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PubMed1985

Relationship between activation state of pyruvate dehydrogenase complex and rate of pyruvate oxidation in isolated cerebro-cortical mitochondria: effects of potassium ions and adenine nucleotides.

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.

Acetyltransferases↗

PubMed1983

Modulation of pyruvate kinase phosphatase activity in hepatocyte extracts by pyruvate kinase-L ligands.

Hepatic pyruvate kinase phosphatase activity has been assayed in native conditions, in Sephadex G-25 filtered extracts of rat hepatocytes, by measuring the reactivation rate of glucagon-inactivated pyruvate kinase-L. The ionic requirements for this reaction, as well as the possible regulatory role of some pyruvate kinase ligands, have been investigated. Pyruvate kinase phosphatase activity was dependent on divalent cations (Mg2+, Mn2+ or Co2+). Mg2+ ions highly enhanced the reactivation rate of pyruvate kinase, while the presence of 100 mM KF inhibited this process. Physiological concentrations of phosphoenolpyruvate or fructose 1,6-bisphosphate inhibited pyruvate kinase phosphatase activity. These inhibitory effects were partially antagonized by the presence of L-alanine. Our results suggest that ligands of pyruvate kinase could play a role in the control of pyruvate kinase phosphatase activity(ies), possibly by modifying the conformational state of the substrate protein.

Animals↗

PubMed1997

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase.

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90 degrees C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80 degrees C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

Aldehyde Oxidoreductases↗

PubMed1994

Structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate.

The molecular structure of rabbit muscle pyruvate kinase, crystallized as a complex with Mn2+, K+, and pyruvate, has been solved to 2.9-A resolution. Crystals employed in the investigation belonged to the space group P1 and had unit cell dimensions a = 83.6 A, b = 109.9 A, c = 146.8 A, alpha = 94.9 degrees, beta = 93.6 degrees, and gamma = 112.3 degrees. There were two tetramers in the asymmetric unit. The structure was solved by molecular replacement, using as the search model the coordinates of the tetramer of pyruvate kinase from cat muscle [Muirhead, H., Claydon, D. A., Barford, D., Lorimer, C. G., Fothergill-Gilmore, L. A., Schiltz, E., & Schmitt, W. (1986) EMBO J.5, 475-481]. The amino acid sequence derived from the cDNA coding for the enzyme from rabbit muscle was fit to the electron density. The rabbit and cat muscle enzymes have approximately 94% sequence identity, and the folding patterns are expected to be nearly identical. There are, however, three regions where the topological models of the cat and rabbit pyruvate kinases differ. Mn2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. These two residues are strictly conserved in all known pyruvate kinases. In addition, the density for Mn2+ is connected to that of pyruvate, consistent with chelation through a carboxylate oxygen and the carbonyl oxygen of the substrate. The epsilon-NH2 of Lys 269 and the OH of Thr 327 lie on either side of the methyl group of bound pyruvate. Spherical electron density, assigned to K+, is located within a well-defined pocket of four oxygen ligands contributed by the carbonyl oxygen of Thr 113, O gamma of Ser 76, O delta 1 of Asn 74, and O delta 2 of Asp 112. The interaction of Asp 112 with the side chains of Lys 269 and Arg 72 may mediate, indirectly, monovalent cation effects on activity.

Amino Acid Sequence↗

PubMed2002

Time-dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1-(13) C]glucose metabolism.

[1-(13) C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono- and dicarboxylic amino acids were determined from (1) H-observed/(13) C-edited and (13) C-NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate-glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re-evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.

Amino Acids↗

PubMed1981

Phosphorylation-mediated changes in pyruvate dehydrogenase activity influence pyruvate-supported calcium accumulation by brain mitochondria.

Changes in the activity of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1, PDH], elicited by inhibition of the phosphorylation of its 40,000 Mr alpha-subunit, were compared with changes in pyruvate-supported calcium accumulation by rat brain mitochondria. Dichloroacetate (DCA) produces concentration-dependent inhibition of the phosphorylation of intramitochondrial PDH alpha-subunit, which is accompanied by stimulation of PDH activity and calcium accumulation. DCA did not affect succinate- or ATP-supported mitochondrial calcium accumulation. The concentration of DCA giving half-maximal inhibition of the phosphorylation was almost identical to that giving half-maximal stimulation of PDH activity and calcium accumulation. PDH activity and pyruvate-supported calcium accumulation showed similar dependence on pyruvate concentration with respective apparent affinities for pyruvate of 40 microM and 30 microM, and both activities exhibited positive cooperativity. DCA modified only the maximal activity of PDH or the maximal calcium DCA modified only the maximal activity of PDH or the maximal calcium accumulation without changing either the apparent affinities for pyruvate or calcium or the Hill coefficients. These data provide evidence that calcium accumulation by mitochondria is tightly linked to PDH activity and that changes in the phosphorylation of the PDH alpha-subunit can be reflected in changes in the calcium-buffering ability of mitochondria. This suggests a possible mechanism by which a variety of manipulations, such as repetitive synaptic stimulation, can alter the regulation of internal calcium levels.

Adenosine Triphosphate↗

PubMed2004

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production.

AIMS: To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. METHODS AND RESULTS: Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. CONCLUSIONS: The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. SIGNIFICANCE AND IMPACT OF THE STUDY: Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.

Acetates↗

PubMed1986

Utilization of pyruvate and pyruvate precursors by normal and carcinogen-altered rat tracheal epithelial cells in culture.

The metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen-altered cells derived from dimethylbenz(a)anthracene-exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate-supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three- to four-fold higher incorporation of radioactivity from [2-14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U-14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two-fold greater glucose consumption and two-fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two-fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U-14C]glutamine or [U-14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2 was distinctly different between NPC and SPC except for the 1.7-fold higher utilization of [1-14C]glucose along the oxidative arm of the pentose cycle in the normal cells.

Alanine↗

PubMed1987

Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

Acetoin↗

PubMed1996

Pyruvate inhibition of pyruvate dehydrogenase kinase. Effects of progressive starvation and hyperthyroidism in vivo, and of dibutyryl cyclic AMP and fatty acids in cultured cardiac myocytes.

Both prolonged starvation and hyperthyroidism evoke stable increases in cardiac pyruvate dehydrogenase kinase (PDHK) activity. Pyruvate inhibits PDHK in rat heart mitochondria with activation of PDHC. The sensitivity of PDHK to inhibition by pyruvate declines after prolonged starvation. In the present study, pyruvate concentrations giving 50% active complex (PDHa) in mitochondria from fed, control and fed, hyperthyroid rats were 0.3 and 0.8 mM, respectively, compared with 1.0 and 2.8 mM, respectively in mitochondria from 24-h-starved and 48-h-starved rats. The results demonstrate that altered pyruvate sensitivity is not of necessity linked with altered PDHK activity. PDHK activities in mitochondria prepared from cardiac myocytes from fed rats were increased after culture for 24 h with dibutyryl cyclic AMP (50 microM) plus n-octanoate (1 mM), with a concomitant decline in sensitivity of PDHK to pyruvate inhibition, suggesting that changes in sensitivity of PDHK to pyruvate inhibition in vivo may be secondary to increased fatty acid supply and cyclic AMP concentrations.

Animals↗

PubMed1980

Effect of streptozotocin-induced diabetes mellitus on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques were used to study the effect of streptozotocin-induced diabetes on the amounts of pyruvate carboxylase and pyruvate dehydrogenase and on their rates of synthesis and degradation. Livers from diabetic rats had twice the pyruvate carboxylase activity of livers from normal rats when expressed in terms of DNA or body weight. The changes in catalytic activity closely paralleled changes in immunoprecipitable enzyme protein. Relative rates of synthesis determined by pulse-labelling studies showed that the ratio of synthesis of pyruvate carboxylase to that of average mitochondrial protein was increased 2.0-2.5 times in diabetic animals over that of control animals. Other radioisotopic studies indicated that the rate of degradation of this enzyme was not altered significantly in diabetic rats, suggesting that the increase in this enzyme was due to an increased rate of synthesis. Similar experiments with pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex, showed that livers from diabetic rats had approximately the same amount of immunoprecipitable enzyme protein as the control animals, but a larger proportion of the enzyme was in its inactive state. The rates of synthesis and degradation of pyruvate dehydrogenase were not affected significantly by diabetes.

Animals↗

PubMed1993

Metabolism of pyruvate by pre-elongation sheep embryos and effect of pyruvate and lactate concentrations during culture in vitro.

In the first of two experiments, utilization of [1-14C]pyruvate by 8-cell and blastocyst-stage embryos derived in vivo was examined during a 3-h incubation in HEPES-buffered synthetic oviduct fluid (SOF) medium in the presence or absence of other substrates. In the second, a factorial design examined the effect of pyruvate (0, 0.33, 1.0 and 3.3 mM) and lactate (3.3, 10 and 33 mM) on development of 1- and 2-cell sheep embryos cultured in vitro in a modified SOF medium (containing glucose, glutamine and modified Eagle's medium non-essential amino acids). Peak utilization of [1-14C]pyruvate was unaffected by the presence or absence of other energy substrates. In contrast, rate of utilization was affected by the addition of other energy substrates, with half maximal utilization occurring at either 0.4 +/- 0.2 mM or 1.2 +/- 0.2 mM for 8-cells and either 0.2 +/- 0.2 mM or 1.3 +/- 0.3 mM for blastocysts when incubated in the absence or presence of other energy substrates respectively. In the second experiment the proportion of embryos developing to blastocysts was inhibited by high lactate levels (P < 0.001), but was generally not affected by pyruvate concentration. However, there was a significant interaction (P < 0.001) between pyruvate and lactate when both were present in the medium. At 0.33 mM pyruvate, 3.3 mM lactate supported good development (83 +/- 8% blastocysts) whereas 10 mM lactate supported less development (50 +/- 11%). However, at the higher levels of pyruvate this effect was lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

beta-Cell adaptation to insulin resistance. Increased pyruvate carboxylase and malate-pyruvate shuttle activity in islets of nondiabetic Zucker fatty rats.

The beta-cell biochemical mechanisms that account for the compensatory hyperfunction with insulin resistance (so-called beta-cell adaptation) are unknown. We investigated glucose metabolism in isolated islets from 10-12-week-old Zucker fatty (ZF) and Zucker lean (ZL) rats (results expressed per mg/islet of protein). ZF rats were obese, hyperlipidemic, and normoglycemic. They had a 3.8-fold increased beta-cell mass along with 3-10-fold increases in insulin secretion to various stimuli during pancreas perfusion despite insulin content per milligram of beta-cells being only one-third that of ZL rats. Islet glucose metabolism (utilization and oxidation) was 1.5-2-fold increased in the ZF islets despite pyruvate dehydrogenase activity being 30% lowered compared with the ZL islets. The reason was increased flux through pyruvate carboxylase (PC) and the malate-pyruvate and citrate-pyruvate shuttles based on the following observations (% ZL islets): increased V(max) of PC (160%), malate dehydrogenase (170%), and malic enzyme (275%); elevated concentrations of oxaloacetate (150%), malate (250%), citrate (140%), and pyruvate (250%); and 2-fold increased release of malate from isolated mitochondria. Inhibition of PC by 5 mm phenylacetic acid markedly lowered glucose-induced insulin secretion in ZF and ZL islets. Thus, our results suggest that PC and the pyruvate shuttles are increased in ZF islets, and this accounts for glucose mitochondrial metabolism being increased when pyruvate dehydrogenase activity is reduced. As the anaplerosis pathways are implicated in glucose-induced insulin secretion and the synthesis of glucose-derived lipid and amino acids, our results highlight the potential importance of PC and the anaplerosis pathways in the enhanced insulin secretion and beta-cell growth that characterize beta-cell adaptation to insulin resistance.

Animals↗

PubMed2004

Muscle pyruvate availability can limit the flux, but not activation, of the pyruvate dehydrogenase complex during submaximal exercise in humans.

While lowering muscle glycogen availability to an extent that would reduce muscle pyruvate formation during intense exercise, we investigated the importance of muscle pyruvate availability to pyruvate dehydrogenase complex (PDC) activation during intense exercise in human skeletal muscle. The present study demonstrated that regardless of whether pre-exercise muscle glycogen content was at a habitual resting concentration (412 +/- 30 mmol (kg dry muscle)(-1)) or depleted (60 +/- 3 mmol (kg dry muscle)(-1)), the increase in PDC activation from its resting value (5.46 +/- 0.96 and 3.67 +/- 0.34 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively) during 10 min of exercise at 75% of the maximum rate of oxygen consumption (VO2max) (delta12.82 +/- 1.72 and delta13.24 +/- 1.42 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively) was the same, despite pyruvate accumulation during exercise being 3-fold lower in the glycogen depleted state (delta0.34 +/- 0.04 and delta0.11 +/- 0.06 mmol (kg dry muscle)(-1), P < 0.001). However, as a result of the reduction in pyruvate availability, calculated flux through the PDC reaction was at least 2-fold lower in the glycogen depleted state compared with normal (21.81 +/- 2.62 and 9.41 +/- 0.63 nmol acetyl-CoA min(-1) (mg protein)(-1), respectively; P < 0.001). It is therefore pertinent to conclude that whilst muscle pyruvate availability appears to be important to the rate of flux through the PDC reaction during in vivo contraction, it is not of primary importance to the control of PDC activation under these conditions, which is probably principally regulated by muscle calcium availability. The proposed central role of pyruvate in muscle PDC activation during in vivo contraction may therefore have been over stated.

Adult↗

PubMed1996

Pyruvate and hepatic pyruvate dehydrogenase levels in rat strains sensitive and resistant to dietary obesity.

This study compared the effects of exogenous pyruvate and lactate on the serum levels of pyruvate, lactate, glucose, alanine, and insulin, as well as the activity of hepatic pyruvate dehydrogenase (PDH) in strains of rat that were either sensitive [Osborne-Mendel (OM)] or resistant (S5B/Pl) to high-fat diet-induced obesity. Serum pyruvate and lactate were significantly higher and glucose lower in ad libitum-fed OM rats, but these differences disappeared after an 18-h fast. The increase in pyruvate and lactate after exogenous pyruvate administration was significantly greater in S5B/Pl rats than OM rats. There were no differences in serum alanine with strain or diet. The total PDH activity was similar across strains and diets but the proportion of PDH in its activated form (PDHa) was decreased in ad libitum-fed S5B/Pl rats. Pyruvate injection increased insulin and hepatic PDHa activity in OM rats fed both high- and low-fat diets, but these responses were greatly attenuated or absent in S5B/Pl rats. The data are consistent with the hypothesis that modulation of carbohydrate oxidation by PDH may be related to susceptibility to obesity when rats are fed a high-fat diet.

Animals↗

PubMed1984

Friedreich's ataxia: intravenous pyruvate load to demonstrate a defect in pyruvate metabolism.

Nine patients with typical Friedreich's ataxia had normal levels of pyruvate and lactate in blood, urine, and CSF. Glucose, pyruvate, and lactate levels were also within the reference ranges after oral glucose loading. After intravenous pyruvate loading, the relative increase in blood pyruvate and lactate levels was significantly higher in Friedreich's ataxia patients than in controls. Significant differences between the two groups were also observed in lactate-pyruvate ratios at various times after infusion. The pyruvate loading test can be useful in demonstrating a defect in pyruvate metabolism in Friedreich's ataxia patients.

Adolescent↗

PubMed1999

Regulation of pyruvate dehydrogenase complex activity in Ehrlich ascites carcinoma cells by Ca2+ and pyruvate.

The dependence of pyruvate dehydrogenase complex (PDC) activity on [Ca2+] was determined in Ehrlich ascites carcinoma cells at different pyruvate concentrations. The resulting family of curves had the following characteristics: a) bell-shaped appearance of all curves with maximum activity at 600 nM Ca2+; b) unchanged position of maxima with changes in pyruvate concentration; c) nonmonotonous changes in PDC activity with increasing pyruvate concentration at fixed [Ca2+]. Feasible mechanisms involving Ca2+-dependent phosphatase and kinase which are consistent with the experimental findings are discussed. To determine the steps in the chain of PDC reactions which determine the observed phenomena, a mathematical model is suggested which is based on the known data on the structural--functional relationships between the complex components--pyruvate dehydrogenase (E1), dihydrolipoyl acetyl transferase (E2), dihydrolipoyl dehydrogenase (E3), protein X, kinase, and phosphatase. To adequately describe the non-trivial dependence of PDC activity on [Ca2+] at different pyruvate concentrations, it was also necessary to consider the interdependence of some steps in the general chain of PDC reactions. Phenomenon (a) is shown to be due only to the involvement of protein X in the PDC reactions, phenomenon (b) to be due to changes in the activity of kinase, and phenomenon (c) to be due to dependence of acetylation and transacetylation rates on pyruvate concentration.

Animals↗

PubMedDate not supplied

Effects of pyruvate on pyruvate dehydrogenase kinase of rat heart.

Sensitivity of rat heart pyruvate dehydrogenase kinase (PDHK) to pyruvate inhibition was tested under various conditions using pyruvate dehydrogenase complex (PDC) in mitochondria (mPDC) and in a high speed precipitate of whole tissue homogenates (hPDC). In the latter preparation pyruvate in the range of concentration 1-10 mM caused increasing inhibition of PDHK when the enzyme was prepared from animals fed ad libitum but had no effect when the enzyme was prepared from 48 h starved animals. Similar behaviour was observed in mPDC from fed and starved animals when rotenone was present, pyruvate at 1 mM concentration stimulated PDHK from hearts of fed animals but was without effect at 10 mM. When mPDC or hPDC from hearts of starved animals was incubated at 30 degrees C for 30 min, inhibition of PDHK by pyruvate was restored.

Animals↗