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At least 73 records · Page 4Linked to original sources
PubMed1985

Creatine-kinase and pyruvate-kinase activities in normal children: implications in Duchenne muscular dystrophy carrier detection.

Serum creatine-kinase (CK) and pyruvate-kinase (PK) levels were determined in 201 boys and girls less than 15 years old to establish values and to investigate a possible correlation between enzyme activity, sex, and age. It was observed that the mean CK activity in boys was significantly higher than in females (of all ages), whereas it did not differ statistically between girls and women. A slight but significant correlation between CK activity and age was found only in females. The mean PK activity in children was significantly greater than in adult women and it decreased significantly with age in children of both sexes. Furthermore, a slight correlation between CK and PK was observed only in girls. Based on these results, we suggest that the results of CK and PK determinations of females at risk for Duchenne muscular dystrophy should be compared with controls of comparable age and sex.

Adolescent↗
PubMed1983

Molecular cloning of DNA complementary to rat L-type pyruvate kinase mRNA. Nutritional and hormonal regulation of L-type pyruvate kinase mRNA concentration.

Rat liver L-type pyruvate kinase mRNA was enriched from total polysomes by immunoprecipitation with a specific antibody and Staphylococcus aureus cells. Double-stranded cDNA synthesized from the enriched mRNA was inserted into the PstI site of pBR322, and the resultant recombinant DNA molecules were used to transform Escherichia coli. Three clones containing DNA complementary to L-type pyruvate kinase mRNA were identified by colony hybridization, hybrid-selected translation, and dot blot hybridization. A partial restriction endonuclease map of cDNA inserts was constructed covering about 1.86 kilobase pairs. The cDNA insert of recombinant plasmid pLPK-14 was used as a hybridization probe to quantitate L-type pyruvate kinase mRNA in rat liver after various treatments. The level of hybridizable L-type enzyme mRNA was markedly increased by a high carbohydrate diet. Diabetes greatly reduced the mRNA level in the liver of rats maintained on a high carbohydrate diet, but insulin administration resulted in restoration of the mRNA level to normal within 24 h. These changes were approximately proportional to the changes in the level of translatable L-type pyruvate kinase mRNA. Thus, we conclude that nutritional and hormonal regulation of synthesis of hepatic L-type pyruvate kinase occurs at the pretranslational level.

Animals↗
PubMed1991

Biochemical and molecular characterization of variant pyruvate kinase enzymes and genes from three patients with red blood cell pyruvate kinase deficiency.

Pyruvate kinase (PK) from red blood cells (RBC) of three patients with nonspherocytic hemolytic anemia due to PK deficiency was characterized according to internationally standardized methods. The variant enzymes, which were designated PK 'Memphis', PK 'Bartlett', and PK 'Pontotoc', had 11, 60, and 61%, respectively, of the normal enzyme activity. All variant PK enzymes had increased thermolability. Compared with control, Km (PEP) were 200-300% greater for PK 'Memphis', 50% less for PK 'Bartlett' and 300-400% greater for PK 'Pontotoc'. The Km (ADP) were 40 and 300% greater than normal for PK 'Bartlett' and PK 'Pontotoc', respectively. All variants required higher than normal concentrations of the allosteric modifier, fructose-1,6-diphosphate, to achieve 50% activation of maximal enzyme activity. To define the molecular basis of the gene defect, DNA samples from these patients were examined for restriction-fragment-linked polymorphisms. No differences were observed in the structure of the patients' PK genes compared with a normal control. These results are consistent with a mutation in coding sequences, rather than a large insertion, deletion or rearrangement of genetic information, as the underlying genetic defect that accounts for the altered enzyme properties in these PK-deficient patients.

Enzyme Stability↗
PubMed1988

Discrimination between neuronal and glial cell tumours by pyruvate kinase electrophoresis.

Pyruvate kinase is a tetrameric enzyme, of which different isozymes are known (M, K and L). In normal human brain of adults mainly the M4 type is found with little or none of type K4 or the hybrid K3M. In gliomas of adults as well as in children a shift in the synthesis of isozymes of pyruvate kinase occurs from the M-towards the K-type. In these tumours the hybrid K2M2 is not expressed. In gliomas of children only a spur of K2M2 is found. There exists a peculiar difference in isozyme distribution of pyruvate kinase between gliomas on the one hand and neuro-, retino- and medulloblastomas on the other. In the latter group of tumours often the whole isozyme set is observed and in all cases the hybrids K2M2 and KM3 are found. These findings provide enzymological criteria for the discrimination of neuronal tumours from glial-cell tumours.

Cerebellar Neoplasms↗
PubMed1978

Homogeneous pyruvate kinase isolated from yeast by two different methods is indistinguishable from pyruvate kinase in cell-free extract.

In this report, we have compared homogeneous yeast (Saccharomyces cerevisiae) pyruvate kinase to enzyme from cell-free extracts in several different ways: 1) isoelectric focusing of cell-free extracts indicates one peak of pyruvate kinase activity whose isoelectric point is the same as that of the pure enzyme; 2) antibody prepared to the pure enzyme produces a single, fused precipitin line against enzyme in the cell-free extract and pure enzyme; 3) immunoelectrophoresis of cell-free extract produces one precipitin arc which has the same mobility as that of the pure enzyme; and 4) immunoprecipitation of the pure enzyme from cell-free extract with subsequent solubilization in 1% sodium dodecyl sulfate and electrophoresis on sodium dodecyl sulfate-polyacrylamide gels produces a single protein band attributable to pyruvate kinase which co-migrates with the purified enzyme. Within the limits of the sensitivity of the methods employed, we conclude that the homogeneous pyruvate kinase prepared from yeast lysed either by Manton-Gaulin homogenization (Aust, A., Yun, S.-L., and Suelter, C. (1975) Methods Enzymol. 42, 176-182) or by toluolysis (Yun, S.-L., Aust, A.E., and Suelter, C.H. (1977) J. Biol. Chem. 251, 124-128) is identical with pyruvate kinase in cell-free extract.

Immunodiffusion↗
PubMed1979

Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase.

1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Tris-maleate shows a loss of positive homotropic interactions, as compared to the same preparation in Tris-HCl. Hill coefficients (n) of n = 1.0-1.2 and n = 1.6-1.8 are obtained in Tris-maleate and Tris-HCl respectively. Half saturation [S] 0.5 and Vmax remain unchanged. Pyruvate kinase in Tris-maleate is slightly more stable to heating at 60 degrees C than in Tris-HCl. Incubation of the enzyme in Tris-maleate for one h with high concentrations of dithiotreitol restores the positive homotropic interactions. 3. It is proposed, that the abnormalities of the pyruvate kinase of some patients with acquired pyruvate kinase deficiency, obtained from a study in Tris-maleate, may partly be induced by the buffer itself.

Erythrocytes↗
PubMed1977

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.

Adenosine Diphosphate↗
PubMed1980

[Localization of pyruvate kinase M1 (ATP pyruvate phosphotransferase-2.7.1.40) in nervous tissue of the rat. Preliminary results].

The M1 pyruvate kinase has been localized in macroneuron's cytoplasm of nervous tissue of adult Rat, in situ as well as in tissue culture. Three methods have been used: histoenzymology, immunoenzymology and immunofluorescence, combined with Nomarski optics. This enzymatic localization may suggest a metabolic or differenciated fonctionnal state of the nerve cell.

Animals↗
PubMed1987

Elevated pyruvate kinase activity in patients with hemolytic anemia due to red cell pyruvate kinase "deficiency".

Two patients with non-spherocytic hemolytic anemia were found to have elevated red blood cell pyruvate kinase activities commensurate with the decreased mean red cell age, but the residual pyruvate kinase had marked kinetic abnormalities. Accumulation of metabolic intermediates before pyruvate kinase and reduced levels of activity of the red blood cells of the parents of both patients supported the diagnosis of an inherited abnormal pyruvate kinase causing hemolytic anemia. Although it was observed in two unrelated persons, review of enzyme assays performed on the red blood cells of 651 patients with hereditary non-spherocytic hemolytic anemia suggests that this occurrence is rare.

Adolescent↗
PubMed1991

Localization of the carbohydrate response element of the rat L-type pyruvate kinase gene.

Pyruvate kinase is a major regulatory enzyme of glycolysis. Transcription of the L-type pyruvate kinase (L-PK) gene in rat liver is induced by feeding a carbohydrate-rich diet. To investigate the regulatory DNA sequences required for this response, primary hepatocytes were transfected with plasmids containing the 5'-flanking sequence of the rat L-PK gene fused to the chloramphenicol acetyltransferase (CAT) gene. Sequences from -4300 to +12 of the L-PK gene directed an increase in CAT activity when hepatocytes were switched from media containing 10 mM lactate to 25 mM glucose. Average induction was 17-fold (n = 13; S.E. = 2.9). Addition of fructose to the media also induced CAT activity. Carbohydrate regulation of the L-PK promoter was retained with 5'-deletions to -197, but constructs deleted to -96 were completely unresponsive. The 101-base pair fragment from -197 to -96 of the L-PK gene can confer carbohydrate regulation when fused in either orientation to the heterologous thymidine kinase promoter, thus defining a carbohydrate response element in this region. Expression of the transfected gene was regulated by insulin and glucagon in a pattern similar to that seen for the endogenous L-PK gene, suggesting that control of L-PK promoter activity was responsible for carbohydrate-mediated changes in L-PK mRNA production.

Animals↗
PubMed1993

Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli. Evidence that the genes for phosphofructokinase and pyruvate kinase constitute an operon.

Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate. The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined. The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da. The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin. Similarly to the E. coli enzymes, the enzyme does not contain an N-terminal domain, in contrast to the eukaryotic pyruvate kinases. However, the Bacillus stearothermophilus enzyme had an extra C-terminal sequence consisting of about 110 amino acid residues. A phosphoenolpyruvate-binding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C-terminal sequence. There was an open reading frame upstream of the pyruvate kinase gene. The homology of the sequence showed that the gene encodes phosphofructokinase. Both phosphofructokinase and pyruvate kinase were expressed in E. coli cells, and the evidence suggesting that both genes constitute an operon is presented.

Amino Acid Sequence↗
PubMed1993

Fetal anaemia due to pyruvate kinase deficiency.

Pyruvate kinase deficiency was diagnosed in an infant by umbilical vessel sampling at 30 weeks' gestation. Although three previous hydropic siblings had been stillborn or died in the neonatal period, this infant survived with transfusion dependent haemolytic anaemia. Prompt fetal diagnosis of pyruvate kinase deficiency is feasible and allows better management of hydrops fetalis due to this disorder.

Anemia, Hemolytic, Congenital↗
PubMed1978

The active site of rabbit muscle pyruvate kinase. Evidence for a site common to the oxalacetate decarboxylase and pyruvate kinase reactions.

Rabbit muscle pyruvate kinase has been shown to catalyze the decarboxylation of oxalacetate (Creighton, D.J. and Rose, I.A. (1976) J. Biol. Chem. 251, 61). Noncovalent and covalent modifiers of the enzyme have been used to assess whether the decarboxylase and kinase reactions take place at a common site. Phosphoenol-alpha-ketobutyrate, an analog of the substrate phosphoenol-pyruvate, inhibits decarboxylase and kinase competitively and with nearly identical Ki values (5.7 micron and 4.8 micron, respectively). Oxalate, an analog of enol-pyruvate, inhibits each competitively and with similar Ki values (11 micron and 4.7 micron, respectively). Both activities are lost in parallel upon reaction with dithionitrobenzoate, which is active-site specific. These results indicate that the two activities share a common site on the enzyme. But, effects of the following modifiers suggest that different amino acid residues at that site participate in the two reactions: phenylalanine inhibition and fructose 1,6-bisphosphate activation are more effective with the decarboxylase; iodoacetamide preferentially inactivates decarboxylase while trinitrobenzenesulfonate preferentially inactivates kinase.

Animals↗