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PubMed1983

Studies with human erythrocyte pyruvate kinase (PK): effects of modification of sulfhydryl groups.

Cysteinyl residues of red cell pyruvate kinase (PK; ATP: pyruvate phosphotransferase, EC 2.7.1.40) were modified with methylmethanethiosulfonate (MMTS), p-nitrophenoxycarbonyl methyl disulfide (NPCMD), and sodium tetrathionate (NaTT). At pH greater than 7 . 0, K0.5 s phosphoenol-pyruvate (PEP) was markedly increased. Fructose-1,6-diphosphate (FDP) increased affinity for PEP, but K0.5 s (PEP) remained elevated and hyperbolic kinetics were not achieved. Inhibition by negative effectors ATP and alanine was not reversed by PEP and FDP concentrations far greater than those abolishing inhibition of unmodified enzyme. At pH less than 7 . 0, PEP affinity was reduced, and FDP markedly increased Vmax and diminished K0.5 s (PEP). MMTS greatly impaired the thermostability of PK. Acid pH alone and the simultaneous presence of Mg++, K+ and PEP prior to MMTS treatment protected against the effects on PEP kinetics, but did not alter the induction of thermolability. No MMTS effect on the FDP binding site, on ADP kinetics or on the relative effectiveness of GDP, UDP or CDP cofactors was demonstrated. The MMTS-induced alterations closely resembled those observed with certain PK mutants associated with haemolytic anaemia.

Adenosine Diphosphate↗
PubMed2001

Structural and regulatory properties of pyruvate kinase from the Cyanobacterium synechococcus PCC 6301.

Pyruvate kinase (PK) from the cyanobacterium Synechococcus PCC 6301 was purified 1,300-fold to electrophoretic homogeneity and a final specific activity of 222 micromol of pyruvate produced/min/mg of protein. The enzyme was shown to have a pI of 5.7 and to exist as a 280-kDa homotetramer composed of 66-kDa subunits. This PK appears to be immunologically related to Bacillus PK and a green algal chloroplast PK, but not to rabbit muscle PK, or vascular plant cytosolic and plastidic PKs. The N-terminal amino acid sequence of the Synechococcus PK exhibited maximal (67%) identity with the corresponding region of a putative PK-A sequence deduced from the genome of the cyanobacterium, Synechocystis PCC 6803. Synechococcus PK was relatively heat-labile and displayed a broad pH optimum around pH 7.0. Its activity was not influenced by K(+), but required high concentrations of Mg(2+), and was relatively nonspecific with respect to the nucleoside diphosphate substrate. Potent allosteric regulation by various effectors was observed (activators: hexose monophosphates, ribose 5-phosphate, glycerol 3-phosphate, and AMP; inhibitors: fructose 1,6-bisphosphate, inorganic phosphate, ATP, and several Krebs' cycle intermediates). The enzyme exhibited marked positive cooperativity for phosphoenolpyruvate, which was eliminated or reduced by the presence of the allosteric activators. The results are discussed in terms of the phylogeny and probable central role of PK in the control of cyanobacterial glycolysis.

Amino Acid Sequence↗
PubMed1981

Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose-1,6-diphosphate.

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The Km for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a pKa of 6.5-6.7. The cooperative interaction of the multiple phosphoenolpyruvate (PEP) binding sites is independent of pH in the range of 5.7-7.8. Variation of the Vmax value with pH indicates the presence of a dissociated group (pKa 6.2-6.3) and of an undissociated group (pKa 7.5-7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S0.5 value for this substrate, as is an undissociated enzyme group with a pKa in the range 6.7-7.0. It is shown that PEP and frucotse-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme, FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.

Adenosine Diphosphate↗
PubMed1981

Red-cell enzyme activities and properties of mutant pyruvate kinase after 1-year storage in a -80 degrees C freezer.

Red cells from 5 healthy adults and an unstable mutant pyruvate kinase (PK), PK Maebashi, were stored for 12 months in a -80 degrees C freezer. With an exception of triosephosphate isomerase and phosphoglycerate kinase activities, normal red-cell enzyme activities remained essentially unchanged. Mutant PK was characterized by the methods recommended by the International Committee for Standardization in Haematology (ICSH). Normal control and PK Maebashi remained essentially unchanged in their characteristics after storage in a -80 degrees C freezer for 12 months. These results indicate that the red cells stored over a long term in a -80 degrees C freezer can be used for the study of unstable mutant PK.

Blood Preservation↗
PubMed1980

pH-dependent amino acid induced conformational changes of rabbit muscle pyruvate kinase.

The interactions of L-Phe and L-Ala with rabbit muscle pyruvate kinase dependend upon the nature of divalent metal ions sutdied: Mg(II), Co(II), Mn(II), and Ni(II). L-phe inhibited all metal derivatives of the enzyme except Mn(II)--enzyme. L-Ala inhibited only Ni(II)--enzyme and had no effect on other metal derivatives. The inhibition by L-Phe could be partially or completely reversed by L-Ala for all metal derivatives. The mode of inhibition of pyruvate kinase by L-Phe depended upon pH as well as the nature of activating divalent metal ions. The sigmoidal response increased with increasing pH for all metal derivatives inhibited by L-Phe. L-Phe and L-Ala strongly perturbed the coordination sphere of enzyme bound Co(II), but not Ni(II). There were poor correlations between visible circular dichroic (cd) spectral changes and the corresponding kinetic changes. However, L-Phe and (or) L-Ala induced ultraviolet cd and difference absorption spectral changes, on the other hand, corresponded remarkably well with the kinetic observations.

Alanine↗
PubMed1994

Tight linkage of pyruvate kinase (PKLR) and glucocerebrosidase (GBA) genes.

Two polymorphisms, one in the liver-type pyruvate kinase gene (PKLR) and one in the glucocerebrosidase gene (GBA), both of which are on band q21 of chromosome 1, were found to be tightly linked. Each of three Gaucher disease mutations in 112 chromosomes studied was associated with a unique haplotype. With a conservative assumption about the length of time that the Gaucher disease mutation has been present in the Jewish population, we deduce that the genetic distance between these two loci is probably under 0.2 centimorgans. Four haplotypes are produced by these polymorphic loci, but two of these are relatively uncommon because the polymorphic sites are in linkage disequilibrium. Nonetheless these markers are potentially useful in the prenatal diagnosis of pyruvate kinase deficiency in families who have at least one affected child and may also be helpful in heterozygote detection in families with Gaucher disease where a specific mutation producing the disease in unknown.

Base Sequence↗
PubMed1986

Insulin dependence of M2 pyruvate kinase in primary culture of human liver.

In the basal state, the presence of L pyruvate kinase (LPK) was constantly observed in primary human liver cell cultures initiated from explants, when cells were examined by immunofluorescence and double labelling. After short-term insulin incubation, M pyruvate kinase (MPK) appeared. Therefore, both LPK and MPK were located in the same cells. We previously obtained the same results in isolated rat hepatocytes in which we demonstrated that short-term regulation of M2PK by insulin was a function of dose and/or incubation time. The present work established that similar conditions govern the regulation of this isozyme in vitro in human hepatocytes.

Cells, Cultured↗
PubMedDate not supplied

Decrease of aldolase and pyruvate kinase activity in erythrocytes of individuals with male hypogonadism as an expression of lack of androgen influence on the bone marrow.

The authors have demonstrated the statistically significant decrease of aldolase and pyruvate kinase activity in erythrocytes in a group of men with hypogonadism and with a significant defect of gonad endocrinologic function. A similar phenomenon appeared in erythrocytes in a group of sexually mature rabbits after castration. In the course of substitutional treatment with testosterone esters given intramuscularly, the increase of activity characteristic for both examined glycolytic enzymes was observed in both experimental groups, i.e. the patients and the animals. The pyruvate kinase increase, however, was not statistically significant in the examined group of men, which resulted from the small size of the group. The authors suggest the existence of testosterone influence on regular activity of bone marrow glycolytic enzymes, and consequently on the mature erythrocytes. The decrease of aldolase and pyruvate kinase activity in erythrocytes of patients with male hypogonadism may decrease the erythrocytes valence. This ist another biochemical argument for the need of conducting long-term substitutional therapy in cases of male hypogonadism.

Adult↗
PubMed1975

Extraction of a factor from Ehrlich ascites tumor cells that increases the activity of the fetal isozyme of pyruvate kinase in mouse liver.

Isoelectrofocusing studies of mouse tissue extracts show mice to have a pyruvate kinase isozyme pattern very similar to that of the rat. Moreover, electrofocusing or kinetic assays conducted on liver extracts from normal mice and from mice bearing Ehrlich ascites tumors show that the latter have a higher proportion of the fetal K-isozyme of pyruvate kinase. Serial injection of the supernatant remaining after centrifugation of homogenized tumor cells at 100,000 x g, or of the phenolic extracts from the latter, produced a similar shift in the liver isozyme pattern involves both a decrease in L-isozyme activity and an increase in K-isozyme activity. However, only the increase in activity of the K-isozyme appears to be a specific response to injection of the extracts. The presence of a specific factor in these extracts was confirmed by the observation that similar extracts prepared from normal adult tissues did not increase activity of the K-isozyme. On the other hand, phenolic extracts from fetal mice did increase K-isozyme activity as did injections of serum from tumor-bearing mice or of the cell-free ascites fluid. Evidence is presented supporting the concept that the factor is proteinaceous in a nature, and that it acts by deprepressing synthesis of the K-isozyme.

Animals↗
PubMed1976

Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid.

4-Hydroxypentanoic acid alanine thioether was synthesized and characterized by n.m.r. spectroscopy. This derivative corresponded to the modified amino acid obtained by allowing 5-chloro-4-oxo[3,5-3H]pentanoic acid to react with rabbit muscle pyruvate kinase. Performic acid oxidation of 4-oxo[3,5-3H]pentanoic acid alanine thioether in pyruvate kinase gave [3H]succinate (67%) and [3H]carboxymethylcysteine (33%) as expected. Evidence is presented to show that NaBH4 reduction followed by periodate oxidation and analysis of radioactive formaldehyde production may provide a convenient method for distinguishing between thiol and amino alkylation by halogenomethyl ketone compounds. Peptide 'mapping' confirms that the modification by 5-chloro-4-oxopentanoic acid occurs primarily at one region of pyruvate kinase.

Alkylation↗
PubMedDate not supplied

Tumor M2-pyruvate kinase in lung cancer patients: immunohistochemical detection and disease monitoring.

BACKGROUND: Lung cancer is one of the main causes for cancer death. A reliable diagnosis and follow-up of patients is important support for a successful therapy. The diagnostic efficiency of a new marker Tumor M2-pyruvate kinase (Tumor M2-PK) is evaluated. MATERIALS AND METHODS: In this report immunohistochemical detection of pyruvate kinase M2 in tissue sections of lung cancer specimens is presented. Furthermore Tumor M2-PK was quantified in EDTA plasma of lung cancer patients in order to monitor the disease, especially under chemotherapy. RESULTS: Immunohistochemical detection of pyruvate kinase M2 in tissue sections of lung cancer specimens showed selective staining of tumor cells, independent of the histological classification of the tumor. In EDTA plasma of lung cancer patients, the marker concentrations correlated well with the tumor load during follow-up, showing significantly increasing concentrations with progressive tumor stages and decreased concentrations during tumor remission. Again, this effect was independent of the histological tumor type. CONCLUSION: The present data indicate that Tumor M2-PK in EDTA-plasma could be a valuable tumor marker for monitoring lung cancer.

Aged↗
PubMed1983

Appearance of the liver form of pyruvate kinase in differentiating cultured foetal hepatocytes.

From about the 16th day of gestation three forms of pyruvate kinase are present in foetal rat liver (L, R, and M2). Hepatocytes isolated from 15-day-old foetuses do not possess the liver form of pyruvate kinase, but after three days in culture this enzyme can be detected. No effect on the appearance of the enzyme could be seen by administration of insulin and fructose. Hepatocytes isolated from 19-day-old foetuses exhibit three forms of the enzyme (L, R, and M2) on day 1 of culture but thereafter only two forms are detectable (L and M2). A decrease in activity of the L form is observed. This could be retarded by administration of insulin and fructose.

Animals↗
PubMed2001

Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate.

Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.

Adenosine Diphosphate↗
PubMed1976

Pyruvate kinase isozymes among human organs and blood cells.

In extracts of human tissues only three different pyruvate kinases were demonstrated by acetate gel electrophoresis. Treatment with reduced glutathione resulted in an alteration of electrophoretic mobility of extracts from kidney and testis. The existence of interconvertible forms of M2 pyruvate kinase (PK) was suggested. Lymphocytes, polymorphonuclear cells, platelets were M2 type PK. An electrophoretic slow form was demonstrated in myeloblasts and lymphoblasts.

Electrophoresis, Cellulose Acetate↗
PubMed1975

Kinetic properties of pyruvate kinase hybrids formed with native type L and inactivated type M subunits.

Bovine type M pyruvate kinase, which normally has hyperbolic kinetics with its substrates, was inactivated by treatment with trinitrobenzenesulfonic acid. The inactivation probably occurs through trinitrophenylation of the epsilon-amino group of a lysine residue in or near the ADP binding site. Although 90 to 95% of the enzymatic activity is lost by this treatment, the molecular weight and sedimentation coefficient of the trinitrophenylated enzyme are quite similar to values obtained with the native enzyme. The inactivated, trinitrophenylated type M pyruvate kinase was hybridized in vitro with the native bovine type L enzyme, which has sigmoidal kinetics with phosphoenolpyruvate but can be activated by fructose 1,6-diphosphate to give hyperbolic kinetics. Four enzymatically active species were produced, designated L4, L3M, L2M2, and LM3, according to their subunit composition. L4 and L3M have sigmoidal kinetics with phosphoenolpyruvate and are activated by fructose diphosphate. Little or no sigmoidicity was seen for L2M2, although this species is activated to a moderate degree by fructose diphosphate. LM3 appears to have hyperbolic kinetics and is activated only slightly by fructose diphosphate. The kinetic results obtained with hybrids containing trinitrophenylated type M subunits are quite similar to the results previously reported by Dyson and Cardenas ((1973) J. Biol. Chem. 248, 8482-8488) using native type M and type L subunits, indicating that the properties of a type L subunit are profoundly affected by the nature of the other subunits present in the tetramer. In fact, type L and type M subunits in a given hybrid seem to have similar kinetic responses toward phosphoenolpyruvate and fructose diphosphate.

Animals↗
PubMed1982

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

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.

Breast Neoplasms↗
PubMed1969

The adenosine triphosphate inhibition of the pyruvate kinase reaction and its dependence on the total magnesium ion concentration.

1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.

Adenine Nucleotides↗
PubMed1987

Kinetic properties of pyruvate kinase from the epaxial muscle of the marine fishes Mugil lisa and Chaetoditerus faber.

Kinetic studies were carried out on the reaction catalyzed by pyruvate kinase (ATP:pyruvate phosphotransferase, E.C. 2.7.1.40) purified from white striated (epaxial) muscle of two marine fish Mugil lisa (Brazilian mullet) and Chaetoditerus faber (harvest fish). This included the establishment of kinetic parameters. Attention was given to the effect of fructose 1,6-bisphosphate (Fru-P2) on PK activity. Effects of ATP, alanine and the divalent ions, Mg2+, Mn2+, Cu2+, Be2+ and Co2+, on the fish muscle enzyme were also studied.

Adenosine Diphosphate↗