Characteristics of thiamine thiazolone diphosphate-induced inhibition of a pyruvate dehydrogenase complex in vitro and in intact mitochondria

Thiamine thiazolone diphosphate (TTPP) was capable of penetrating through the mitochondrial membrane and of inhibiting the pyruvate dehydrogenase complex (PDC) in intact mitochondria. TTPP depressed the activity of mammalian PDC in a mixed manner (Ki = 5.10(-8) M) and yeast pyruvate decarboxylase (Ki = 5.10(-6) M) via a competitive mechanism with respect to thiamine diphosphate. It was shown that decarboxylation of pyruvate in intact and disrupted mitochondria of rat liver and brain is less inhibited by TTPP than the overall activity of PDC determined by the formation of acetyl-CoA. It was assumed that TTPP as a transition state analog participates only in oxidative reactions (but not in simple decarboxylation of pyruvate).     

Prostaglandins and the rat seminal vesicle: effects of mating and adrenergic stimulation

The concentrations of PGE, PGF, and 6-keto-PGF1 alpha were increased in rat seminal vesicle tissue following mating activity. Likewise, synthesis of PGE and PGF was stimulated by epinephrine (3 X 10(-7) to 3 X 10(-6) M) in tissues and media from in vitro incubations of intact rat seminal vesicles. The in vitro stimulation was inhibited by phentolamine, an alpha-adrenoreceptor blocking agent. Carbamylcholine (2 X 10(-6) M) and bradykinin (1 X 10(-6) M) had no effect on PGE or PGF synthesis, even though both compounds stimulated contractility of the rat seminal vesicle at these concentrations. These data suggest that mating and adrenergic stimulation increase prostaglandin synthesis in the rat seminal vesicle, probably through an alpha-adrenergically mediated mechanism.     

Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.

Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.     

Role of pyruvate in enhancing pyruvate decarboxylase stability towards benzaldehyde

Biotransformation of benzaldehyde and pyruvate into (R)-phenylacetylcarbinol (PAC) catalysed by Candida utilis pyruvate decarboxylase (PDC) at low buffer concentration (20 mM MOPS) was enhanced by maintenance of neutral pH through acetic acid addition. PDC was very stable in this buffer (half-life 138 h at 6 degrees C), however a benzaldehyde emulsion (400 mM) caused rapid deactivation. The inclusion of 2M glycerol did not protect PDC from inactivation by benzaldehyde but initial rates were increased by 50% and the final PAC level was enhanced from 40 to 51 g l(-1). Low levels of by-products acetaldehyde (0.1-0.15 g l(-1)) and acetoin (1.1-1.3 g l(-1)) were formed in both the presence and absence of 2 M glycerol. Interestingly PDC was more stable towards benzaldehyde when pyruvate was present: no activity was lost during the first hour of biotransformation (2 M glycerol, benzaldehyde concentration decreased from 400 to 345 mM, pyruvate from 480 to 420 mM) but PDC was completely inactivated in less than 30 min when exposed to the same concentrations of benzaldehyde in the absence of pyruvate. Thus the enzyme in catalytic action was more stable than the resting enzyme.     

二氢硫辛酰转乙酰基酶通过乙酰化磷酸葡糖酸脱氢酶促进核酸合成

丙酮酸脱氢酶复合物(pyruvate dehydrogenase complex,PDC)是位于线粒体内的多酶复合物,催化丙酮酸不可逆地氧化脱羧转为乙酰辅酶A,二氢硫辛酰转乙酰基酶(dihydrolipoyl acetyltransferase,DLAT)是PDC的1个亚基.PDC在细胞线粒体呼吸中发挥关键作用.但是D...     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.     

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.     

Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.     

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.     

Calcitonin increases pyruvate carboxylase activity in hepatic mitochondria of rats

The effect of calcitonin (CT) on calcium content and enzyme activity in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant increase in the content of calcium, the activity of pyruvate carboxylase, succinate dehydrogenase and ATPase 15 min after the hormone treatment. The significant increases in calcium content and pyruvate carboxylase activity were also observed 30 min after CT administration, while succinate dehydrogenase and ATPase activity began to decrease. A physiological dose of CT (20 MRC mU/100 g BW) caused a marked increase in calcium content and pyruvate carboxylase activity but not succinate dehydrogenase of ATPase-activity. The removal of calcium by 10 mM EGTA washing of the mitochondria produced a remarkable reduction in pyruvate carboxylase activity increased by CT administration. The addition of calcium ion of 2.5 x 10(-2) - 2.5 x 10(1) nmoles Ca2+ per mg mitochondrial protein produced a marked increase in pyruvate carboxylase activity. The present results suggest that calcium taken up by the hepatic mitochondria after CT administration activates pyruvate carboxylase.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.     

Regulation of hepatic glycine catabolism by glucagon

Glucagon stimulates 14CO2 production from [1-14C] glycine by isolated rat hepatocytes. Maximal stimulation (70%) of decarboxylation of glycine by hepatocytes was achieved when the concentration of glucagon in the medium reached 10 nM; half-maximal stimulation occurred at a concentration of about 2 nM. A lag period of 10 min was observed before the stimulation could be measured. Inclusion of beta-hydroxybutyrate (10 mM) or acetoacetate (10 mM) did not affect the magnitude of stimulation suggesting that the effects of glucagon were independent of mitochondrial redox state. Glucagon did not affect either the concentration or specific activity of intracellular glycine, thus excluding the possibilities that altered concentration or specific activity of intracellular glycine contributes to the observed stimulation. The stimulation of decarboxylation of glycine by glucagon was further studied by monitoring 14CO2 production from [1-14C]glycine by mitochondria isolated from rats previously injected with glucagon. Glycine decarboxylation was significantly stimulated in the mitochondria isolated from the glucagon-injected rats. We suggest that glucagon is a major regulator of hepatic glycine metabolism through the glycine cleavage enzyme system and may be responsible for the increased hepatic glycine removal observed in animals fed high-protein diets.     

Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase

Norepinephrine and epinephrine, in the presence of the beta-adrenergic antagonist propranolol (10(-5) M), stimulated adipocyte pyruvate dehydrogenase at low concentrations but inhibited the enzyme at higher concentrations. The alpha-adrenergic agonist, phenylephrine, rapidly stimulated pyruvate dehydrogenase activity in a dose-dependent manner with maximal stimulation observed at 10(-6) M. The stimulation of pyruvate dehydrogenase by phenylephrine was mediated via alpha 1-receptors. Inhibition of pyruvate dehydrogenase by catecholamines was mediated via beta-adrenergic receptors, since the beta-agonist, isoproterenol, and dibutyryl cAMP produced similar effects. Like insulin, alpha-adrenergic agonists increased the active form of pyruvate dehydrogenase without changing the total enzyme activity and cellular ATP concentration. The effects induced by maximally effective concentrations of insulin and alpha-adrenergic agonists were nonadditive. The ability of phenylephrine and methoxamine to stimulate pyruvate dehydrogenase and phosphorylase and to inhibit glycogen synthase was not affected by the removal of extracellular Ca2+. Similarly, the stimulation of pyruvate dehydrogenase and glycogen synthase by insulin was also observed under the same conditions. However, when intracellular adipocyte Ca2+ was depleted by incubating cells in a Ca2+-free buffer containing 1 mM ethylene glycol bis(beta-amino-ethyl ether)-N,N,N' -tetraacetic acid, the actions of alpha-adrenergic agonists, but not insulin, on pyruvate dehydrogenase were completely abolished. Vasopressin and angiotensin II also stimulated pyruvate dehydrogenase in a dose-dependent manner with enhancement of glucose oxidation and lipogenesis. Our results demonstrate that the Ca2+ -dependent hormones stimulate pyruvate dehydrogenase and lipogenesis in isolated rat adipocytes, and the action is dependent upon intracellular, but not extracellular, Ca2+.     

Stimulatory effect of ADP, ATP, NAD(P) on pyruvate production from malate by uncoupled human placental mitochondria

It has been shown that ADP, ATP, NAD(P), and NAD(P)H significantly stimulate pyruvate production from malate by intact uncoupled human term placental mitochondria. No stimulation by ADP was observed when mitochondria were incubated in the presence of NAD(P) or NAD(P)H or when mitochondrial membrane had been disrupted. Atractyloside and oligomycin were without effect on ADP- and ATP-stimulated pyruvate production. Other dinucleotides tested such as GDP, UDP, and CDP, stimulated pyruvate production only slightly when mitochondria were incubated in the absence of phosphate. The rate of pyruvate production by intact mitochondria is commensurate with partly purified NAD(P)-linked malic enzyme activity as measured by NAD(P) reduction as far as the effects of pH of hydroxymalonate on these both processes is concerned. It is concluded that pyruvate production by intact human placental mitochondria is catalyzed by NAD(P)-linked malic enzyme and that this process is stimulated by ADP and ATP.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Enzymatic (R)-phenylacetylcarbinol production in benzaldehyde emulsions

(R)-Phenylacetylcarbinol [(R)-PAC)] is the chiral precursor for the production of the pharmaceuticals ephedrine and pseudoephedrine. Reaction conditions were improved to achieve increased (R)-PAC levels in a simple batch biotransformation of benzaldehyde emulsions and pyruvate, using partially purified pyruvate decarboxylase (PDC) from the filamentous fungus Rhizopus javanicus NRRL 13161 as the catalyst. Lowering the temperature from 23 degrees C to 6 degrees C decreased initial rates but increased final (R)-PAC concentrations. Addition of ethanol, which increases benzaldehyde solubility, was not beneficial for (R)-PAC production. It was established that proton uptake during biotransformation increases the pH above 7 thereby limiting (R)-PAC production. For small-scale studies, biotransformations were buffered with 2-2.5 M MOPS (initial pH 6.5). High concentrations of MOPS as well as some alcohols and KCl stabilised PDC. A balance between PDC and substrate concentrations was determined with regards to ( R)-PAC production and yields on enzyme and substrates. R. javanicus PDC (7.4 U/ml) produced 50.6 g/l (337 mM) ( R)-PAC in 29 h at 6 degrees C with initial 400 mM benzaldehyde and 600 mM pyruvate. Molar yields on consumed benzaldehyde and pyruvate were 97% and 59%, respectively, with 17% pyruvate degraded and 24% converted into acetaldehyde and acetoin; 43% PDC activity remained, indicating reasonable enzyme stability at high substrate and product concentrations.     

The regulation of pyruvate oxidation during membrane depolarization of rat brain synaptosomes.

Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.