Showing papers on "Pyruvate kinase published in 1982"

Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets.

Abstract: 1. The physiological response of rainbow trout (Salmo gairdneri) reared on different levels of available carbohydrate in practical trout diets having the same levels of energy and nitrogen for 16-24 weeks was determined. 2. Weight gain was significantly reduced in trout reared on the highest level of available carbohydrate, 210 g cerelose (alpha-glucose) kg, and there was a significant linear regression (R2 0.88 of dietary carbohydrate on weight gain. 3. Liver: body-weight values and liver glycogen levels increased in relation to increased dietary carbohydrate. 4. Liver glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity increased and liver phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity decreased per kg body-weight of fish with increasing dietary carbohydrate. However, no significant effect was noted on the activity of these liver enzymes above a dietary cerelose level of 140 g/kg. 5. Liver fructose diphosphatase (EC 3.1.3.11) activity increased with increasing dietary carbohydrate has been interpreted as meaning a recycling of triosephosphate to glucose-6-phosphate. 6. Dietary carbohydrate level had no significant effect on the liver pyruvate kinase (EC 2.7.1.40) activity, the rate of glucose utilization or the percentage conversion of [14C]alanine to glucose in the plasma of trout. 7. The results indicate that rainbow trout have a limited ability to adapt to increased dietary carbohydrate and a level in excess of 140 g/kg of the diet is not efficiently utilized.

Pyruvate kinase isozymes from rat

Abstract: Publisher Summary This chapter describes the tissue distribution, methods of assay, electrophoretic behavior, purification, and characterization of pyruvate kinase (PK) isozymes. The isozymes are called, L, R, M 1 , and M 2 . Two different assay methods are presented in the chapter: one is the simple colorimetric 2,4-dinitrophenylhydrazone method, in which pyruvic acid formed from phosphoenol pyruvate (PEP) by PK is determined with the reagent for carbonyl residues, 2,4-dinitrophenylhydrazine. The other method is the NADH–LDH coupled method in which PK activity is measured by following the oxidation of NADH at 340 nm using a recording spectrophotometer with a coupled assay system. M 1 is the main type in specifically differentiated tissues, such as adult skeletal muscle, heart, and brain. L is predominant in gluconeogenic tissues, especially liver, but is a minor type in kidney, while R is present in erythrocytes and hematopoietic tissues. The four types of PK differ clearly in electrophoretic mobilities. However, L and R, and M 1 and M 2 , respectively, are closely similar in certain properties, including their immunological and kinetic properties, phosphorylations by cAMP-dependent protein kinase, amino acid compositions, and peptide maps of limited proteolysis products.

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

Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics.

Abstract: 1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.

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.

Intermediary carbohydrate metabolism in protoscoleces of Echinococcus granulosus (horse and sheep strains) and E. multilocularis.

Abstract: With few exceptions, the specific activities of the glycolytic enzymes and the steady-state content of glycolytic and associated intermediates in protoscoleces of the horse (E.g.H) and sheep (E.g.S) strains of Echinococcus granulosus and the closely related E. multilocularis (E.m.) are very similar. Phosphorylase, hexokinase, phosphofructokinase and pyruvate kinase catalyse non-equilibrium reactions and the patterns of activity for pyruvate kinase, phosphoenolpyruvate carboxykinase and malic enzyme are similar in the three organisms. The levels of tricarboxylic acid cycle intermediates in E.g.H., E.g.S. and E.m. are of the same order as those reported in tissues with an active cycle. Each has a complete sequence of cycle enzymes but there are substantial differences between the three parasites with regard to the activity of individual enzymes, The activities of NAD and NADP-linked isocitrate dehydrogenases are significantly lower in E.g.H. than in E.g.S. and particularly in E.m. which suggests that the tricarboxylic acid cycle may play a more important role in carbohydrate metabolism and energy production in the latter parasites. Nevertheless, the three organisms utilize fermentative pathways for alternative energy production, fix carbon dioxide via phosphoenolpyruvate carboxykinase and have a partial reversed tricarboxylic acid cycle. It is speculated that in vivo more carbon will be channelled towards oxaloacetate than pyruvate at the phosphoenolpyruvate branch point. The steady state content of ATP and the ATP/AMP ratios are low in the three organisms, suggesting a low rate of ATP utilization in each.

Phosphoenolpyruvate synthetase inMethanobacterium thermoautotrophicum

Abstract: The thermophilic autotrophMethanobacterium thermoautotrophicum assimilates CO2 via a novel pathway rather than via the Calvin cycle. The central intermediate of this pathway is acetyl CoA which is reductively carboxylated to pyruvate. Cell extracts of the organism contained phosphoenolpyruvate synthetase with a specific activity of 100 nmol min-1 mg-1 protein (65°C). Pyruvate kinase and pyruvate, phosphate dikinase were not detected. Phosphoenolpyruvate synthetase was partially purified (50-fold) and the following reaction stoichiometry was established: $${\text{Pyruvate + ATP + H}}_{\text{2}} {\text{O }} \to {\text{ Phosphoenolpyruvate + AMP + P}}_{\text{i}} $$ The enzyme activity was depedent on free Mg2+ ions, NH 4 + or K+ ions, and SH-groups. Mn2+, but not Ca2+, could partially substitute for Mg2+; Na+ could not substitute for K+ or NH 4 + . The pH-optima,V max-values and the apparentK M-values for the substrates of the enzyme in both directions were determined. Thermodynamic, kinetic and regulatory features indicate that, in vivo, the enzyme functions in the direction of phosphoenolpyruvate synthesis from pyruvate. Not only is the synthesis of phosphoenolpyruvate via the PEP synthetase reaction energetically favorable; the enzyme also catalyzed this synthesis 100 times faster than the reverse reaction, the apparentK M value for pyruvate (40 μM) being low and the apparentK M value for phosphate (100 mM) being high. Furthermore, AMP, ADP, PP and α-ketoglutarate were inhibitors of PEP synthesis, indicating that the enzyme activity may be controlled in vivo. The role of phosphoenolpyruvate synthetase in autotrophic CO2 assimilation pathway ofMethanobacterium, as expected from previous labelling studies, is confirmed.

Expression of multimolecular forms of pyruvate kinase in normal, benign, and malignant human breast tissue

Abstract: The levels of multimolecular forms of pyruvate kinase present in four normal human breast specimens, nine benign tissues, and 13 malignant breast carcinomas were determined. The different enzymatic forms were separated by isoelectrofocusing, quantitated photometrically, and characterized further by kinetic studies using phosphoenolpyruvate as the variable substrate in the presence of different effectors. A correlation between specific activity and malignancy was found. The mean specific activities (+/- S.E.) of the normal, benign, and malignant tissues were: 0.078 +/- 0.006, 0.36 +/- 0.072, and 3.50 +/- 0.696 IU/mg protein, respectively. A form of pyruvate kinase with an isoelectric point (pI) of 7.0 predominated in the breast tissues. The properties of this form were consistent with it being the K4 isozyme, known to be widely distributed in mammalian tissues. Higher pI forms were also found. The M4 isozyme, expressed by normal muscle and brain, has a pI value similar to the highest pI form found in the breast tissues. Therefore, the pI data alone suggest that the breast specimens also express some M-type subunits. This conclusion was not supported by the kinetic data. The higher pI forms are thought to be a posttranslationally modified K isozyme. Although this modified form is found in normal specimens, it seems more prevalent in neoplasms.

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.

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

Characterization of Some Glycolytic Enzymes from Human Retina and Retinoblastoma

Abstract: Glycolytic enzymes were studied from normal human retinas (both fetal and adult) and from retinoblastomas of eight patients and an established retinoblastoma cell line. No significant differences were found between the enzyme activities in the tissues investigated except for hexokinase and pyruvate kinase, which were significantly decreased in the tumor cells. In fetal retina, five different forms of pyruvate kinase could be detected by electrophoresis (K 4 , K 3 M, K 2 M 2 , KM 3 , and M 4 ). In adult retina the K 4 isozyme is almost absent, while in retino-blastoma the M 4 isozyme is hardly present. In the retinoblastoma cell line, the M 4 isozyme is completely absent. Alanine inhibition of pyruvate kinase is in agreement with the electrophoretic pattern. Pyruvate kinase from the retinoblastoma cell line is more inhibited compared to the pyruvate kinase of fetal retina and retinoblastoma and is even more inhibited compared to adult retina. Electrophoresis of aldolase from adult retina revealed the presence of all potential A–C hybrids (A 4 , A 3 C, A 2 C 2 , AC 3 , and C 4 ). Fetal retina, however, is characterized by the predominance of the A type. The same patterns were observed in the retinoblastoma cell line and retinoblastoma. However, in other brain tumors, e.g., gliomas of adults, a five-membered A–C hybrid set is found. Electrophoresis of hexokinase from normal fetal and adult retina revealed the predominance of hexokinase type I; retinoblastoma and retinoblastoma cell line are both characterized by the presence of considerable amounts of hexokinase type II. The isozyme shifts in retinoblastoma result in an enzyme pattern identical to that of fetal retina except for the presence of hexokinase type II.

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.

The Deficient Carbohydrate Metabolic Pathways and the Incomplete Tricarboxylic Acid Cycle in an Obligately Autotrophic Hydrogen-oxidizing Bacterium

Abstract: The activities of enzymes of carbohydrate metabolism, enzymes of the tricarboxylic acid cycle and some related enzymes were measured in cell-free extracts of strain TK-6, an extremely thermophilic, obligately autotrophic, aerobic hydrogen-oxidizing bacterium. Activities of phosphofructokinase, aldolase, pyruvate kinase, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the Embden-Meyerhof and the Entner-Doudoroff pathways were not found in the extracts. All of the tricarboxylic acid cycle enzymes except α-ketoglutarate dehydrogenase, and reduced nicotinamide adenine dinucleotide oxidase were present. These metabolic defects are considered to be one of the reasons for the obligate autotrophy of strain TK-6.

Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle

Abstract: Phosphoenolpyruvate carboxykinase activity in crude extracts of muscle has frequently been determined by using a continuous spectrophotometric method, which is shown to grossly overestimate enzyme activity. NADH oxidation attributed to phosphoenolpyruvate carboxykinase activity in the assay is due to lactate production. Under the normal assay conditions. Na+ ions stimulate pyruvate kinase, providing pyruvate for lactate formation by lactate dehydrogenase and sufficiently to account for most of the observed NADH oxidation.