Showing papers on "Pyruvate dehydrogenase kinase published in 1970"

Regulation of Liver Pyruvate Kinase and the Phosphoenolpyruvate Crossroads

Abstract: Certain kinetic properties of the pyruvate kinase activity in different tissues of the rat have been studied in fresh extracts with particular attention to their possible value for the regulation of gluconeogenesis and glycolysis. The pyruvate kinase activity in fresh extracts of the gluconeogenic tissues liver and kidney cortex has allosteric properties very marked in near physiological conditions, as follows: (a) co-operativity in the kinetics with respect to the concentration of phosphoenolpyruvate; (b) strong allosteric inhibition by alanine and ATP, each of which raises the [S]0.5 value and increases the sigmodicity respect to the concentration of phosphoenolpyruvate; and (c) very strong activation by fructose diphosphate, which greatly lowers the [S]0.5 value and shifts the kinetics from markedly sigmoid to hyperbolic, and can fully counteract the inhibitory effects of alanine and ATP. Keeping the extracts of liver and kidney at low temperature (0–2°) leads to desensitization of the pyruvate kinase to the homotropic cooperativity of the phosphoenolpyruvate substrate and to the allosteric inhibition by alanine and ATP and the activation by fructose diphosphate. This cold desensitization is reversible. These facts and their time dependence make it possible to understand previous difficuties to obtain reproducible allosteric effects with the pyruvate kinase of rat liver. The pyruvate kinase of other tissues examined, including heart and adipose tissue, did not exhibit any of the allosteric properties above described for the enzyme of the gluconeogenic tissues. The μmolar range of fructose diphosphate concentrations required for activation of liver pyruvate kinase in the presence of the two physiological inhibitors and within the physiological range of concentrations of phosphoenolpyruvate fits well in order of magnitude with the calculated range of concentrations of free fructose diphosphate prevailing in liver. These results strongly support the hypothesis that an isoenzyme of pyruvate kinase present in liver and kidney has evolved with built-in specific regulatory mechanisms in order to prevent the diversion of phosphoenolpyruvate from its way to glucose in glucoenogenic situations. An efficient regulation in the reversible switch over from glycolysis to gluconeogenesis seems to be feasible by the interplay of two feedback inhibitors, alanine and ATP, and a forward activator, fructose diphosphate.

Interconversion of phospho- and dephospho- forms of pig heart pyruvate dehydrogenase.

Abstract: Pyruvate dehydrogenase from pig heart exists in active and inactive forms. Interconversion from the active (dephospho) form into the inactive (phospho) form is catalyzed by an ATP-dependent kinase. Conversely the enzyme is reactivated by a phosphatase which removes the phosphate group from the protein. By gradient centrifugation pyruvate dehydrogenase was prepared free of phosphatase but still containing the kinase. Reactivation of pyruvate dehydrogenase is stimulated by adenosine 3′,5′-cyclic phosphate. There is incorporation of 32P from γ-32P-ATP into the protein fraction containing the phosphatase and this phosphorylation reaction is also stimulated by adenosine 3′,5′-cyclic phosphate. The participation of this phosphate in the pyruvate dehydrogenase interconversion system suggests that, in heart muscle, pyruvate oxidation may be under hormonal control by a mechanism similar to that involved in the regulation of glycogen synthesis and breakdown.

Metabolic differentiation of rabbit skeletal muscle as induced by specific innervation.

Abstract: Cross-innervations and re-innervations were performed on the slow (“red”) m. soleus and the fast (“white”) m. extensor digitorum of the rabbit. Innervation of the slow soleus muscle with “fast” motor neurons causes a significant change of the metabolic type of the muscle. This transformation consists in a parallel increase of the activity levels of glycogen phosphorylase, of all the investigated glycolytic enzymes (phosphofructokinase, triosephosphate isomerase, triosephosphate dehydrogenase, phosphoglycerate kinase, glycerate phosphomutase, phosphopyruvate hydratase, lactate dehydrogenase), of adenylate kinase and mitochondrial glycerolphosphate dehydrogenase. A concomitant and parallel decrease is found in the activity levels of extramitochondrial hexokinase, and alanine aminotransferase, of all the investigated enzymes of the citric acid cycle (intramitochondrial citrate synthase, and succinate dehydrogenase, extra- and intramitochondrial malate dehydrogenase, and NADP isocitrate dehydrogenase), of intramitochondrial 3-hydroxyacyl-CoA dehydrogenase and glutamate dehydrogenase, and of extra- and intramitrochondrial aspartate aminotransferase. In spite of the marked changes in absolute activities, no alterations occur with regard to the ratios of enzymes and enzyme groups which by comparison of functionally different muscles have been classified as constant. Changes in the activity ratios concern the same enzymes which in previous studies have been shown to be characteristic of the metabolic type (phosphorylase/hexokinase, triosephosphate dehydrogenase/citrate synthase, lactate dehydrogenase/citrate synthase, triosephosphate dehydrogenase/3-hydroxyacyl-CoA dehydrogenase, adenylate kinase/creatine kinase). The alterations show that a conversion of the metabolic type from “slow” to “fast” takes place and indicate a specific influence of the innervation on metabolic differentiation in muscle, that is the control of a specific pattern of enzyme synthesis. Due to the obvious failure of innervation of the fast extensor muscle with “slow” motor neurons from the tibial nerve, no significant changes were observed in this muscle.

Regulation of Pyruvate Dehydrogenase Complex Synthesis in Escherichia coli K12

Abstract: 1 The synthesis of the pyruvate dehydrogenase complex in Escherichia coli K12 is inducible, and pyruvate is the metabolite causing induction. In a mutant lacking the activities of phosphoenol pyruvate synthase and dihydrolipoamide transacetylase (a component of the pyruvate dehydrogenase complex) pyruvate is no longer significantly metabolized under certain growth conditions. In such a mutant pyruvate as well as α-ketobutyrate induces pyruvate dehydrogenase synthesis. Decreasing inducing activity was found with increasing chain length (or branching) of homologous α-ketoacids. Apo-pyruvate dehydrogenase is produced when thiamine requiring strains are grown in the absence of thiamine, i. e., the pyruvate dehydrogenase complex formed is inactive. Such strains, possessing wild type pyruvate dehydrogenase complex, accumulate pyruvate when growing without thiamine. Only with thiamine starvation could the synthesis of wild type pyruvate dehydrogenase complex be fully induced. Also, in merodiploid strains homogenotic for the wild type ace locus a gene dosage effect was found only upon thiamine deprivation. Inducibility is thus separable from enzymatic activity; enzymatically active pyruvate dehydrogenase complex removes the effector inducing its synthesis thus preventing full induction during normal growth conditions. No evidence was found for an indirect action by pyruvate, i. e., pyruvate does not induce by inhibition or activation of some other reaction (in the citric acid cycle or glycolysis), thereby causing an increased concentration of another metabolite which is the true inducer. 2 It is possible that pyruvate serves not only as inducing metabolite for pyruvate dehydrogenase complex synthesis but also as repressing metabolite for isocitric lyase synthesis (or for a glyoxylate cycle operon). 3 Both ace mutants and wild type possess pyruvate oxidase, an acetate producing enzyme system independent of the pyruvate dehydrogenase complex. It appears that the activity of the oxidase is too low to provide enough acetate which can replace that normally produced via pyruvate dehydrogenase complex. A physiological role for the oxidase has not been found.

Fluctuation of hepatic enzymes important in glucose metabolism in relation to thyroid function.

Abstract: Enzyme activities of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, pyruvate kinase and of mitochondrial glycerolphosphate dehydrogenase were studied in the livers of rats under the following states of thyroid function: 131I-thyroidectomized rats, 131I-thyroidectomized rats treated with triiodothyronine, normal rats treated with excess of triiodothyronine, and normal untreated rats. In the thyroidectomized group all of the enzyme activities listed were significantly diminished. After treatment with a single dose of triiodothyronine the activities of pyruvate carboxylase and of pyruvate kinase were restored to normal whereas phosphoenolpyruvate carboxykinase and — most markedly — glycerolphosphate dehydrogenase were raised above the normal level. After repeated administration of triiodothyronine to normal rats a further increase in the activity of pyruvate carboxylase, phosphoenolpyruvate carboxykinase and of glycerolphosphate dehydrogenase was observed. The activities of glycerolphosphate dehydrogenase and of pyruvate carboxylase 70 h following triiodothyronine injection into hypothyroid rats were more than 50% lower after treatment with actinomycin C suggesting that the increase in enzyme activities was due to increased synthesis of enzyme protein. Phosphoenolpyruvate carboxykinase activity was not changed significantly by actinomycin C. The possible significance of these thyroid hormone dependent enzyme fluctuations in controlling the overall rate of hepatic gluconeogenesis is discussed.

The Oxidation of Malate by Mitochondria Isolated from Cauliflower Buds

Abstract: 1Pyruvate is the major product of malate oxidation by isolated cauliflower bud mitochondria when pyruvate dehydrogenase is inhibited. Pyruvate formation, which requires Mg++, Mn++ or Co++, is stimulated by added NAD. 2The effects of ADP, 2,4-dinitrophenol and respiratory inhibitors on pyruvate formation indicate that the reaction is linked to mitochondrial electron transport. 3No enzymic decarboxylation of oxaloacetate to pyruvate by the mitochondria can be observed. With added malate oxaloacetate breaks down in the mitochondria, probably to malate using NADH generated by a mitochondrial malic enzyme. 4Acetyl-CoA and glutamate, which compete for oxaloacetate in the mitochondria, do not inhibit pyruvate formation from malate. 5A solubilised NAD requiring malic enzyme is present in sonicates of the mitochondria. 6It is concluded that oxaloacetate is not an intermediate in pyruvate formation during malate oxidation by cauliflower bud mitochondria, and that a direct conversion of malate to pyruvate by a mitochondrial NAD requiring malic enzyme occurs.

Immunochemical studies with soluble and mitochondrial pyruvate carboxylase activities from rat tissues.

Abstract: 1. Pyruvate carboxylase (EC 6.4.1.1), purified from rat liver mitochondria to a specific activity of 14 units/mg, was used for the preparation of antibodies in rabbits. 2. Tissue distribution studies showed that pyruvate carboxylase was present in all rat tissues that were tested, with considerable activities both in gluconeogenic tissues such as liver and kidney and in tissues with high rates of lipogenesis such as white adipose tissue, brown adipose tissue, adrenal gland and lactating mammary gland. 3. Immunochemical titration experiments with the specific antibodies showed no differences between the inactivation of pyruvate carboxylase from mitochondrial or soluble fractions of liver, kidney, mammary gland, brown adipose tissue or white adipose tissue. 4. The antibodies were relatively less effective in reactions against pyruvate carboxylase from sheep liver than against the enzyme from rat tissues. 5. Pyruvate carboxylase antibodies did not inactivate either propionyl-CoA carboxylase or acetyl-CoA carboxylase from rat liver. 6. It is concluded that pyruvate carboxylase in lipogenic tissues is similar antigenically to the enzyme in gluconeogenic tissues and that the soluble activities of pyruvate carboxylase detected in many rat tissues do not represent discrete enzymes but are the result of mitochondrial damage during tissue homogenization.

Physiological concentrations of lactate dehydrogenases and substrate inhibition.

Abstract: Lactate dehydrogenases at physiological concentrations are inhibited by high concentrations of pyruvate when the enzyme and the pyruvate are incubated in the presence of oxidized nicotinamide-adenine dinucleotide before assay. The inhibition is much more pronounced with the H-type than with the M-type lactate dehydrogenase. These results suggest that substrate inhibition may be operative in vivo.

Catalytic properties of the lactate dehydrogenase isozyme "X" from mouse testis.

Abstract: Lactate dehydrogenase (LDH) isozyme 1 (B4), 5 (A4), and “X” (Testis or Sperm type) have been partially purified from mouse tissues. The following studies were carried out on the three isozymes: Km and optimum substrate concentration for pyruvate, α-oxo-butyrate, α-oxo-valerate, lactate, α-OH-butyrate, and α-OH-valerate, inhibition by substrate and product, effect of malate, N-(4-carboxy-2-hydroxyphenyl) maleimide, some citric acid cycle metabolites, urea, trypsin and pH. The mouse testicular LDH isozyme clearly differs from the common lactate dehydrogenases and from the tesficular isozymes from other species. It shows distinct sensitivity to inhibition by substrate or product whether the direct (pyruvate lactate) or reverse reactions are studied. There is no effect of increasing concentrations of pyruvate or lactate on the direct reaction, while a clear inhibition by lactate or pyruvate is demonstrated on the reverse reaction. Citric acid cycle metabolites, specially malate and succinate, inhibit the direct reaction catalyzed by the “X” isozyme. These peculiar characteristics suggest a high degree of specialization for the principal testicular isozyme of lactate dehydrogenase.

Triglyceride hydrolysis and assay

Abstract: GLYCEROL ESTERS PRESENT IN AQUEOUS MEDIA, SUCH AS SERUM TRIGLYCERIDE AND MILK FAT, ARE ASSAYED BY COMPLETE ENZYMATIC HYDROLYSIS USING BOTH A LIPASE AND A PROTEASE, WHEREUPON THE LIBERATED GLYCEROL IS DETERMINED. A NUMBER OF ALTERNATIVE ENZYMATIC PROCEDURES ARE PROVIDED FOR THE GLYCEROL ASSAY, ALL IN THE ORGINAL AQUEOUS MEDIUM, SUCH AS CONVERSION TO GLYCEROL-1-PHOSPHATE WITH GLYCEROL KINASE; CONVERSION OF ATP TO ADP BY THE SAME RECTION FOLLOWED BY CONVERSION OF THE ADP PLUS PHOSPHOENOLPYRUVATE USING PYRUVATE KINASE TO ATP AND PYRUVATE ION FOLLOWED BY DETERMINING THE LATTER; AND ALTERNATIVELY CONVERTING THE PYRUVATE ION SO FORMED WITH NADH USING LACTATE DEHYDROGENASE TO LACTATE ION AND NAD AND DETERMINING THE LATTER BY OPTICAL MEASUREMENT.

Properties and regulation of pyruvate carboxylase from Bacillus stearothermophilus

Abstract: Pyruvate carboxylase has been purified 400-fold from the thermophile, Bacillus stearothermophilus; it resembles pyruvate carboxylases purified from mesophilic organisms in its general kinetic and regulatory properties. The enzyme is virtually inactive in the absence of acetylcoenzyme A ; this activating effect is antagonized by L-aspartate. Kinetic studies show that these two compounds act as allosteric effectors. ADP inhibits the enzyme activity competitively with ATP. Although the thermophile enzyme is appreciably more thermostable than similar mesophile enzymes, it is quite labile at the temperature at which the organism grows optimally, but can be stabilized by the two allosteric effectors and by some of the reactants.

The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

Abstract: The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved–re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved–re-fed animals. NNN′N′-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved–re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved–re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved–re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.

Characterization and Properties of the Pyruvate Phosphorylation System of Acetobacter xylinum

Abstract: The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.