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PubMed1984

The Pk-3 gene determines both the heart, M1, and the kidney, M2, pyruvate kinase isozymes in the mouse; and a simple electrophoretic method for separating phosphoglucomutase-3.

We have found that in mice carrying Pk-3r, an allele leading to loss or activity of kidney pyruvate kinase, the activity of heart pyruvate kinase is also diminished. Electrophoretic studies on tissues from mice carrying Pk-3r and/or Pk-3b, an allele determining an electrophoretically detectable variant, show that Pk-3 affects the expression of both the heart, M1, and the kidney, M2, pyruvate kinase isozymes. These results, together with linkage data, indicate that both isozymes are determined by the same structural gene, Pk-3. We also report a simple method for separating phosphoglucomutase-3 (PGM-3) by electrophoresis on cellulose acetate plates.

Animals↗
PubMed1981

The regulatory properties of rabbit muscle pyruvate kinase. The effect of pH.

The regulatory behavior of rabbit pyruvate kinase has been studied as a function of pH. The initial velocity of the enzyme-catalysed reaction as a function of ADP concentration was analysed with the exponential model for a regulatory enzyme. The analysis of the exponential model parameters as functions of pH provided pK values of 6.6 and 8.08 for the free enzyme in its fully ADP-bound conformation. By contrast, the binding of ADP to the ADP-free conformation of the free enzyme did not involve groups that ionize within the pH range (6.2-8.5) of these experiments. The results suggest that homotropic allosteric interactions actually alter the mode of ADP binding. The pK values of 6.63 and 9.00 determined from the analysis of V as a function of pH are readily interpreted in terms of a direct phosphoryl-transfer mechanism in which the beta-phosphoryl group of ADP (pK 6.63) acts as the nucleophile and a lysine epsilon-amino group (pK 9.0) acts as the proton donor in the pyruvate kinase reaction.

Adenosine Diphosphate↗
PubMed1979

Significance of the electrophoretic modifications of defective pyruvate kinase variants. Study of six new observations.

Six new defective pyruvate kinase variants have been characterized in patients suffering from chronic hemolysis. Partially purified enzyme variants exhibited various anomalies from immunological, kinetic, stability and electrophoretic points of view. The significance of the electrophoretic anomalies has been interpreted in view of the normal post-synthetic maturation of the precursor enzyme L'4 into L2L'2 and L4, and the ability of trypsin to induce in vitro the transition L'4 leads to L4 has been tested. One defective enzyme existed in a single L'4 form and could not be transformed by trypsin into L4. In three cases slow-moving L'4 and L2L'2 forms were transformed by trypsin into an abnormal slow-moving L4 form. In the last two observations the L'4 and L2L'2 forms exhibited normal mobility and were normally transformed by trypsin into L4. The relevance of these data to the functional anomalies of the defective variants and to the nature of the primary genetic anomaly giving rise to the congenital defects in erythrocyte pyruvate kinase is discussed.

Anemia, Hemolytic, Congenital↗
PubMed1992

Pyruvate kinase from Trichomonas vaginalis, an allosteric enzyme stimulated by ribose 5-phosphate and glycerate 3-phosphate.

Trichomonas vaginalis pyruvate kinase was purified over 1750 fold to a specific activity greater than 100 mumol min-1 (mg protein)-1. The enzyme is a tetramer of M(r) 266,000, consisting of subunits of M(r) 53,000 and 56,000 in equivalent amounts. Its activity was dependent on the presence of magnesium but was not stimulated by potassium or ammonium. The enzyme exhibited positive cooperativity towards phosphoenolpyruvate and was inhibited by inorganic phosphate, which increased the sigmoidicity of the saturation curve for phosphoenolpyruvate without affecting maximal activity. It was heterotropically stimulated by ribose 5-phosphate and glycerate 3-phosphate, not previously known to act on eukaryotic pyruvate kinases, but was unaffected by known effectors of most pyruvate kinases, including fructose 1,6-bisphosphate and fructose 2,6-bisphosphate.

Allosteric Regulation↗
PubMed1980

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase.

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.

Animals↗
PubMedDate not supplied

Quantification of tumor type M2 pyruvate kinase (Tu M2-PK) in human carcinomas.

Proliferating and tumor cells express a certain isoenzyme of pyruvate kinase, called PK type M2. This isoenzyme can be isolated in an active tetrameric and an inactive dimeric form. We have termed this form tumor type M2-PK. This tumor type pyruvate kinase can be quantified by a specific ELISA in blood sera and tumor homogenates. In this study we have compared 26 normal colon mucosa and colon cancer specimens from the same patients. The total specific pyruvate kinase activity and the amount of the tumour type M2-PK measured by ELISA was increased in the tumor samples compared to the normal colon mucosa of the same patient. In normal colon mucosa the specific PK-activity ranged between 0.21 and 1.25 U/mg protein whereas in colon carcinoma we found activities between 0.99 and 7.08 U/mg. The amount of tumor M2-PK measured by ELISA ranged between 0.82 and 27.10 U/mg protein in normal colon mucosa and between 1.96 and 242.40 U/mg protein in colon carcinoma. The tumor M2-PK content in the serum of 666 healthy blood donors was measured by ELISA and compared to sera from 15 colon carcinoma patients and showed a highly significant difference (Mann-Whitney rank sum test, p < 0.001). The values for the 50%-percentiles (median) of blood donors were 10.8 U/ml and 55.0 U/ml for colon carcinoma.

Adenocarcinoma↗
PubMed1980

Properties of pyruvate kinase and flagellar ATPase in rabbit spermatozoa: relation to metabolic strategy of the sperm cell.

Rabbit sperm pyruvate kinase remains bound to the cell structure of hypotonically treated mature rabbit epididymal spermatozoa (HTRES). It displays kinetic behavior very similar to that of rabbit muscle pyruvate kinase with regard to KM values for substrates, activation by monovalent and divalent cations, inhibition by phenylalanine which is reversed by alanine, and lack of activation by fructose-1,6-biphosphate. The flagellar ATPase also remains bound to the cell structure of HTRES, whose motility may be reactivated by a source of ATP. It requires Mg+2 for activity; the KM for both ATP and MG+2 is 0.2 mM, implying that MgATP is the substrate. The ATPase activity is not inhibited by ouabain, oligomycin, or vanadate, which also do not affect reconstituted motility, and is not affected by cyclic AMP in the presence of an inhibitor of phosphodiesterase. The activities of pyruvate kinase and the flagellar ATPase in a given preparation of HTRES are comparable. Rabbit spermatozoa have a metabolic strategy which is very similar to muscle cells. This suggests that the major use of the sperm cell's metabolic machinery is maintenance of energy for the contractile work of motility and that only minor amounts of metabolic energy appear to be consumed in other reactions, including those involved in fertilization.

Adenosine Triphosphatases↗
PubMed1988

Plasma pyruvate kinase activity vs creatine kinase activity as an indicator of the porcine stress syndrome.

Plasma pyruvate kinase (PK) and creatine kinase (CK) activities were increased significantly (P less than 0.001 and P less than 0.05, respectively) in homozygote halothane-reacting pigs (nn), compared with those activities in homozygote nonreacting pigs (NN). Pyruvate kinase activity was less variable within groups than was CK activity, allowing more effective discrimination between nn and NN geno-types. The PK and CK activities in plasma increase with age in halothane-reacting pigs and the nonreacting pigs. Enzyme activities in heterozygote (Nn) nonreacting pigs did not differ significantly (P greater than 0.05) from enzyme activities of homozygote (NN) nonreacting pigs. Although PK was better than CK in identifying stress-susceptible pigs, age-related effects and the failure to identify heterozygotes may restrict the use of plasma PK activity as a diagnostic test for the stress syndrome.

Animals↗
PubMed1990

Kinetic studies of pyruvate kinase during in vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice.

Pyruvate kinase studies in the granulocyte-macrophage lineage during in vitro differentiation have been performed using culture techniques on GM-CFC cells and a study has also been done in bone marrow cells. The enzyme exhibits biphasic behaviour with respect to both of its substrates in cells derived from in vitro cultures at 5 and 7 days of incubation period. However in bone marrow cells these kinetics are only observed for ADP. The different kinetic behaviour of pyruvate kinase toward Fru-1,6-P2, Ala, Phe and ATP in the three cellular populations allows us to conclude that the expression of pyruvate kinase is associated with the differentiation of these cells.

Adenosine Diphosphate↗
PubMed1975

Effect of lipopolysaccharide and lipid A on mouse liver pyruvate kinase activity.

Several investigators have reported lipid A as the biologically active unit in the lipopolysaccharide (LPS) molecule. To determine if lipid A was responsible for the reported increases in pyruvate kinase, mice were injected with endotoxin from Salmonella typhimurium SR-11, the Re mutant of Salmonella minnesota R 595, and lipid A-bovine serum albumin conjugate. The livers were homogenized and the activity of pyruvate kinase was measured. Similar increases in enzyme were obtained with all three preparations. These data imply that the lipid portion of the LPS molecule was responsible for alterations in host enzyme activity. To further determine if the lipid portion was the active unit, a lipid-degraded endotoxin (endotoxoid) prepared by potassium methylate treatment was inoculated into mice. An initial increase in liver pyruvate kinase activity was observed with all preparations. The marked increase observed at 16 h with the native product and lipid A conjugate was not obtained with the endotoxoid. These experiments extend and confirm previous observations that lipid A is responsible for the effects associated with LPS. Animals tolerant to endotoxin from S. typhimurium SR-11 were challenged with endotoxin from the Re mutant. A significant increase in pyruvate kinase activity was not obtained, suggesting that anti-O antibodies are not important in the development of tolerance.

Animals↗
PubMedDate not supplied

Influence of inorganic pyrophosphate on the kinetics of muscle pyruvate kinase: a simple nonallosteric feedback model.

Potassium pyrophosphate was used instead of ATP as a model ligand for magnesium cation for the study of effector influence on the kinetics of pyruvate kinase muscle isozyme M(1). The pyruvate kinase activation by low concentration of pyrophosphate and inhibition by high concentration of pyrophosphate was considered to be the result of reversible reactions of magnesium cation with pyrophosphate, ADP, ATP, and PEP. The apparent K(m) and V(m) or in some cases the pseudo-first order reaction rate constant (instead of K(m) and V(m)) of pyruvate kinase at any given pyrophosphate concentration were analysed as a function of concentration of free magnesium cation and its complexes with all ligands present in an assay mixture. The functions of reaction parameters with respect to concentration of magnesium complexes indicate the coexistence in the reaction mixture of simple and mixed complexes of magnesium cation with substrates, pyrophosphate, and an enzyme-substrate complex. The parameters of the simulated reaction for the proposed interactions fit the measured experimental data. A simple model with nonallosteric feedback has been proposed. According to this model, mutual and simultaneous interactions of reaction products with substrates and with an enzyme result in the coexistence of simple and mixed, labile and inert complexes.

Adenosine Triphosphate↗
PubMed1994

Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate and adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate. New nucleotide affinity labels which react with rabbit muscle pyruvate kinase.

Three new reactive nucleotide analogues with bromo-keto substituents adjacent to a thiophosphate have been synthesized. Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (GMPS-BDB), reacts covalently with rabbit muscle pyruvate kinase with complete inactivation and incorporation of 1.8 mol of reagent/mol of enzyme subunit. By contrast, the mono-keto compound, guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPS-BOP), causes no loss of pyruvate kinase activity. When the analogous adenosyl nucleotide derivatives are incubated with pyruvate kinase, the di-keto compound, adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (AMPS-BDB), rapidly effects inactivation, whereas the mono-keto compound, adenosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (AMPS-BOP), causes no loss of activity. Complete protection against inactivation by GMPS-BDB is provided by phosphoenolpyruvate in the presence of K+ and Mn2+ and the amount of reagent incorporated (0.9 mol/reagent/mol subunit) is reduced to half that observed in the absence of protectants. Gas-phase sequencing of the tryptic peptides purified from inactive GMPS-BDB or AMPS-BDB-modified enzyme gave the cysteine-labeled peptides: C151DENILWLDYK161, and N162IC164K165 as the two major peptide products, with a smaller amount of N43TGIIC48TIGPASR55. Reaction in the presence of the protectants PEP, K+, and Mn2+ yielded Cys164 as the only labeled residue, indicating that inactivation is primarily due to modification of Cys151. We propose that GMPS-BDB (or AMPS-BDB), which may exist in enolized form in aqueous solution, functions as a reactive analogue of phosphoenolpyruvate and GDP (ADP) to target Cys151 in the active site of pyruvate kinase.

Adenosine Diphosphate↗
PubMed1978

Mammalian pyruvate kinase hybrid isozymes: tissue distribution and physiological significance.

The purpose of this study was to examine the pyruvate kinase isozymic patterns of a wide variety of tissues from rats and mice, particularly regarding hybrid isozymes. For these studies, we employed longer electrophoresis times than used in most earlier studies in order to improve the resolution of closely spaced bands. The tissue distributions of types K, L, and M pyruvate kinases were found to be approximately the same as those reported earlier for rats and other mammals. In addition, K-M hybrids could be detected in most tissues examined in relative quantities which differed from one tissue to another in the same organism, in corresponding tissues from different species, and within a single tissue during development. Hybrid isozymes containing type L subunits occur in only a few tissues of either the fetus or the adult of either animal. In earlier studies utilizing L-M hybrid isozymes produced in vitro, we showed that the kinetic properties of a given subunit are profoundly affected by the nature of its neighbors within the tetramer (Dyson and Cardenas, ['73] J. Biol. Chem., 248: 8482-8488). Based on these altered kinetic properties, we suggest that there is little need for anorganism to suppress completely the gene activity for one subunit type of pyruvate kinase during the synthesis of larger quantities of a second subunit type.

Animals↗
PubMed1979

Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase.

Certain phosphocreatine preparations contain a contaminant that inhibits phosphofructokinase and pyruvate kinase assays. The contaminant can be separated from phosphocreatine by anion exchange chromatography. After appropriate purification, phosphocreatine has no effect on phosphofructokinase or pyruvate kinase; thus, there is no evidence that it serves muscle as a regulator of these enzymes. Although the inhibitory preparations of phosphocreatine contain inorganic phosphate and trace amounts of more negatively charged phosphorylated contaminants, the inhibitor is not inorganic phosphate or pyrophosphate. The nature of the inhibitor remains to be determined.

Animals↗
PubMed1990

[Studies on pyruvate kinase from pig dental pulp and brain].

Most of the enzymes involved in glycolysis are readily reversible and are also active in gluconeogenesis. However, three reaction steps are irreversible, i.e., those catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase; for in each of these reactions there occurs a large negative free-energy change, and these are reactions thus bypassed by alternate enzyme-catalyzed reactions. Pyruvate kinase (EC 2.7.1.40, PK) plays an important role in controlling glycolysis and gluconeogenesis. To clarify the characteristics of glycolysis in dental pulp, we examined the enzymatic properties of pyruvate kinase from pig dental pulp and compared them with those of the enzyme from pig brain. 1) Pyruvate kinase from dental pulp and brain were purified by use of ammonium sulphate fractionation, phosphocellulose colum chromatography, and isoelectric focusing. The prepared enzymes showed a single protein band on SDS polyacrylamide gel electrophoresis. 2) The subunit molecular weight of dental pulp and brain enzymes was determined to be 63,000 and 59,000, respectively. 3) Substrate inhibition of dental pulp and brain enzymes by phosphoenolpyruvate was not observed, and the relationship between reaction velocity and substrate concentration at pH 7.2 was explained by the Michaelis-Menten equation. Fructose-1,6-diphosphate had no observable effect on either enzyme. 4) Effect of amino acids on dental pulp and brain enzyme activity were examined, and no significant relationship was observed between the side chain structure of amino acids and their potency in inhibiting dental pulp and brain enzyme activity. Glutamic and aspartic acids markedly inhibited dental pulp and brain enzymes at pH 7.2. 5) Oxalate showed inhibitory activity against dental pulp and brain enzymes, and the Ki value was determined to be 50 microM and 80 microM, respectively. The inhibition of dental pulp and brain enzyme activity by oxalate was competitive with respect to phosphoenolpyruvate. 6) Both dental pulp and brain enzymes were clearly inhibited by malate at concentrations higher than 1.0 mM: 50% and 100% inhibition occurred at 2.2-2.3 mM and 3.0 mM malate, respectively.

Animals↗
PubMed1988

Thermodynamic nonideality as a probe of allosteric mechanisms: preexistence of the isomerization equilibrium for rabbit muscle pyruvate kinase.

Sedimentation velocity studies in the presence and absence of an inert space-filling solute, sucrose, have been used to establish preexistence of the isomerization equilibrium responsible for the allosteric behavior of rabbit muscle pyruvate kinase. Whereas the inclusion of phenylalanine (5 mM) with enzyme gives rise to a decrease of 0.3 S in the sedimentation coefficient of pyruvate kinase, the corresponding effect of phosphoenolpyruvate is to increase the sedimentation coefficient by 0.03 S. Consideration of these findings to signify the existence of an isomeric equilibrium between compact and expanded forms of the enzyme is substantiated by the finding that inclusion of sucrose (0.1 M) also brings about the change in sedimentation coefficient effected by phosphoenolpyruvate. By demonstrating that rabbit muscle pyruvate kinase undergoes isomerization in the absence of substrate, this study removes any necessity to consider the existence of an isomerization equilibrium that is substrate-induced; and thereby provides experimental support for adoption of the Monod model of allostery to interpret enzyme kinetic data for pyruvate kinase [R. W. Oberfelder, B. G. Barisas, and J. C. Lee (1984) Biochemistry 23, 3822-3826].

Animals↗
PubMed1981

Gluconeogenesis in rabbit liver. IV. The effects of glucagon, epinephrine, alpha- and beta-adrenergic agents on gluconeogenesis and pyruvate kinase in hepatocytes given dihydroxyacetone or fructose.

1. Epinephrine, isoproterenol and phenylephrine each increases significantly gluconeogenesis (from dihydroxy-acetone or D-fructose) and glycogenolysis when added to hepatocytes from 48-h fasted rabbits. Such stimulation of both processes by epinephrine, isoproterenol or phenylephrine is negated by the beta-adrenergic antagonist propranolol but remains significant in the presence of the alpha-adrenergic antagonist phentolamine. Conversely, previous data suggest that catecholamine-induced stimulation of glucose formation from L-lactate is both alpha- and beta-adrenergic-sensitive. 2. Glucagon, epinephrine, isoproterenol, phenylephrine and dibutyryl cyclic AMP each inhibits significantly pyruvate kinase activity in rabbit hepatocytes. Inhibition of pyruvate kinase activity by epinephrine, isoproterenol or phenylephrine is negated by propranolol but insensitive to phentolamine. 3. These observations suggest that enhancement by epinephrine of glucose formation from either dihydroxyacetone or D-fructose is solely beta-adrenergic-regulated, just as is its inhibition of pyruvate kinase activity. Stimulation of gluconeogenesis by glucagon, epinephrine, isoproterenol, phenylephrine or dibutyryl cyclic AMP may be at least in part directly related to their ability to inhibit pyruvate kinase.

Animals↗
PubMed1976

Rabbit muscle pyruvate kinase. Amino- and carboxyl-terminal studies.

Amino-terminal analysis of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) failed to detect the presence of any free amino-terminal residues. Acetyl group analysis demonstrated the presence of between 3.7 and 4.0 mol of acetyl groups per mol of enzyme. The acetylated amino-terminal residue was isolated from pronase digests of the enzyme and identified as N-acetylserine. Quantitative recovery experiments indicated that all acetyl residues are found at the amino termini. Carboxyl-terminal analyses using the tritium exchange method suggested the presence of a blocked carboxyl-terminal residue, supporting previous hydrazinolysis and carboxypeptidase studies.

Acetates↗