Showing papers on "Pyruvate dehydrogenase phosphatase published in 1983"
304 citations
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
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
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
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
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
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
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+.
49 citations
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.
48 citations
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.
48 citations
01 Mar 1983
TL;DR: Parameters which are important for industrial application of the enzymes were determined: substrate specifity, pH and temperature optimum, temperature stability, stability at different pH-values, and the storage stability of the enzyme in crude extracts.
Abstract: To initiate studies of the stereospecific reduction of pyruvate and phenylpyruvate to the corresponding d-2-hydroxyacids a limited screening was carried out for microorganisms possessing a high NADH-dependet d-lactate dehydrogenase activity. Lactobacillus confusus was found to produce the desired dehydrogenase, which showed also relatively high activity towards phenylpyruvate, so this strain was selected for large scale production of the enzyme. A procedure for large scale purification of the enzyme starting with 24 kg wet cells is described including liquid-liquid extraction, ultrafiltration and chromatography on DEAE-cellulose, yielding a catalyst with specific activities of 216 U×mg−1 for pyruvate reduction and 15 U×mg−1 for phenyl-pyruvate reduction. A further tenfold purification can be achieved by affinity chromatography on Blue-Sepharose C-6B. Parameters which are important for industrial application of the enzyme were determined: substrate specifity, pH and temperature optimum, temperature stability, stability at different pH-values, and the storage stability of the enzyme in crude extracts.
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.
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.
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.
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.
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.
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.
TL;DR: The branched‐chain 2 oxoacid dehydrogenase complex has been purified from well‐washed ox‐kidney mitochondria together with branching‐chain dehydrogenases kinase and reactivation and dephosphorylation are attributed to a mitochondrial brancEDH phosphatase.
TL;DR: It is concluded that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.
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.
TL;DR: A mutant, No. 1–231, resistant to S-(2-aminoethyl)-l-cysteine (AEC) plus threonine which was previously derived from a low citrate synthase mutant (No. 15–8) of Brevibacterium flavum, produced 41 g/liter of lysine and showed pyruvate kinase and homoserine dehydrogenase activities of about 1/10 and 1/20 as much as those of
Abstract: A mutant, No. 1–231, resistant to S-(2-aminoethyl)-l-cysteine (AEC) plus threonine which was previously derived from a low citrate synthase mutant (No. 15–8) of Brevibacterium flavum, produced 41 g/liter of lysine (as HCl salt, 41% yield) and showed pyruvate kinase and homoserine dehydrogenase activities of about 1/10 and 1/20 as much as those of No. 15–8, respectively, but its aspartokinase was still normally sensitive to the feedback inhibition by lysine plus threonine. Another AEC-resistant mutant, No. 2–190, from No. 15–8 showed aspartokinase insensitive to the feedback inhibition without any change in pyruvate kinase and homoserine dehydrogenase activities. In addition, both the two AEC-resistant mutants and parent No. 15–8 showed partial desensitization of phosphoenolpyruvate carboxylase to the feedback inhibition by l-aspartic acid.β-Fluoropyruvic acid-sensitive mutants No. 22 and No. 2–11 were derived from No. 1–231. These sensitive strains produced 51 g/liter of lysine (51% yield) and were found ...
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.
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.
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.
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.
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.
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.
TL;DR: The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.
Abstract: The activity of pyruvate dehydrogenase in extracts of pig mesenteric lymphocytes was measured under different preincubation conditions. The mitogens concanavalin A and ionophore A23187 both increased pyruvate dehydrogenase activity. In both cases activation required extracellular Ca2+. Digitonin-permeabilized cells required 0.5 microM free Ca2+ for half-maximal activation of pyruvate dehydrogenase. The stimulation by concanavalin A in intact cells was probably not due to changes in effectors of pyruvate dehydrogenase kinase. This evidence suggests that activation of pyruvate dehydrogenase is by Ca2+ activation of pyruvate dehydrogenase phosphatase and supports the view that the cytoplasmic free [Ca2+] rises to something less than 1 microM on stimulation with mitogens.
TL;DR: The α‐subunit of the E1 component of branched‐chain 2‐oxoacid dehydrogenase is phosphorylated at 3 sites by an endogenous kinase, and the similarities with the covalent regulation of pyruvate dehydrogen enzyme complex are discussed.
TL;DR: The existence of this mutant, in conjunction with earlier results, strongly suggests that the two pyruvate kinases in this bacterium are distinct forms and not interconvertible.