Showing papers on "Pyruvate dehydrogenase kinase published in 1982"

Effects of altered [ADP-ribose]n metabolism on expression of fetal functions by adult hepatocytes

Abstract: The nuclear enzyme ADP-ribosyltransferase (ADPRT) catalyses the postsynthetic modification of chromatin proteins1 The significance of [ADP-ribose]n-modified chromatin proteins for the regulation of chromatin function is not understood1 Recently, [ADP-ribose]n biosynthesis has been demonstrated to participate in the repair of some types of DNA damage2–4 Here we demonstrate that another function of [ADP-ribose]n biosynthesis is related to the regulation of the expression of two fetal functions of cultured hepatocytes—the K-type (III) isozyme of pyruvate kinase, and γ-glutamyltranspeptidase, which is also a marker of early neoplastic transformation of liver cells5

Genetic analysis of the pyruvate decarboxylase reaction in yeast glycolysis.

Abstract: Six different pyruvate decarboxylase mutants of Saccharomyces cerevisiae were isolated. They belong to two unlinked complementation groups. Evidence is presented that one group is affected in a structural gene. The fact that five of the six mutants had residual pyruvate decarboxylase activity provided the opportunity for an intensive physiological characterization. It was shown that the loss of enzyme activity in vitro is reflected in a lower fermentation rate, an increased pyruvate secretion, and slower growth on a 2% glucose medium. The different effects of antimycin A on leaky mutants grown on ethanol versus the same mutants grown on glucose support the view that glucose induces some of the glycolytic enzymes, especially pyruvate decarboxylase.

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney.

Abstract: Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

Purification of rat kidney branched-chain oxo acid dehydrogenase complex with endogenous kinase activity.

Abstract: A method was devised to purify branched-chain oxo acid dehydrogenase (BCOAD) from rat kidney which retains endogenous kinase activity. Incorporation of 32P into purified enzyme parallels the time course of enzyme inhibition by ATP. Phosphorylation occurs on a serine residue(s) of the 46000-mol.wt. subunit of the enzyme complex. Endogenous phosphatase activity is not present after purification, and added pyruvate dehydrogenase phosphate phosphatase does not re-activate BCOAD or liberate 32P from previously labelled enzyme. These results demonstrate that BCOAD can be regulated by an endogenous protein kinase and that the phosphorylation-cycle enzymes regulating BCOAD appear to be distinct from those associated with pyruvate dehydrogenase complex.

AMP- and fructose 1,6-bisphosphate-activated pyruvate kinases from Escherichia coli

Abstract: Publisher Summary This chapter presents an assay method, purification, and properties of AMP- and fructose 1,6-bisphosphate-activated pyruvate kinases from Escherichia coli . Two noninterconvertible forms of pyruvate kinase are detected in Escherichia coli. Both show positive cooperative effects with respect to the substrate phosphoenolpyruvate; one of them is activated by fructose 1,6-bisphosphate and inhibited by ATP and succinyl-CoA, whereas the second is activated by AMP and by several intermediates of the hexose phosphate pathway. The approaches successfully used for the continuous assay of E. coli pyruvate kinase include both direct and coupled methods. The chapter discusses the lactate dehydrogenase coupled assay, because it is the most widely used and requires minimal amount of enzyme. The different physical and kinetic properties of the two forms of pyruvate kinase from E. coli allow differential assay in crude extracts containing both. The purification steps involved include preparation of the crude extract and diethylaminoethyl (DEAE)-cellulose chromatography. Further purification of type 1 pyruvate kinase is done following heat treatment, affinity chromatography on phosphocellulose, Sephacryl S-200 chromatography, and DEAE-Sephadex chromatography. Further purification of type II pyruvate kinase involves affinity chromatography on phosphocellulose and DEAE-Sephadex chromatography.

Putative mediators of insulin action: regulation of pyruvate dehydrogenase and adenylate cyclase activities

Abstract: Recent evidence suggests that certain actions of insulin may be mediated by the selective generation of chemically undefined intracellular substances. Incubation of rat liver particulate fraction with low concentrations of insulin enhances the release into the supernatant of a substance that stimulates mitochondrial pyruvate dehydrogenase. Higher concentrations of insulin release less stimulating activity. It is possible to resolve activities that stimulate and inhibit pyruvate dehydrogenase by differential ethanol extraction of the supernatant solutions. The elaboration of both factors is dependent upon the presence of insulin in a dose-dependent manner. Moreover, fractions that contain the pyruvate dehydrogenase-inhibiting activity also inhibit adipocyte basal and hormonally stimulated adenylate cyclase. The production of this adenylate cyclase inhibitory activity is also stimulated by insulin. Cyclase inhibition is virtually abolished when the nonhydrolyzable ATP analog, 5'-adenylyl imidodiphosphate, is included in the assay. These results indicate that the bimodal effects of insulin on certain functions may be ascribed to the generation of at least two distinct chemical substances that show opposing activities, which may operate by regulating phosphorylation reactions.

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

Abstract: 1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.

Pyruvate dehydrogenase kinase/activator in rat heart mitochondria, Assay, effect of starvation, and effect of protein-synthesis inhibitors of starvation.

Abstract: Purified pig heart pyruvate dehydrogenase complex is denuded of its intrinsic pyruvate dehydrogenase kinase activity by sedimentation from dilute solution (60 munits/ml). Kinase activity is restored by a supernatant fraction prepared by high-speed centrifugation of rat heart mitochondrial extracts; the factor responsible is referred to as kinase/activator. Kinase/activator was also assayed by its ability to accelerate NgATP-induced inactivation in dilute solutions of unprocessed complex (50 munits/ml). With this assay it has been shown that the activity of kinase/activator in heart mitochondria is increased 3-6 fold by starvation of rats for 48 h. This increase was prevented completely by cycloheximide treatment and prevented partially by puromycin treatment of rats during starvation. The concentration of kinase/activator in heart mitochondria fell during 20 h of re-feeding of 48 h-starved rats; this fall was correlated with an increase in the proportion of complex in the active form. Kinase/activator was also extracted from ox kidney mitochondria, and on gel filtration (Sephadex G-100, superfine grade) was eluted close to the void volume. Kinase/activator (ox kidney or rat heart) was thermolabile, non-diffusable on dialysis, and inactivated by trypsin. The results of this study appear to show increased cytoplasmic synthesis in starvation of pyruvate dehydrogenase kinase and/or of an activator of the kinase.

Studies on the pyruvate dehydrogenase complex in brain with the arylamine acetyltransferase-coupled assay.

Abstract: A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCl2, and partially on thiamine pyrophosphate. Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal to maximum recorded rates of pyruvate flux for brain in vivo. Activities as low as 0.6 nmol/min could be measured. Use of ArAT at different purities (I--2-fold and II--55-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4 degrees C or at -20 degrees C for 3 days or at +20 degrees C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.

[65] Pyruvate dehydrogenase complex from bovine kidney and heart

Abstract: Publisher Summary In eukaryotic cells, the pyruvate dehydrogenase complex is located in mitochondria, within the inner membrane-matrix compartment The complex consists of three catalytic components: pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase These three enzymes, acting in sequence, catalyze the overall reaction The Activity of this multienzyme complex is assayed spectrophotometrically by the measurement of nicotinamide adenine dinucleotide dehydrogenase (NADH) production The assay is used routinely at all the levels of purity of the enzyme complex, beginning with mitochondrial extracts prepared by the freeze-thaw procedure However, the assay is less satisfactory with crude extracts that contain lactate dehydrogenase or NADH oxidase With these preparations, 14 CO 2 production from pyruvate is measured, or the overall reaction is coupled with the arylamine acetyltransferase-catalyzed reaction The acetylation of an appropriate arylamine is measured spectrophotometrically The α subunit of pyruvate dehydrogenase and the subunit of dihydrolipoyl transacetylase are very sensitive to proteolysis The steps involved in the purification of pyruvate dehydrogenase are also discussed in the chapter

Pyruvate kinase: is the mechanism of phospho transfer associative or dissociative?

Abstract: To test for the possibility that pyruvate kinase proceeds via a dissociative path, we have investigated whether the complex enzyme . ADP . metaphosphate is transiently formed from the complex enzyme . ATP. It is shown that when highly purified pyruvate kinase is used, the rate of positional oxygen isotope exchange in ATP (beta, gamma bridge and beta nonbridge) is about 10(4) times slower in the absence of the cosubstrate pyruvate than it is in the presence of pyruvate. Further, the rate of racemization of the gamma-phospho group of [gamma (S)-16O,17O,18O]-ATP is undetectable, being at least 30 times slower even than the rate of positional isotope exchange. These tests thus provide no evidence that pyruvate kinase follows a dissociative mechanism. Indeed, it is argued that the available data are more consistent with an associative path. Evidence is presented that the single, associative, transition state is symmetrical, in which bond making and bond breaking processes are rather precisely balanced.

Pyruvate kinases from human erythrocytes and liver

Abstract: Publisher Summary This chapter describes an assay method and the purification procedure for pyruvate kinases from human erythrocytes and liver. Pyruvate kinase activity is measured by coupling with lactate dehydrogenase, which transforms pyruvate into lactate and oxidizes NADH into NAD; the oxidation of NADH is followed at 340 nm. The purification procedure involves preparation of the Dextran Blue–Sepharose column, isolation of human erythrocyte pyruvate kinase, hemolysate, ammonium sulfate fractionation, and Dextran Blue–Sepharose 4B chromatography. Erythrocytes contain two active forms of pyruvate kinase that can be electrophoretically distinguished. On isoelectric focusing, nondissociated enzymes exhibit multiple active forms with isoelectric pH values between 5.85 and 6.69; liver enzyme forms are slightly more acidic than the erythrocyte forms. Erythrocyte and liver L-type pyruvate kinases are allosteric enzymes with fructose-l,6-P 2 as the major allosteric activator and adenosine triphosphate (ATP) and alanine as the major allosteric inhibitors.

Regulation of glycolysis and fatty acid synthesis from glucose in sheep adipose tissue

Abstract: 1. The following were measured in adipose-tissue pieces, obtained from 7–9 month-old sheep, before or after the tissue pieces had been maintained in tissue culture for 24 h: the rates of synthesis from glucose of fatty acids, acylglycerol glycerol, pyruvate and lactate; the rate of glucose oxidation to CO2; the rate of glucose oxidation via the pentose phosphate pathway; the activities of hexokinase, glucose 6-phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, pyruvate dehydrogenase and ATP citrate lyase; the intra- and extra-cellular water content; the concentration of various metabolites and ATP, ADP and AMP. 2. The proportion of glucose carbon converted into the various products in sheep adipose tissue differs markedly from that observed in rat adipose tissue. 3. There was a general increase in the rate of glucose utilization by the adipose-tissue pieces after maintenance in tissue culture; largest changes were seen in the rates of glycolysis and fatty acid synthesis from glucose. These increases are paralleled by an increase in pyruvate kinase activity. There was no change in the activities of the other enzymes as measured, although the net flux through all the enzymes increased. 4. Incubation of fresh adipose-tissue pieces for 2–6h led to an increase in the affinity of pyruvate kinase for phosphoenolpyruvate. 5. The rate of pyruvate production by glycolysis was greater than the activity of pyruvate dehydrogenase of the tissue. 6. The results suggest that both pyruvate kinase and pyruvate dehydrogenase have important roles in restricting the utilization of glucose carbon for fatty acid synthesis in sheep adipose tissue.

Role of pyruvate carboxylation in the energy-linked regulation of pool sizes of tricarboxylic acid-cycle intermediates in the myocardium.

Abstract: The increase in the metabolite pool size of the tricarboxylic acid cycle in the isolated perfused rat heart after a decrease in the ATP consumption by KCl-induced arrest was used to study the anaplerotic mechanisms. During net anaplerosis the label incorporation into the tricarboxylic acid-cycle intermediates from [1-14C]pyruvate increased and occurred mainly by pathways not involving prior release of the label to CO2. A method for determination of the specific radioactivity of mitochondrial pyruvate was devised, and the results corroborated the notion that tissue alanine can be used as an indicator of the specific radioactivity of intracellular pyruvate.

Purification of Bovine Kidney and Heart Pyruvate Dehydrogenaseb Phosphatase on Sepharose Derivatized with the Pyruvate Dehydrogenase Complex

Abstract: Pyruvate dehydrogenase phosphatase has been purified to apparent homogeneity from mitochondrial extracts of both beef heart and beef kidney. An essential step in this three-step purification is affinity chromatography of a largely purified phosphatase fraction using Sepharose beads to which pyruvate dehydrogenase complex is covalently bound through the lipoic acid residues of the dihydrolipoyl transacetylase component of the complex. The purified phosphatase, which has a native relative molecular mass, Mr, of about 140000, is composed of two nonidentical subunits of Mr 89000 and 49000.

Insulin mediates the stimulation of pyruvate kinase by a dual mechanism

Abstract: A radioimmunoassay specific for liver pyruvate kinase was used to determine the mechanism(s) involved in the insulin stimulation of this enzyme activity in chronically diabetic rats. Rats, made diabetic with alloxan, were fed on a high-carbohydrate (50%-sucrose) fat-free diet and treated with insulin for 12, 36 or 60 h. Livers were removed at the various times, a piece was kept for determination of glycogen, and the remainder was homogenized. The 100000 g supernatant was prepared and used for determination of pyruvate kinase activity and quantity. Glycogen increased to a maximum of approx. 7% by 12 h after insulin treatment, and was maintained at this elevated value for 60 h. Liver pyruvate kinase activity, which is depressed in diabetes, did not respond to insulin until 36 h of treatment, with a more substantial increase occurring by 60 h. Radioimmunoassay data indicated that the increase in activity was concomitant with a substantial increase in the quantity of the enzyme and a moderate increase in its specific activity. These results demonstrate that a dual mechanism, i.e. an increase in both the quantity and specific activity of the enzyme, regulates the insulin-mediated stimulation of liver pyruvate kinase in the diabetic rat.

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase.

Abstract: Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle

[61] Lactate dehydrogenase isozymes from mouse

Abstract: Publisher Summary This chapter describes the assay method and properties of lactate dehydrogenase isozymes isolated from the mouse. Three homotetrameric isozymes of lactate dehydrogenase, designated as A 4 , B 4 , and C 4 or X, are found in mice. Each isozyme is expressed by a separate structural gene. All the lactate dehydrogenase isozymes require nicotinamide adenine dinucleotide kinase (NAD + ) or nicotinamide adenine dinucleotide dehydrogenase (NADH) as the coenzyme and catalyze the same chemical reactions. The purification of these enzymes is achieved by general ligand affinity chromatography in combination with ion-exchange chromatography to separate the multiple forms of lactate dehydrogenase. The activity of lactate dehydrogenase isozymes is determined at 25°C spectrophotometrically by following the changes in absorbance at 340 nm. The reaction mixture in a total volume of 1 ml contains 0.1 M Tris-HCl, pH 8.0, 1.0 m M pyruvate, 0.15 m M NADH, and a suitable amount of enzyme to produce a decrease in the absorbance of 0.05–0.l per minute at 340 nm. Lactate dehydrogenase isozymes are virtually inactive with α-ketoglutarate as the substrate. Thin-layer chromatography of the product is performed on cellulose plates with a fluorescent indicator.

Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange

Abstract: Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffective. Hydrogen peroxide and t-butyl hydroperoxide were inactive. The data indicate pyruvate dehydrogenase kinase (EC 2.7.1.99) contains a thiol group (or groups) that is involved in maintaining a conformation of the enzyme that facilitates phosphorylation and inactivation of its protein substrate, pyruvate dehydrogenase (EC 1.2.4.1). These findings suggest that modulation of pyruvate dehydrogenase kinase activity by thiol-disulfide exchange may be an important physiological mechanism for regulation of kinase activity and, hence, activity of the pyruvate dehydrogenase complex.

Purification and properties of pyruvate kinase from Streptococcus mutans.

Abstract: Pyruvate kinase (EC 2.7.1.40) from Streptococcus mutans strain JC2 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 180,000 to 190,000, and the enzyme was considered to consist of four identical subunits. This enzyme was completely dependent on glucose 6-phosphate for activity, and the saturation curve for activation by glucose 6-phosphate was sigmoidal. In the presence of 0.5 mM glucose 6-phosphate, the saturation curves for the substrates phosphoenolpyruvate and ADP were hyperbolic, and the Km values were 0.22 and 0.39 mM, respectively. GDP, IDP, and UDP could replace ADP, and the Km for GDP (0.026 mM) was 0.067 of that for ADP. The enzyme required not only divalent cations, Mg2+ or Mn2+, but also monovalent cations, 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 glucose 6-phosphate increased. However, the enzyme was immediately inactivated in a solution without Pi. The intracellular concentration of glucose 6-phosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. mutans.