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Showing papers on "Pyruvate dehydrogenase kinase published in 1983"



Journal ArticleDOI
TL;DR: Reperfusion resulted in a rapid increase in mitochondrial ATP/ADP ratio and the increased availability of ATP as substrate for the kinase coupled with continued high levels of NADH and acetyl CoA which stimulate kinase activity may have accounted for the early inactivation of PDH with reperfusion.

107 citations


Journal ArticleDOI
TL;DR: Control of gluconeogenesis from lactate was studied by titrating rat liver cells with lactate and pyruvate in a ratio of 10:1 in a perifusion system and it can be concluded that pyruVate carboxylase limits maximal gluconeogenic flux.

95 citations


Journal ArticleDOI
TL;DR: The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.

94 citations


Journal ArticleDOI
TL;DR: Observations of pyruvate dehydrogenase kinase and the results of peptide mapping indicate that the two subunits are distinctly different proteins, and it is proposed that the beta subunit is a regulatory subunit.

83 citations


Journal ArticleDOI
TL;DR: The increase in active PDH with higher levels of cardia work was associated most closely with reduced mitochondrial NADH/NAD ratios and with decreased acetyl CoA/CoA ratios when insulin or pyruvate were present.

82 citations


Journal ArticleDOI
TL;DR: It was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability.
Abstract: The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.

71 citations


Journal ArticleDOI
TL;DR: A more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken, finding that the results can be reconciled with a metabolic model containing two cytOSolic functional pyruVate pools.
Abstract: 1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.

69 citations


Journal ArticleDOI
TL;DR: Pyruvate (2 to 60 mM), acting alone and in conjunction with insulin and epidermal growth factor (EGF), enhances DNA synthesis in primary monolayer cultures of adult rat liver parenchymal cells.
Abstract: Pyruvate (2 to 60 mM), acting alone and in conjunction with insulin and epidermal growth factor (EGF), enhances DNA synthesis in primary monolayer cultures of adult rat liver parenchymal cells. Lactate can replace pyruvate in stimulating DNA synthesis. Several other intermediary metabolites (oxaloacetate, α-ketoglutarate, α-ketobutyrate, succinate, fumarate, and malate), though less potent than pyruvate and lactate, also elevate DNA synthesis, whereas alanine at similar concentrations is inhibitory.

65 citations


Journal Article
TL;DR: The results document a decreased activity of the pyruvate translocator in tumor mitochondria which seems to be correlated with the growth rate of the tumor cells.
Abstract: A comparative study of the transport of pyruvate in mitochondria isolated from normal rat liver and from three tumors has been carried out. The Km for net pyruvate uptake in mitochondria isolated from Ehrlich ascites tumor cells is practically equal to that measured in normal rat liver mitochondria while, on the other hand, it is higher in Morris hepatomas 44 and 3924A. The Vmax of pyruvate uptake is depressed in all three types of tumor mitochondria as compared to that in the rat liver mitochondria, with the depression being higher in Morris hepatoma 3924A mitochondria. The lower activity of pyruvate translocator in mitochondria isolated from tumor cells as compared to that in rat liver mitochondria is also shown by depression of the rate of pyruvate-supported oxygen uptake. The results document a decreased activity of the pyruvate translocator in tumor mitochondria which seems to be correlated with the growth rate of the tumor cells.

55 citations


Journal ArticleDOI
TL;DR: The changes in metabolite concentrations suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.
Abstract: Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.

Journal ArticleDOI
TL;DR: In isolated rat hepatocytes phenylephrine promotes a rapid increase in the amount of pyruvate dehydrogenase present in its active form (PDHa), which could be explained by an increase in mitochondrial free Ca2+.

Journal ArticleDOI
TL;DR: The shared subunit was actively regulated to accommodate its demand in both enzymes under most growth conditions, and Lipoamide dehydrogenase, the subunit shared by the two complexes, was found to be in significant excess of its stoichiometric demand in the two enzymes.
Abstract: The oxidative decarboxylations of pyruvate and 2-oxoglutarate in Escherichia coli are carried out by two large, multienzyme complexes: pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The enzyme complexes each contain three subunits: two are unique to the individual complexes, the third is shared between them. Resolution of the polypeptide subunits on two-dimensional gels allowed quantitative analysis of their cellular levels and patterns of synthesis in growing cells. Cells growing in glucose-salts medium were found to contain roughly 85 to 136 pyruvate dehydrogenase complexes and 73 2-oxoglutarate complexes. Lipoamide dehydrogenase, the subunit shared by the two complexes, was found to be in significant excess of its stoichiometric demand in the two enzyme complexes under most growth conditions. The subunits unique to each of the complexes were coordinately regulated over a wide variety of growth conditions and a broad range of expression. The two complexes responded to different, but partially overlapping, regulatory signals. Most importantly, the shared subunit was actively regulated to accommodate its demand in both enzymes. These results are discussed with regard to possible mechanisms of regulation of the enzyme complexes in general and of the shared subunit specifically.

Journal ArticleDOI
TL;DR: Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.

Journal ArticleDOI
TL;DR: It is concluded that the decreased flux through pyruvate dehydrogenase during t-butyl hydroperoxide metabolism results from oxidation of critical thiol group(s) of the enzyme complex consequential to a decrease in mitochondrial GSH/GSSG.
Abstract: Addition of t-butyl hydroperoxide to isolated perfused rat liver leads to a decreased flux through pyruvate dehydrogenase, shown by a decreased 14CO2 release from [1-14C]pyruvate. The effect is observed at rates of infusion of t-butyl hydroperoxide exceeding 0.7 mumol per min per g liver in normal livers and at significantly lower rates in glutathione-depleted livers. The effect is absent in livers from Se-deficient rats in which the hepatic Se-dependent glutathione peroxidase activity is very low, indicating that reduction of t-butyl hydroperoxide by glutathione peroxidase is a necessary prerequisite for the inhibition. With isolated mitochondria, decreased 14CO2 release from [1-14C]pyruvate during t-butyl hydroperoxide metabolism correlates with decreased GSH and increased GSSG contents, respectively. The addition of various disulfide compounds, including GSSG, inhibits activity of the enzyme in mitochondrial extracts. In both mitochondria and perfused liver, t-butyl hydroperoxide-mediated decrease of pyruvate dehydrogenase flux is relieved by thiol reductants. The active (dephospho)form of pyruvate dehydrogenase as measured in freeze-stopped liver samples is actually increased from 46% to 72% during t-butyl hydroperoxide metabolism. The tissue levels of ATP and ADP and perfusate beta-hydroxybutyrate/acetoacetate ratio are not markedly perturbed by addition of the hydroperoxide (10 min). It is concluded that the decreased flux through pyruvate dehydrogenase during t-butyl hydroperoxide metabolism results from oxidation of critical thiol group(s) of the enzyme complex consequential to a decrease in mitochondrial GSH/GSSG.

Journal ArticleDOI
TL;DR: During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated, indicating that the phosphate originated from the beta-position of ADP as indicated by the labelling of the enzymes during inactivation in the presence of [beta-32P]ADP.

Journal ArticleDOI
TL;DR: The observation that isobutyryl residues are incorporated in the enzyme during the simultaneous oxidation of both of these substrates seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase sub unit.

Journal ArticleDOI
TL;DR: Experiments with active enzyme gel chromatography indicate that citrate synthase also associates with pyruvate dehydrogenase complex in its functioning state, raising the possibility of the dynamic compartmentation of acetyl-CoA in the mitochondria which results in the direction of acetylene from pyruVate towards citrate.

Journal ArticleDOI
TL;DR: The presence of a highly symmetrical, eight-stranded α/β-barrel structure in several unrelated enzymes seems to have significance for substrate binding, which may indicate whether these homologies reflect convergent or divergent evolution.

Journal ArticleDOI
TL;DR: PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K‐phosphate, which support the validity of the measurements of the activity of this enzyme in brain homogenates.
Abstract: The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.

Journal ArticleDOI
TL;DR: A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydration complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes during differentiation.
Abstract: The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation.

Journal ArticleDOI
TL;DR: It is contention that the inactivation of pyruvate dehydrogenase complex at low propionate levels may be due to an increase in the mitochondrial acyl-CoA/CoASH ratios, whereas the activation of the enzyme complex demonstrated at high propionATE levels is due to the inhibition of the pyruVate dehydrogensase kinase in a manner similar to that caused by pyruviate or dichloroacetic acid.

Journal ArticleDOI
TL;DR: Radiolabeling pyruvate carboxylase with [14C]biotin and [3H]leucine demonstrated that the turnover of biotin associated with the enzyme was identical to that of the enzymatic protein.

Journal ArticleDOI
TL;DR: The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenases complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation, and the phosphatase reaction is activated by Ca2+ and possibly by uncharacterized factors mediating insulin action in adipocytes.
Abstract: The pyruvate dehydrogenase and branched-chain 2-oxoacid dehydrogenase complexes of animal mitochondria are inactivated by phosphorylation of serine residues, and reactivated by dephosphorylation. In addition, phosphorylated branched-chain complex is reactivated, apparently without dephosphorylation, by a protein or protein-associated factor present in liver and kidney mitochondria but not in heart or skeletal muscle mitochondria. Interconversion of the branched-chain complex may adjust the degradation of branched-chain amino acids in different tissues in response to supply. Phosphorylation is inhibited by branched-chain ketoacids, ADP and TPP. The pyruvate dehydrogenase complex is almost totally inactivated (99%) by starvation or diabetes, the kinase reactions being accelerated by products of fatty acid oxidation and by a protein or protein-associated factor induced by starvation or diabetes. There are three sites of phosphorylation, but only sites 1 and 2 are inactivating. Site 1 phosphorylation accounts for 98% of inactivation except during dephosphorylation when its contribution falls to 93%. Sites 2 and 3 are only fully phosphorylated when the complex is fully inactivated (starvation, diabetes). Phosphorylation of sites 2 and 3 inhibits reactivation by phosphatase. The phosphatase reaction is activated by Ca2+ (which may mediate effects of muscle work) and possibly by uncharacterized factors mediating insulin action in adipocytes.

Journal ArticleDOI
TL;DR: Pyruvate dehydrogenase complexes were purified from two ace (acetate-requiring) mutants of B. subtilis and found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification.
Abstract: A simple procedure is described for the purification of the pyruvate dehydrogenase complex and dihydrolipoamide dehydrogenase from Bacillus subtilis. The method is rapid and applicable to small quantities of bacterial cells. The purified pyruvate dehydrogenase complex (s0(20),w = 73S) comprises multiple copies of four different types of polypeptide chain, with apparent Mr values of 59 500, 55 000, 42 500 and 36 000: these were identified as the polypeptide chains of the lipoate acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3) and the two types of subunit of the pyruvate decarboxylase (E1) components respectively. Pyruvate dehydrogenase complexes were also purified from two ace (acetate-requiring) mutants of B. subtilis. That from mutant 61142 was found to be inactive, owing to an inactive E1 component, which was bound less tightly than wild-type E1 and was gradually lost from the E2E3 subcomplex during purification. Subunit-exchange experiments demonstrated that the E2E3 subcomplex retained full enzymic activity, suggesting that the lesion was limited to the E1 component. Mutant 61141R elaborated a functional pyruvate dehydrogenase complex, but this also contained a defective E1 component, the Km for pyruvate being raised from 0.4 mM to 4.3 mM. The E1 component rapidly dissociated from the E2E3 subcomplex at low temperature (0-4 degrees C), leaving an E2E3 subcomplex which by subunit-exchange experiments was judged to retain full enzymic activity. These ace mutants provide interesting opportunities to analyse defects in the self-assembly and catalytic activity of the pyruvate dehydrogenase complex.

Journal ArticleDOI
TL;DR: In cell-free translation programmed with free and membrane-bound polysomes, activity of mRNA coding for the precursor of the enzyme was much higher in freepolysomes than in membrane- bound polysome, and its translation product was detected as a putative precursor larger than the mature subunit.

Journal ArticleDOI
TL;DR: The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ and in vitro and were similar for both yeast cells and germ-tube forming cells.
Abstract: Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Journal ArticleDOI
TL;DR: The remarkable fluctuation of intracellular levels of fructose 1,6-bisphosphate and phosphoenolpyruvate observed in the cells growing under glucose limitation and nitrogen limitation implies that the intrace cellular concentration of fructose1,6,bisph phosphate, in cooperation with that of Pi, may regulate pyruvATE kinase activity in S. sanguis in vivo.
Abstract: It was found that pyruvate kinases with two different regulatory characteristics were distributed among oral streptococci. The pyruvate kinases of Streptococcus mutans, Streptococcus salivarius, and Streptococcus bovis were activated by glucose 6-phosphate, whereas the enzymes of both Streptococcus sanguis and Streptococcus mitis were activated by fructose 1,6-bisphosphate. Pyruvate kinase (EC 2.7.1.40) from S. sanguis NCTC 10904 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 250,000 to 260,000 and consisted of four identical subunits. Whereas the pyruvate kinase from S. mutans was completely dependent on glucose 6-phosphate (K. Abbe and T. Yamada, J. Bacteriol. 149:299-305, 1982), the enzyme from S. sanguis was activated by fructose 1,6-bisphosphate. In the presence of 0.5 mM fructose 1,6-bisphosphate, the saturation curves for the substrates, phosphoenolpyruvate and ADP, were hyperbolic, and the Km values were 0.13 and 0.30 mM, respectively. Without fructose 1,6-bisphosphate, however, saturation curves for both substrates were sigmoidal. GDP, IDP, and UDP could replace ADP. Like the enzyme from S. mutans, the enzyme from S. sanguis required a divalent cation, Mg2+ or Mn2+, and a monovalent cation, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for fructose 1,6-bisphosphate increased. The remarkable fluctuation of intracellular levels of fructose 1,6-bisphosphate and phosphoenolpyruvate observed in the cells growing under glucose limitation and nitrogen limitation implies that the intracellular concentration of fructose 1,6-bisphosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. sanguis in vivo.

Journal Article
TL;DR: It is postulated that binding of endotoxin to the plasma membrane triggers a sequence of events that lead to an initial decline in intracellular calcium concentration and that this latter event may be responsible for the inhibition of PDH phosphatase and consequent conversion of the complex to its inactive phosphorylated form.
Abstract: Intramitochondrial substrate metabolism was examined in cultured neuroblastoma NB41A3 cells exposed to endotoxin in order to elucidate possible causes for the changes in [ATP]/[ADP][Pi] and [NAD+]/[NADH] reported by us previously in these cells [1]. Flux through pyruvate dehydrogenase (PDH), measured with [1-14C]-pyruvate, was inhibited by 54% within 10 min in endotoxin-treated cells (0.99 nmol/min/mg dry wt vs 0.46 nmol/min/mg dry wt). In contrast, flux through 2-oxoglutarate dehydrogenase, measured with [1-14C]-glutamate was unaltered (0.79 nmol/min/mg dry wt). Dichloroacetate, an inhibitor of PDH kinase, restored flux through PDH to control levels. In endotoxin-treated cells, only 44% of the total PDH complex was in the active (nonphosphorylated) form as compared to 72% in control cells. Equilibrium uptake studies with 45Ca2+ and atomic absorption measurements showed that intracellular [Ca2+] in endotoxin-treated cells was about 20% lower than in control cells. It is postulated that binding of endotoxin to the plasma membrane triggers a sequence of events that lead to an initial decline in intracellular calcium concentration and that this latter event may be responsible for the inhibition of PDH phosphatase and consequent conversion of the complex to its inactive phosphorylated form.

Journal ArticleDOI
TL;DR: Circular dichroic spectrum suggests that the optical properties around the active site of the enzyme are similar to those of La-amino acid transaminase.