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At least 19 recordsLinked to original sources
PubMed1975

Normalisation of red blood cell pyruvate kinase in pyruvate kinase deficiency by riboflavin treatment.

A patient with an erythrocyte glutathione reductase activity of 50% of the normal value and an abnormal pyruvate kinase (PK) was given 36 mg riboflavin daily for 6 months. The glutathione reductase activity was restored and the abnormal pyruvate kinase was converted to normal. The clinical state of the patient improved. It can be concluded that the abnormality of PK, at least with this patient, is a secondary effect. Therefore, it is suggested that other abnormalities be searched for when an altered PK is detected. Treatment of this abnormality will help the patients more efficiently.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1978

Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.

Animals↗
PubMed1975

Regulation of glycolysis in Neurospora crassa. Kinetic properties of pyruvate kinase.

Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40), extracted from the mycelium of Neurospora crassa has been purified 560-fold by precipitation with ammonium sulphate, chromatography with DEAE-Sephadex, and gel filtration with Sephadex G-200. Potassium and magnesium are required for enzyme activity. Fructose, 1,6-diphosphate is the only physiological activator found for the enzyme. In decreasing order of potency, citrate, oxalacetate, calcium, and ATP are inhibitors. Phosphoenolpyruvate is cooperatively bound by the enzyme and the cooperatively is reduced by ATP and completely eliminated by fructose-1,6-diphosphate. Lowering of pH from 7-5 to 5-5 changes the Hill coefficient from 2-7 to 1-0. Substitution of ADP by other nucleotides reduces enzyme activity. Manganese can substitute for the cofactor magnesium, but the reaction velocity is then reduced. MgADP- is cooperatively bound by the enzyme and inhibition of the enzyme occurs only when either magnesium or ADP is in excess of the other beyond the optimum concentration. These kinetics properties of pyruvate kinase are compatible with the role of a regulator of glycolysis in Neurospora crassa.

Adenosine Triphosphate↗
PubMed1977

A monomeric form of pyruvate kinase in human pyruvate kinase deficiency.

A mutant pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human erythrocytes which is easily separated into monomers and dimers by gel chromatography is described. Tht mutant enzyme shows almost the same pH optimum and thermostability as normal enzyme, but has a decreased stability on shaking with air, a decreased Km for phosphoenolpyruvate and a loss of allosteric properties. The apparent Km values for phosphoenolpyruvate of tetramers and monomers were the same. The tetrameric enzyme was slightly activated by fructose-1,6-diphosphate but the monomeric form was not. The tetrameric enzyme was found to dissociate spontaneously to dimeric and monomeric forms.

Adenosine Diphosphate↗
PubMed1975

Effect of desdanine on nucleoside diphosphate kinase and pyruvate kinase of Escherichia coli.

Kinetic analysis demonstrated that the irreversible inhibition of nucleoside diphosphate kinase of Escherichia coli by desdanine proceeds via a reversible Enzyme-Inhibitor complex. It is known that pyruvate kinase of E. coli becomes inactive upon prolonged dialysis in the absence of a reducing reagent, such as dithiothreitol and that the inactive enzyme is reactivated if dithiothretiol is added. Desdanine inhibits this reactivation process. The effect is discussed in relation to inhibition in relation to the inhibition of growth of E. coli by desdanine under anaerobic conditions. Pyruvate kinase of E. coli changes not only in intracellular quantity but in its kinetic characteristics depending on growth conditions, aerobic or anaerobic. The enzyme shows a broad specificity for nucleside diphosphates, especially in the presence of AMP, and thus resembles closely nucleoside diphosphate kinase. The in vivo role of pyruvate kinase in supplying nucleoside triphosphates under anaerobic conditions is discussed.

Acrylamides↗
PubMedDate not supplied

Serum creatine kinase and pyruvate kinase in Duchenne muscular dystrophy carrier detection.

The incidence of elevated serum creatine kinase (CK) and pyruvate kinase (PK) activities was compared in 20 definite carriers of Duchenne muscular dystrophy (DMD), 47 possible carriers, and 42 female controls. When adult age was not regarded as a variable, 70% of the definite carriers had elevated PK, 55% had elevated CK, and 75% had elevated PK or elevated CK or both, 38% of the possible carriers had elevated PK, 19% had elevated CK, and 40% had elevated PK or elevated CK or both. The detection efficiency of the CK test was influenced by the age of the subjects: the upper normal limit of serum CK in the adult controls was at the minimum between 21 and 35 years of age, and CK activity in some carriers declined from elevated to normal levels with increasing age. With these considerations, 70% of definite carriers had elevated CK and 80% had elevated PK and/or CK; 34% of the possible carriers had elevated CK and 43% had elevated PK and/or CK. On the basis of the PK and CK measurements, only 16 of 24 possible carrier mothers were likely to be DMD carriers, implying that the other 8 were non-carrier mothers of new mutant sons.

Adolescent↗
PubMed1991

Estimate of the intrafamilial correlation for serum creatine kinase and pyruvate kinase in females at risk for Duchenne and Becker muscular dystrophies.

In order to verify the possibility of nonrandom X-inactivation in females heterozygous for Duchenne (DMD) and Becker (BMD) muscular dystrophies, intrafamilial correlations and the heritabilities for serum creatine kinase (CK) and pyruvate kinase (PK) were estimated in a large sample of females belonging to families with affected patients. The results of the present investigation suggest that the apparent intrafamilial correlations for serum CK reported in previous studies in DMD families are not related with the presence of the DMD/BMD gene. Our data do not seem to support the hypothesis of a gene leading to a preferential inactivation of the X-chromosome in females at risk for the dystrophin gene.

Adolescent↗
PubMed1979

Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.

Acetate Kinase↗
PubMed1977

Studies on the cyclic 3':5'-AMP-stimulated pig liver protein kinase reaction with pyruvate kinase as substrate.

The phosphorylation of pig liver pyruvate kinase by cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been studied. For comparison, mixed histone and a synthetic heptapeptide were also used as substrates. Protein kinase was purified by chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-200. The enzyme was stimulated by cyclic AMP with apparent Ka values of 2.5 and 0.8 x 10-7 M for pyruvate kinase and histone substrates, respectively. Divalent cations were essential for the activity of the protein kinase. Variation of the concentration of ATP resulted in approximately straight lines in Lineweaver-Burk plots for the phosphorylation of both pyruvate kinase and mixed histone. The apparent Km values for ATP were 21 and 11 muM, respectively. The phosphorylation rate increased with the concentration of pyruvate kinase even at a concentration of 2 muM pyruvate kinase. At a high ionic strength, the phosphorylation rate of both pyruvate kinase and histone decreased. The phosphorylation rate varied markedly with pH in imidazole/HC1 and Tris/HC1 buffers. At slightly alkaline pH values, pyruvate kinase was phosphorylated at a much higher rate than pH7, but this was not the case for histone. At pH 8.5, the phosphorylation rate of pyruvate kinase was 3.5 times the rate at pH 7, while the corresponding increase for the histone phosphorylation was 50 per cent. In potassium phosphate buffers, the phosphorylation rate of both substrates did not change significantly over the pH range studied. Arrhenius' plots of the protein kinase reaction resulted in a break at about 10 degrees when pyruvate kinase was used as substrate, whereas a straight line was obtained when using histone. The negative allosteric effectors of pyruvate kinase, alanine, and phenylalanine, increased the phosphorylation rate of pyruvate kinase at pH 8 by 50 and 120 per cent, respectively. The same effectors did not influence the phosphorylation rate of mixed histone or a synthetic heptapeptide. It is concluded that the conformations adopted by pyruvate kinase in the presence of allosteric inhibitors make it a better substrate for the protein kinase.

Adenosine Triphosphate↗
PubMed1975

Membrane-localized pyruvate kinase of red blood cells in hemolytic anemia associated with pyruvate kinase deficiency.

Pyruvate kinase activity of red blood cell membranes, which is normally masked, has been determined after mechanical disruption of the membranes in normal individuals and in three homozygous patients with pyruvate kinase deficiency. Although patients 1 and 2, who were siblings, had relatively high enzyme activities in their hemolysates, they had the severest form of the disorder. The activities of their membrane fragments were decreased to seven per cent of fragments of normal membranes. Patient 3 had a mild form of hemolytic anemia despite a low enzyme activity of his hemolysates. The membrane fragments of this patient contained 28 per cent of the pyruvate kinase activity of normal fragments. The data suggest a relationship between the amount of membrane-localized pyruvate kinase and the severity of the clinical disorder. The reduced production of ATP by the enzyme portion localized within the membrane may cause an impairment of membrane functions in pyruvate kinase deficiency.

Aged↗
PubMed1978

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver.

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.

Erythrocytes↗
PubMed1977

Purification and characterization of pig kidney pyruvate kinase (type A).

Pyruvate kinase type A was purified from pig kidney with a yield of 9%. The final enzyme fraction had a specific activity of 500 units/mg of protein. The enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis in detergent and in ultracentrifugation experiments. The molecular weight of the enzyme was found to be 210,000 with the use of ultracentrifugation and 249,000 at gel chromatography. The sedimentation coefficient (S degrees 20, w) was calculated to be 9.8 S. For the reduced and alkylated pyruvate kinase, a molecular weight of 60,000 was found with the use of several methods. The Stokes radius for the enzyme was calculated to be 56 A. No NH2-terminal amino acid was detected in the enzyme, and the only findings in carbohydrate analyses of the kidney pyruvate kinase were trace amounts of glucose. The isoelectric point of the enzyme was estimated to be pH 5.6. Pig kidney pyruvate kinase type A was not phosphorylated on incubation with ATP and cyclic 3':5'-AMP-dependent protein kinase. The amino acid compositions of pig kidney and pig muscle pyruvate kinases were very similar and differed clearly from that of pig liver pyruvate kinase.

Amino Acid Sequence↗
PubMed1975

Electrophoretic, immunologic and kinetic characterization of erythrocyte pyruvate kinase in the Basenji dog with pyruvate kinase deficiency.

The electrophoretic mobility and the immunologic specificity of erythrocyte pyruvate kinase (PK) of the homozygous Basenji dog with PK deficiency were identical to those of normal M2-type PK isozyme seen in the white cell but not to those of the erythrocyte PK isozyme. Kinetic properties and stability were also consistent with the M2-type PK isozyme. Defective PK in the homozygous red cell was due to the absence of the erythrocyte PK isozyme and the compensatory presence of M2-type PK isozyme, as seen in the severe classical type PK deficiency in man.

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↗
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↗