-603. 14. MCGINNIS, J. F., AND DE VELLIS, J. (1974) Nature (London), 250, 422-424. 15. MCGINNIS, J. F., AND DE VELLIS, J. (1976) Fed. Proc. 35, 1636. D. L., AND FONDY, T. P. (1973) Eur. 16. WARKENTIN, J. Biochem. 36, 97-109. 17. PAIGEN, K. P. (1971) in Enzyme Synthesis and Degradation in Mammalian Systems (Rechcigl, M., ed.), pp. l-49, Karger, Basel.