Showing papers on "Pyruvate dehydrogenase kinase published in 1979"
TL;DR: Material in a chromatographic fraction from an extract of insulin-treated muscle stimulated pyruvate dehydrogenase activity in addipocyte mitochondria, similar to insulin's activation of the enzyme in a plasma membrane-mitochondria mixture.
Abstract: Material in a chromatographic fraction from an extract of insulin-treated muscle stimulated pyruvate dehydrogenase activity in addipocyte mitochondria. This action was similar to insulin's activation of the enzyme in a plasma membrane-mitochondria mixture. Neither the chromatographic fraction nor insulin required adenosine triphosphate or magnesium ion (Mg2+), suggesting that both agents acted through a calcium-sensitive phosphatase. This fraction may contain a chemical mediator of insulin action.
221 citations
TL;DR: A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to growing on acetate or pyruvate, and was found to lack alpha-ketoglutarate dehydrogenase activity, and revertants of normal growth phenotype contained the activity again.
Abstract: A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.
171 citations
TL;DR: Enzymological observations point to an in vivo defect in the activation mechanism of the pyruvate dehydrogenase complex as the biochemical disturbance in SNE, and suggest that dichloroacetate may be beneficial in treating SNE.
Abstract: Autopsy examination confirmed the diagnosis of subacute necrotizing encephalomyelopathy (SNE) in a 7-month-old male infant who underwent several metabolic studies before death. Intermittent lactic acidemia and fumaric aciduria, an extreme hyperglycemic response to an intravenous bolus of alanine, and an elevated total body flux rate of glucose (58.4 mumoles . kg-1 . min-1) suggested a disturbance in the oxidative decarboxylation of pyruvate. Enzymological studies of postmortem samples revealed low nonactivated pyruvate dehydrogenase activity in liver (19.4%) and brain (53.8%). The lowest brain pyruvate dehydrogenase activities were noted in the midbrain and pontine regions. Supramaximal activation of the hepatic pyruvate dehydrogenase complex (135% of control values) occurred in vitro. Spontaneous reactivation following in vitro inactivation of the complex with adenosine triphosphate was significantly less (p less than 0.02) in the patient's samples compared to controls. The biochemical defect was not apparent in fibroblasts. These enzymological observations point to an in vivo defect in the activation mechanism of the pyruvate dehydrogenase complex as the biochemical disturbance in SNE. The findings suggest that dichloroacetate may be beneficial in treating SNE.
155 citations
TL;DR: The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.1.1) was investigated, and high activity was found in Bacillus species, and the enzyme is stable over a wide pH range.
Abstract: 1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.
124 citations
TL;DR: Results show that pyruvate inhibition of PDH, kinase-catalyzed inactivation of the pyruVate dehydrogenase complex is tightly integrated with the level of intramitochondrial ADP and would be greatly enhanced at low ATP:ADP ratios.
118 citations
TL;DR: Standard methods for the analysis of PK variants would make it possible to compare results obtained in different laboratories, and to identify variants on the basis of biochemical parameters as well as clinical manifestations, and comprise the major part of this report.
Abstract: Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40, PK) deficiency associated with hereditary nonspherocytic haemolytic anaemia was first reported in 1961 (Valentine et al, 1961). I t has become increasingly apparent that most, if not all, cases of PK deficiency are caused by the production of mutant enzymes with abnormal characteristics. A variety of techniques and methods have thus far been used in different laboratories for characterizing PK variants. The use of standard methods for the analysis of PK variants would make it possible to compare results obtained in different laboratories, and to identify variants on the basis of biochemical parameters as well as clinical manifestations. Such methods have been developed through a collaborative effort among members of a working group of the ICSH Expert Panel on Red Cell Enzymes, and comprise the major part of this report. As far as possible, a common buffer system, substrate and co-factor solution are proposed in order to simplify the methodology as well as the interpretation of data. The number of essential parameters for abnormal PK are minimized because of the limitation of both time required for the studies and volume of blood obtainable from a patient. This does not imply that other procedures such as urea stability (Koster et a l , 1971; Miwa et al , 1975), alanine inhibition (Staal et al, 1976), immunological methods (Lincoln et al , 1975; Miwa et al , 1975; Kahn et a l , 1976), isoelectric focusing (lbsen & Trippet, 1971; Kahn et al , 1976; Oda et a l , 1976), etc., are unimportant. Rather, it is suggested that these other studies be done optionally for further understanding of the abnormal characteristics of the variant enzymes.
104 citations
TL;DR: Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruVate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants.
Abstract: Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg(2+) and thiamine pyrophosphate.Chloroplast-associated pyruvate dehydrogenase complex provides a direct link between pyruvate metabolism and chloroplast fatty acid biosynthesis by providing the substrate, acetyl-CoA, necessary for membrane development in young plants.
100 citations
TL;DR: It is shown that glycerol kinase and acetate kinase proceed by pathways that result in inversion of the configuration at phosphorus, which provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
Abstract: We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.
95 citations
TL;DR: Enhanced oxidation of acetyl-CoA from alpha-ketoisocaproate by lactate addition is caused by a low activity of pyruvate dehydrogenase combined with increased flux through the citric acid cycle in response to the energy requirements for gluconeogenesis.
94 citations
TL;DR: In this paper, the regulation of pyruvate kinase in isolated hepatocytes from fasted rats was studied where the intracellular level of fructose 1,6-bisphosphate was elevated 5-fold by the addition of 5 mM dihydroxyacetone.
85 citations
TL;DR: The levels of 6-phosphogluconate dehydrogenase and glucose 6- phosphate dehydrogensase are subject to metabolic regulation; they increased three- to fivefold with increasing growth rate.
Abstract: The levels of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase are subject to metabolic regulation; they increased three- to fivefold with increasing growth rate.
TL;DR: The results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site) during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.
Abstract: 1. When pig heart pyruvate dehydrogenase complex was phosphorylated to completion with [gamma-32P]ATP by its intrinsic kinase, three phosphorylation sites were observed. The amino acid sequences around these sites were: sequence 1, Tyr-Gly-Met-Gly-Thr-Ser(P)-Val-Glu-Arg; and sequence 2, Tyr-His-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Val-Ser(P)-Tyr-Arg. 2. When phosphorylated to inactivation by repetitive additions of limiting quantities of [gamma-32P]ATP, phosphate was incorporated mainly (more than 90%) into Ser-5 of sequence 2. Phosphorylation of this site thus results in activation of pyruvate dehydrogenase. 3. If Ser-5 is phosphorylated with ATP and the enzyme then incubated with [gamma-32P]ATP, phosphorylation of the remaining sites occurred. Ser-12 of sequence 2 is phosphorylated about twice as rapidly as Ser-6 of sequence 1. 4. Incubation of pyruvate dehydrogenase with excess [gamma-32P]ATP with termination of phosphorylation at about 50% complete inactivation showed that Ser-5 of sequence 2 was phosphorylated most rapidly, but also that Ser-12 of sequence 2 was significantly (15% of total) phosphorylated. Ser-6 sequence 1 contained about 1% total P. 5. These results suggest that addition of limiting amounts of ATP produces primarily phosphorylation of Ser-5 of sequence 2 (inactivating site). This also occurs during incubation with excess ATP before complete inactivation occurs, but a greater occupancy of other sites also occurs during this treatment.
TL;DR: The observation that catecholamines do not increase gluconeogenesis to the same extent as glucagon was not due to a differential effect on mitochondrial CO2 fixation, but to a smaller inhibition of pyruvate kinase activity than did glucagon.
TL;DR: Evidence is presented that dephosphorylation of the three phosphorylation sites on bovine kidney pyruvate dehydrogenase by pyruVate dehydrogensase phosphatase is random, and the presence of phosphoryl groups at sites 2 and 3 did not significantly affect the rate of deph phosphatelation at site 1 or the rates of reactivation of the enzyme by the phosphat enzyme.
TL;DR: Investigation of the kinetics of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate found that incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.
Abstract: 1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.
TL;DR: Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruVate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status.
TL;DR: Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography, finding the plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides.
Abstract: Isoenzymes of pyruvate kinase from green leaves of castor bean and etiolated leaves of pea plants have been separated by ion filtration chromatography. One of the isoenzymes is localized in the plastid, whereas the other is in the cytosol. The cytosolic enzyme has a pH optimum from pH 7 to pH 9, and is able to utilize nucleotides other than ADP as the phosphoryl acceptor. The plastid enzyme has a much sharper optimum at pH 8, and is less efficient at using alternative nucleotides. The plastic pyruvate kinase, unlike the cytosolic enzyme, requires the presence of dithiothreitol or 2-mercaptoethanol during isolation and storage to stabilize the activity.
TL;DR: Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.
Abstract: 1. Pyruvate carboxylase is present in brown adipose tissue mitochondria. 2. In isolated mitochondria, pyruvate, bicarbonate and ATP, the substrates for pyruvate carboxylase, are able to replace added malate in supplying a condensing partner for acetyl-CoA formed from beta-oxidation of fatty acids. 3. In brown adipocytes, pyruvate and CO2 increase the rate of norepinephrine-stimulated respiration synergistically. 4. The norepinephrine-stimulated respiration in brown adipocytes is diminished when pyruvate transport into the mitochondria is inhibited. 5. Pyruvate carboxylation increases the intramitochondrial level of citric acid cycle intermediates, as shown by titrations of malonate inhibition of respiration. 6. Pyruvate carboxylation can continuously supply the mitochondria with citric acid cycle intermediates, as evidenced by its ability to maintain respiration when oxoglutarate conversion to glutamate is stimulated. 7. Pyruvate carboxylation is necessary for maximal oxygen consumption even when drainage of the citric acid cycle for amino acid synthesis is eliminated. 8. Pyruvate carboxylation explains observed effects of CO2 on respiration in brown adipocytes, and may also explain the increased glucose uptake by brown adipose tissue during thermogenesis in vivo.
TL;DR: It is suggested that further studies on the treatment of hyperglycemia and lactic acidosis with pyruvate dehydrogenase activators be carried out with 2-chloropropionate rather than dichloroacetate, since oxalic acid stone formation and renal dysfunction may prove to be side effects of any therapeutic application of dichlorOacetate.
TL;DR: Brain octopine dehydrogenase, like H-type lactate dehydrogen enzyme, displays properties which may poise the enzyme for a major role in the oxidation of octopines in vivo.
Abstract: Octopine dehydrogenase, the terminal enzyme of anaerobic glycolysis in the cuttlefish, Sepia officinalis, displays kinetically distinct tissue-specific isozymic forms. An initial survey of octopine dehydrogenase from eleven tissues of Sepia revealed that only brain octopine dehydrogenase showed significant substrate inhibition by pyruvate. This property, which is characteristic of H-type lactate dehydrogenase, was used as the basis for a study comparing and contrasting the kinetic properties of the mantle muscle and brain isozymes of octopine dehydrogenase. Compared to the mantle muscle enzyme, brain octopine dehydrogenase displayed: (a) a higher apparent affinity for the reactants of the reverse reaction (Km for octopine was 10-fold lower than that of the muscle enzyme), (b) stronger substrate inhibition by both pyruvate and octopine, (c) greater inhibition by the product of the forward reaction, octopine, (d) an increased activity with the hypoxanthine derivative of NADH, and (e) an inhibition of enzyme activity in an incubate containing NAD+, pyruvate, and arginine (indicative of the formation of an inhibitory complex, enzyme-pyruvate-arginine-NAD+). The kinetic properties of the mantle muscle and brain isozymes of octopine dehydrogenase and the differences between these two forms appear analogous to the well-known properties of the M4 versus H4 isozymes of lactate dehydrogenase. Mantle muscle octopine dehydrogenase, with kinetic properties resembling the M form of lactate dehydrogenase, appears geared for the rapid synthesis of octopine under conditions of muscular work. Brain octopine dehydrogenase, like H-type lactate dehydrogenase, displays properties which may poise the enzyme for a major role in the oxidation of octopine in vivo.
TL;DR: Purified Pisum sativum chloroplasts showed the presence of all three components of the pyruvate-dehydrogenase complex (PDC), and acetyl coenzyme A generated internally via pyruVate may serve as the main source of carbon for chloroplastic fatty-acid synthesis.
TL;DR: Kinetic and Sephadex gel filtration epxeriments indicate that in the presence of palmitoyl-CoA, glutamate dehydrogenase forms a complex with mitochondrial malate dehydrogenases, and if the two enzymes are incubated in polyethylene glycol, there is a marked increase in the amount of both enzymes precipitated.
TL;DR: The pyruvate dehyhdrogenase complex has been isolated from Ascaris muscle mitochondria and purified to near homogeneity by differential centrifugation, (NH4)2SO4 fractionation and calcium phosphate gel-cellulose chromatography, suggesting regulation by the more reduced state of the pyridine nucleotide pool in Ascari mitochondria.
TL;DR: The results of this investigation demonstrate that selective changes in the metabolic response of the hepatocyte to various adrenergic agonists occur as the rat matures; however, similar changes are not observed with glucagon.
TL;DR: By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation.
Abstract: Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed ‘inactivase’) obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex.
By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogenase and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.
TL;DR: Adaptations in diet that enhanced fatty acid synthesis on meal feeding led to an increased steady-state concentration of pyruvate kinase protein, and the rate-determining step in adjusting enzyme concentration seems to be proteolysis.
Abstract: Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20--100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30--45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0--30%- and 45--85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.