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PubMed1979

Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in leukocytes and fibroblasts from a patient with pyruvate carboxylase deficiency.

Normal values are given for the activities of pyruvate carboxylase (E.C.6.4.1.1), mitochondrial phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32, PEPCK), and citrate synthase (E.C. 4.1.3.7) in fibroblasts, lymphocytes, and leukocytes. Also given are values for these enzymes in the leukocytes and fibroblasts from a severely mentally and developmentally retarded patient with proximal renal tubular acidosis and hepatic, cerebral, and renal cortical pyruvate carboxylase deficiency. In normals, virtually all of the mitochondrial PEPCK and pyruvate carboxylase activity was present in the mononuclear leukocyte fraction of whole venous blood. Cellular fractionation studies with human lymphocytes and fibroblasts demonstrated that all of the PEPCK activity in these cells is mitochondrial. Normal values for pyruvate carboxylase in leukocytes were 0.092 (0.070--0.208) mU/mg protein (n=5), in lymphocytes 0.154 (0.092--0.262) mU/mg protein (n=5), and in fibroblasts 1.36 (0.778--2.19) mU/mg protein (n=5). The patient with hepatic, renal, and cerebral pyruvate carboxylase deficiency had no detectable activity (less than 0.009 mU/mg protein) in his leukocytes and 0.018 mU/mg protein in his fibroblasts. Data from an assay for pyruvate carboxylase activity in the patient's fibroblasts show that the activity observed is significant but very close to the lower limits of the assay. Values for PEPCK in normal lymphocytes were 1.42 (0.824--1.88) mU/mg protein (n=5), in leukocytes 1.68 (1.64--1.72) mU/mg protein (n=2), and in fibroblasts 5.49 (3.94--6.33) mU/mg protein (n=6).

Acidosis, Renal Tubular↗

PubMed1983

The substrate-mediated inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex.

Incubation of the pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex with Mg2+, thiamine pyrophosphate and low concentrations of pyruvic acid in the absence of electron acceptors results in irreversible time-dependent inactivation of the enzyme. The rate of the enzyme inactivation is markedly decreased in the presence of high concentrations of pyruvate; in this case acetoin and acetolactate are detected in the reaction mixture. The enzyme activity is stabilized when the artificial electron acceptor, 2,6-dichlorophenolindophenol, is present in the reaction mixture. The substrate-mediated inactivation of the enzyme is accompanied by incorporation of the 2-[14C]-substrate fragment and labelled thiamine pyrophosphate into the protein fraction. The enzyme reactivation by neutral hydroxylamine and the protective effect of dithiothreitol suggest that the SH-group(s) may be involved in the substrate-mediated inactivation of pyruvate dehydrogenase.

Animals↗

PubMed1977

An energy-conserving pyruvate-to-acetate pathway in Entamoeba histolytica. Pyruvate synthase and a new acetate thiokinase.

Under anaerobic conditions, cells of Entamoeba histolytica grown with bacteria produce H2 and acetate while cells grown axenically produce neither. Aerobically, acetate is produced and O2 is consumed by amebae from either type of cells. Centrifuged extracts, 2.4 x 106 x g x min, from both types of cells contain pyruvate synthase (EC 1.2.7.1) and an acetate thiokinase which, together, form a system capable of converting pyruvate to acetate. Pyruvate synthase catalyzes the reaction: pyruvate + CoA leads to CO2 + acetyl-CoA + 2E. Electron acceptors which function with this enzyme are FAD, FMN, riboflavin, ferredoxin, and methyl viologen, but not NAD or NADP. The amebal acetate thiokinase catalyzes the reaction acetyl-CoA + ADP + Pi leads to acetate + ATP + CoA. For this apparently new enzyme we suggest the trivial name acetyl-CoA-synthetase (ADP-forming). Extracts from axenic amebae do not contain hydrogenase, but extracts from cells grown with bacteria do. It is postulated that in bacteria-grown amebae electrons generated at the pyruvate synthase step are utilized anaerobically to produce H2 via the hydrogenase and that the acetyl-CoA is converted to acetate in an energy-conserving step catalyzed by amebal acetyl-CoA synthetase. Aerobically, cells grown under either regimen may utilize the energy-conserving pyruvate-to-acetate pathway since O2 then serves as the ultimate electron acceptor.

Acetate-CoA Ligase↗

PubMed1977

[Study of the kinetic mechanism of the pyruvate-2,6-dichlorophenolindophenol reductase activity of muscle pyruvate dehydrogenase].

The mechanism of pyruvate-2,6-dichlorophenol-indophenol (2,6-CPI) reductase reaction catalyzed by the pyruvate dehydrogenase complex from pigeon breast muscle and by its pyruvate dehydrogenase component was studied. The K'm values for 2,6-DCPI in both cases were found equal to 1.3--1.4-10(-5) M. The double reverse values plots obtained at a fixed concentration of the first substrate and a variable concentration of the second one were linear and had a constant K'm/V'max ratio. The substitution of thiamine pyrophosphate and pyruvate by the substrate decarboxylation product, i.e. 2-oxyethyl thiamine pyrophosphate under similar conditions resulted in kinetic plots, typical for the "ping-pong" mechanism of enzymatic reactions. A mechanism of the pyruvate 2,6-DCPI reductase reaction, providing for the interaction of 2-oxyethyl thiamine pyrophosphate after its binding to the apoenzyme with a certain protein group of the pyruvate dehydrogenase active centre, was postulated. The reaction was shown to result in the production of acetyl-substituted reduced form of the enzyme. Regeneration of free enzyme required the presence of 2,6-DCPI as oxidizing agent.

2,6-Dichloroindophenol↗

PubMed1989

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit.

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.

Acetolactate Synthase↗

PubMed1984

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes.

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.

Adenosine Diphosphate↗

PubMed1984

The carboxybiotin complex of pyruvate carboxylase. A kinetic analysis of the effects of Mg2+ ions on its stability and on its reaction with pyruvate.

The enzyme-[14C] carboxybiotin complex of sheep liver pyruvate carboxylase was isolated and the reaction between this and pyruvate was studied by using the quenched-flow rapid-reaction technique. At 0.5 degrees C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-first-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-first-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the first sub-site, whereas in the slow phase of the reaction with 2-oxobutyrate this dissociation is the rate-limiting step. It is possible, from the reaction scheme proposed, that the inhibition of overall enzymic activity by high concentrations of Mg2+ could be caused by the transfer of the carboxy group from biotin to pyruvate becoming rate-limiting. The efficacy of a substrate as a signal for the movement of carboxybiotin from the first sub-site is reflected by the amount that the effective affinity of the enzyme- carboxybiotin complex for Mg2+ is lowered. In the presence of the substrates tested, the affinities of the carboxybiotin complex can be arranged in order of increasing magnitude, i.e.: (formula; see text). The kinetics of the decay of the enzyme-[14C] carboxybiotin complex at 0 degree C in the absence of substrates are similar to the reaction with pyruvate except that the carboxybiotin is also unstable in the first sub-site, to some degree. This similarity allows for the proposal of a general scheme for the decarboxylation of the enzyme- carboxybiotin complex in the presence or in the absence of substrates.

Biotin↗

PubMed1983

Energy utilization and pyruvate as determinants of pyruvate dehydrogenase in norepinephrine-stimulated heart.

The quantitative effects of norepinephrine (NE) on the active form of the cardiac pyruvate dehydrogenase complex (PDCa) and the rate of oxidative decarboxylation of pyruvate (MVPyr) were compared with those of pyruvate (Pyr). Isolated working guinea pig hearts metabolized pyruvate alone or in combination with alternative energy-providing substrates (ketone bodies, octanoate, glucose). NE produced proportional increases in PDCa and myocardial oxygen uptake (MVO2). Total PDC activity (PDC1) remained constant. The PDCa/PDC1 ratios in NE depleted hearts (reserpine pretreatment) compared well with those in hearts containing endogenous NE, provided myocardial substrate supply and MVO2 were also comparable. No evidence was obtained indicating that NE or perfusate Ca2+ can dissociate PDCa or MVPyr from MVO2, even in presence of the alternative cosubstrates. In contrast, 0.2-10 mM Pyr produced stepwise but only submaximum increases in PDCa and MVPyr, with MVO2 remaining constant. Thus, at all Pyr concentrations tested, NE stimulations of myocaridal energy utilization and MVO2 were followed by further increases in PDCa and MVPyr. Evidently, pyruvate, and particularly cellular respiration are important determinants in the regulation of cardiac PDC, also during adrenergic stimulation of the heart.

Animals↗

PubMed1991

Screening for disorders of pyruvate metabolism by measuring the ratio of the rates of lactate production and pyruvate decarboxylation in cultured skin fibroblasts.

We assayed the rates of lactate production from [1-14C]pyruvate and decarboxylation of [1-14C]pyruvate in cultured skin fibroblasts from 8 patients with disorders of pyruvate metabolism and 16 control subjects. The disorders of pyruvate metabolism could be more readily detected by measuring the ratio between the rates of lactate production and pyruvate decarboxylation by cultured skin fibroblasts than by measuring either the rate in isolation.

Acidosis, Lactic↗

PubMed1974

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate.

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.

Acetyl Coenzyme A↗

PubMed1975

Activation of pyruvate dehydrogenase in adipose tissue by insulin. Evidence for an effect of insulin on pyruvate dehydrogenase phosphate phosphatase.

1. The mechanism by which insulin activates pyruvate dehydrogenase in rat epididymal adipose tissue was further investigated. 2. When crude extracts, prepared from tissue segments previously exposed to insulin (2m-i.u/ml) for 2min, were supplemented with Mg-2+, Ca-2+, glucose and hexokinase and incubated at 30 degrees C, they displayed an enhanced rate of increase in pyruvate dehydrogenase activity compared with control extracts. 3. When similar extracts were instead supplemented with fluoride, ADP, creatine phosphate and creatine kinase, the rate of decrease in pyruvate dehydrogenase activity observed during incubation at 30 degrees C was unaffected by insulin treatment. 4. It is suggested that insulin increases the fraction of pyruvate dehydrogenase present in the tissue in the active dephospho form by increasing the activity of pyruvate dehydrogenase phosphate phosphatase.

Adenosine Diphosphate↗

PubMed1986

Use of toluene-permeabilized mitochondria to study the regulation of adipose tissue pyruvate dehydrogenase in situ. Further evidence that insulin acts through stimulation of pyruvate dehydrogenase phosphate phosphatase.

Rat epididymal-adipose-tissue mitochondria were made selectively permeable to small molecules without the loss of matrix enzymes by treating the mitochondria with toluene under controlled conditions. With this preparation the entire pyruvate dehydrogenase system was shown to be retained within the mitochondrial matrix and to retain its normal catalytic activity. By using dilute suspensions of these permeabilized mitochondria maintained in the cuvette of a spectrophotometer, it was possible to monitor changes of pyruvate dehydrogenase activity continuously while the activities of the interconverting kinase and phosphatase could be independently manipulated. Permeabilized mitochondria were prepared from control and insulin-treated adipose tissue, and the properties of both the pyruvate dehydrogenase kinase and the phosphatase were compared in situ. No difference in kinase activity was detected, but increases in phosphatase activity were observed in permeabilized mitochondria from insulin-treated tissue. Further studies showed that the main effect of insulin treatment was a decrease in the apparent Ka of the phosphatase for Mg2+, in agreement with earlier studies with mitochondria made permeable to Mg2+ by using the ionophore A23187 [Thomas, Diggle & Denton (1986) Biochem. J. 238, 83-91]. No effects of spermine were detected, although spermine diminishes the Ka of purified phosphatase preparations for Mg2+. Since effects of insulin on pyruvate dehydrogenase phosphatase activity are not evident in mitochondrial extracts, it is concluded that insulin may act by altering some high-Mr component which interacts with the pyruvate dehydrogenase system within intact or permeabilized mitochondria, but not when the mitochondrial membranes are disrupted.

Adipose Tissue↗

PubMed1977

Deficient activity of hepatic pyruvate dehydrogenase and pyruvate carboxylase in Reye's syndrome.

The activity of certain hepatic enzymes involved in carbohydrate metabolism was measured in postmortem samples from six cases of Reye's syndrome. The activities of the two exclusively extramitochondrial enzymes, glucose-6-phosphatase and fructose-1,6-diphosphatase, were all within the normal range. Activities of pyruvate carboxylase and pyruvate dehydrogenase, both of which are exclusively mitochondrial enzymes, were below levels, shown by control tissue in every case, the average being 21.7% of the lowest control value for pyruvate carboxylase and 11.6% of that for pyruvate dehydrogenase. Impaired pyruvate metabolism appears to be another feature in Reye's syndrome.

Adolescent↗

PubMed1983

Normal activities of hepatic pyruvate dehydrogenase and pyruvate carboxylase in Leigh's syndrome.

A case of Leigh's syndrome (subacute necrotizing encephalomyelopathy, SNE), proven by autopsy, was reported. The persistent elevation of pyruvate and lactate in blood and hyperalanemia suggested an impairment of pyruvate oxidation, but the enzyme activities of pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) in liver tissues of the patient revealed normal. It is postulated that Leigh's syndrome and both enzyme deficiencies are distinct entities.

Brain↗

PubMed1975

Membrane-localized pyruvate kinase of red blood cells in hemolytic anemia associated with pyruvate kinase deficiency.

Pyruvate kinase activity of red blood cell membranes, which is normally masked, has been determined after mechanical disruption of the membranes in normal individuals and in three homozygous patients with pyruvate kinase deficiency. Although patients 1 and 2, who were siblings, had relatively high enzyme activities in their hemolysates, they had the severest form of the disorder. The activities of their membrane fragments were decreased to seven per cent of fragments of normal membranes. Patient 3 had a mild form of hemolytic anemia despite a low enzyme activity of his hemolysates. The membrane fragments of this patient contained 28 per cent of the pyruvate kinase activity of normal fragments. The data suggest a relationship between the amount of membrane-localized pyruvate kinase and the severity of the clinical disorder. The reduced production of ATP by the enzyme portion localized within the membrane may cause an impairment of membrane functions in pyruvate kinase deficiency.

Aged↗

PubMed1983

Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci.

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.

Cations↗

PubMed1978

Homogeneous pyruvate kinase isolated from yeast by two different methods is indistinguishable from pyruvate kinase in cell-free extract.

In this report, we have compared homogeneous yeast (Saccharomyces cerevisiae) pyruvate kinase to enzyme from cell-free extracts in several different ways: 1) isoelectric focusing of cell-free extracts indicates one peak of pyruvate kinase activity whose isoelectric point is the same as that of the pure enzyme; 2) antibody prepared to the pure enzyme produces a single, fused precipitin line against enzyme in the cell-free extract and pure enzyme; 3) immunoelectrophoresis of cell-free extract produces one precipitin arc which has the same mobility as that of the pure enzyme; and 4) immunoprecipitation of the pure enzyme from cell-free extract with subsequent solubilization in 1% sodium dodecyl sulfate and electrophoresis on sodium dodecyl sulfate-polyacrylamide gels produces a single protein band attributable to pyruvate kinase which co-migrates with the purified enzyme. Within the limits of the sensitivity of the methods employed, we conclude that the homogeneous pyruvate kinase prepared from yeast lysed either by Manton-Gaulin homogenization (Aust, A., Yun, S.-L., and Suelter, C. (1975) Methods Enzymol. 42, 176-182) or by toluolysis (Yun, S.-L., Aust, A.E., and Suelter, C.H. (1977) J. Biol. Chem. 251, 124-128) is identical with pyruvate kinase in cell-free extract.

Immunodiffusion↗

PubMed1983

Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.

Animals↗