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PubMed2005

Interindividual differences in leg muscle mass and pyruvate kinase activity correlate with interindividual differences in jumping performance of Hyla multilineata.

Frog jumping is an excellent model system for examining the structural basis of interindividual variation in burst locomotor performance. Some possible factors that affect jump performance, such as total body size, hindlimb length, muscle mass, and muscle mechanical and biochemical properties, were analysed at the interindividual (intraspecies) level in the tree frog Hyla multilineata. The aim of this study was to determine which of these physiological and anatomical variables both vary between individuals and are correlated with interindividual variation in jump performance. The model produced via stepwise linear regression analysis of absolute data suggested that 62% of the interindividual variation in maximum jump distance could be explained by a combination of interindividual variation in absolute plantaris muscle mass, total hindlimb muscle mass (excluding plantaris muscle), and pyruvate kinase activity. When body length effects were removed, multiple regression indicated that the same independent variables explained 43% of the residual interindividual variation in jump distance. This suggests that individuals with relatively large jumping muscles and high pyruvate kinase activity for their body size achieved comparatively large maximal jump distances for their body size.

Animals↗
PubMed1992

Insulin regulation of pyruvate kinase activity in isolated adipocytes. Crucial role of glucose and the hexosamine biosynthesis pathway in the expression of insulin action.

We recently identified glutamine:fructose-6-phosphate amidotransferase (GFAT) as an insulin-regulated enzyme in adipocytes. Moreover, we found that loss of GFAT activity is not due to a direct action of insulin but rather is mediated by enhanced glucose uptake and the subsequent routing of glucose through the hexosamine biosynthesis pathway. To assess whether other cytosolic enzymes are controlled through formation of hexosamine products, we treated adipocytes for 5 h with physiological concentrations of insulin (ED50 = 0.33 ng/ml), glucose (ED50 = 4.5 mM), and glutamine (ED50 = 4.4 mM) and then measured pyruvate kinase (PK) activity. Combined treatment resulted in a progressive (t 1/2 of 2.5 h) and marked (3-fold) increase in PK activity, whereas omission of one or more of these components failed to alter enzyme activity. Several lines of additional evidence implicated the hexosamine biosynthesis pathway in PK regulation; therefore, it appears that the M2 isoform of pyruvate kinase represents another enzyme regulated by insulin through stimulation of glucose uptake and formation of hexosamine products. Related studies revealed that enhancement of PK activity is dependent upon ongoing mRNA synthesis and de novo protein synthesis and is mediated by an increase in enzyme content. Considered together, these findings provide new insights into the cascade of metabolic events triggered by insulin and implicated a novel metabolic pathway in the pretranslational control of enzyme function.

Adipose Tissue↗
PubMed1984

[Hereditary erythrocyte pyruvate kinase abnormality: another kindred from Aargau Canton].

A Swiss family with abnormal red cell pyruvate kinase is described. In two new-born siblings the red cell defect was responsible for marked hyperbilirubinemia. At the present time both children have compensated hemolysis and are clinically healthy. The abnormal enzyme is unstable and has a decreased Hill coefficient and a decreased substrate affinity. It is different from two other enzyme variants found in the same area.

Erythrocytes↗
PubMedDate not supplied

[Vascular complications in a case of hemolytic anemia due to pyruvate kinase deficiency].

A rare case of congenital non-spherocytic hemolytic anemia due to pyruvate kinase deficiency is reported. Of particular interest is the never before in the literature mentioned complication of migratory phlebitis and acute thrombosis of the left lower limb associated with trophic changes of the foot. Arteriography disclosed thrombosis of all three calf arteries. The patient was treated by lumbar sympathectomy combined with streptokinase fibrinolysis and resulted in healing of the trophic changes. The distal location of the vascular lesions made direct vascular reconstruction illusory. Fogarty thrombectomy is not indicated either due to the thrombotic nature of the lesions in association with thrombocytosis.

Adult↗
PubMed2001

Glucose and cAMP regulate the L-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein.

Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser(196) and Thr(666) were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser(196) in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr(666) in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP.

Amino Acid Sequence↗
PubMed2001

Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules.

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N2-fixing soybean (Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phosphoenolpyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.

Journal Article↗
PubMed1974

Mechanism of renaturation of pyruvate kinase of Saccharomyces carlsbergensis: activation by L-valine and magnesium and manganese ions.

Pyruvate kinase (EC 2.7.1.40) of S. carlsbergensis is a tetrameric enzyme, composed of four identical subunits each of which contains 1 mole of L-valine noncovalently bound. The enzyme readily dissociates into monomeric units. L-Valine and magnesium or manganese ions are specific primers of the renaturation process of the enzyme. The amino acid induces renaturation with a K(0.5) of 17 muM and a pseudo first-order rate constant of 0.019 min(-1) at 25 degrees with respect to the monomeric species, indicating that L-valine influences the folding of the monomeric form from a disordered state to its native conformation being followed by a spontaneous reassociation with formation of the tetrameric enzyme. Independently, magnesium and manganese ions induce the renaturation with a first-order rate constant of the same magnitude.

Enzyme Activation↗
PubMed1976

Electrofocusing and kinetic studies of adult and embryonic chicken pyruvate kinases.

Chicken embryos less than 15 days old contain only the K isozyme of pyruvate kinase, which appears to exist in vivo as an R,T conformational set with pI values of 7.2 and 6.6, respectively. Sets of lower pI and higher pI K-isozyme variants also are obtained. Whole embryos of 15 days or more of development show progressively increasing amounts of higher pI, lower K0.5S enzymatic variants. Tissue distribution and kinetic properties suggest that the highest pI form (pH 8.8-9.0) is an M-isozyme analogue. The intermediate forms are postulated to be hybrids. Adult liver extracts contain only the embryonic K isozyme; no evidence for an L-isozyme analogue was obtained. All major forms of the enzymes are compared with respect to saturation by phosphoenolpyruvate in the absence of effector and in the presence of fructose 1,6-diphosphate, alanine, serine, phenylalanine, tryptophan, and/or Mg-ATP.

Aging↗
PubMed1976

Serum enolase and pyruvate kinase activities in the diagnosis of myocardial infarction.

Sequential determinations of serum enolase and pyruvate kinase (PK) activities were carried out in addition to routine enzyme assays on 191 patients admitted to a coronary care unit. The upper reference values established in 61 healthy laboratory staff members were 21 U/1 for enolase and 76 U/1 for PK. Of 99 patients with a confirmed diagnosis of myocardial infarction (MI), 97 manifested raised serum enolase during the period of observation; 85 of these patients had raised serum PK. The incidence of raised values for serum PK among patients considered not to have sustained infarction was similar to that shown by the routine enzymes, but serum enolase activity was much more frequently raised in these patients. The time-course of elevation of serum enolase and also that of PK are discussed in relation to the changes in other serum enzymes routinely measured for MI.

Clinical Enzyme Tests↗
PubMed1983

Aplastic crisis due to parvovirus infection in pyruvate kinase deficiency.

A thirteen-year-old boy with congenital haemolytic anaemia due to pyruvate kinase (PK) deficiency had an aplastic crisis. A serum parvovirus-like virus (SPLV) was demonstrated in the blood by electron microscopy and, subsequently, IgM and IgG antibodies to the prototype SPLV B19 were detected. In an attempt to define the level of erythropoiesis that is involved in parvovirus-induced bone marrow suppression, the levels of circulating early erythroid progenitors (burst forming units erythroid, BFU-E) were monitored during the crisis and recovery period. The virus-containing plasma inhibited the formation of BFU-Es from non-immune subjects and this effect was neutralised by convalescent serum. Colony forming units granulocyte-macrophage (CFU-GM) were also inhibited but this was probably non-specific since neutralisation did not reverse the effect. These experiments, together with the clinical data, suggest a selective effect of SPLV at the stage of erythroid progenitors.

Adolescent↗
PubMed1990

Subunit level crosslinking of rabbit muscle pyruvate kinase by o-phthaldialdehyde.

o-Phthaldialdehyde caused irreversible inhibition of rabbit muscle pyruvate kinase following preliminary formation of an enzyme-reagent complex. At pH 7.5, 35 degrees C, the dissociation constant for the complex and the maximal pseudo-first-order rate constant for covalent modification were 0.32 +/- 0.08 mM and 2.54 +/- 0.23 min-1, respectively. The inactivation was accompanied by uv-spectral changes pointing to isoindole formation, with a limiting stoichiometry of 1 isoindole linkage per enzyme subunit. Phosphoenolpyruvate, ADP, and ATP effectively protected the enzyme against inactivation, suggesting that the active site is the target of o-phthaldialdehyde action. As native and modified enzymes were indistinguishable with respect to mobility of the major band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was concluded that the crosslinkage was intrasubunit in character, and that the amino acid residues involved must be closely positioned in the polypeptide backbone. Lysine 366, previously shown to be selectively reactive toward 2',3'-dialdehyde ADP (Bezares et al., 1987, Arch, Biochem. Biophys. 253, 133-137), and cysteine 325 or 357 are implicated.

Aldehydes↗
PubMed1998

Delayed onset of hemolytic anemia in CBA-Pk-1slc/Pk-1slc mice with a point mutation of the gene encoding red blood cell type pyruvate kinase.

The Pk-1slc gene encodes a mutant red blood cell (RBC) type pyruvate kinase (PK), and adult CBA-Pk-1slc/Pk-1slc mice show a severe nonspherocytic hemolytic anemia. However, the number of RBCs and the proportion of reticulocytes were comparable between neonatal CBA-Pk-1slc/Pk-1slc mice and control -+/+ mice. Since the age-dependent increase of RBCs was much greater in CBA-+/+ mice than in CBA-Pk-1slc/Pk-1slc mice, significant anemia was observed in the latter mice on day 14 after birth. The increase of RBCs in CBA-+/+ mice was due to the prolongation of their survival time. The half life of RBCs increased in CBA-+/+ mice with ages, but it decreased in CBA-Pk-1slc/Pk-1slc mice. The relatively longer half life of RBCs in neonatal CBA-Pk-1slc/Pk-1slc mice appeared to be due to the delayed switching from M2-type PK that are expressed by undifferentiated erythroid precursor cells to RBC-type PK that are expressed by mature RBCs.

Age of Onset↗
PubMed1987

Differential effects of glucose and fructose on liver L-type pyruvate kinase gene expression in vivo.

Dietary carbohydrates are known to stimulate L-type pyruvate kinase gene expression at the transcriptional level in the liver. However, the short-term effects, the time course, and the mechanism of the gene activation by elemental hexoses in normal fasted rats remain unknown. In the present study, both glucose and fructose were found to stimulate the gene expression at the transcriptional level in liver. However, the kinetics and the extent of the mRNA induction differed according to the carbohydrate given. Fructose stimulated early (2-4 h) and transiently the gene transcription, the RNA precursor, and the mRNA accumulation in 48-h-fasted rats while maximum stimulation of the RNA synthesis by glucose was delayed until the 12th h of refeeding, despite an early rise of plasma insulin. In contrast, insulin release was not required for fructose to trigger the gene transcription, nor did the high cyclic AMP levels in fasted rat liver prevent RNA synthesis by fructose. The agent(s) operating early in fructose-fed animals might be powerful enough to not require insulin for gene activation and to balance the inhibitory action of glucagon in the liver.

Animals↗
PubMed2000

Dominant negative role of the glutamic acid residue conserved in the pyruvate kinase M(1) isozyme in the heterotropic allosteric effect involving fructose-1,6-bisphosphate.

Pyruvate kinase M(1), a nonallosteric isozyme, lacks heterotropic allosteric effect involving fructose-1,6-bisphosphate (FBP). To explore the molecular basis for this, a series of mutants were prepared and characterized, in which the possible candidate, Glu-432, was replaced in the rat M(1) isozyme and its allosteric mutant with the replacement of Ala-398 by Arg. Although these single mutants of Glu-432 remained nearly fully active, similar to the wild type, only the mutants with replacements by Lys and Ala were more efficiently activated by FBP when the enzymes were inhibited by L-phenylalanine. Kinetic analyses and ligand-induced fluorescence quenching studies using the allosteric double mutants indicated that the loss of a negative charge at residue 432 led to a dramatic decrease in the apparent activation constant and apparent K(d) for FBP. Furthermore, this enhancement was found to be associated with the modification of the FBP-binding site rather than the alteration of the subunit assembly. These findings suggest that Glu-432 hinders the heterotropic allosteric effect by preventing the binding of FBP through a repulsive electrostatic interaction and thereby contributes to its unique unregulated properties, independent of the shifted allosteric transition.

Allosteric Regulation↗
PubMed2000

Arsanilic acid-Sepharose chromatography of pyruvate kinase from KB cells.

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)-Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris-HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)-Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)-Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)-Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.

Arsanilic Acid↗
PubMed2004

Ex vivo analysis of aberrant splicing induced by two donor site mutations in PKLR of a patient with severe pyruvate kinase deficiency.

Two single-nucleotide substitutions in PKLR constituted the molecular basis underlying pyruvate kinase (PK) deficiency in a patient with severe haemolytic anaemia. One novel mutation, IVS5+1G>A, abolished the intron 5 donor splice site. The other mutation, c.1436G>A, altered the intron 10 donor splice site consensus sequence and, moreover, encoded an R479H substitution. We studied the effects on PKLR pre-mRNA processing, using ex vivo-produced nucleated erythroid cells from the patient. Abolition of the intron 5 splice site initiated two events in the majority of transcripts: skipping of exon 5 or, surprisingly, simultaneous skipping of exon 5 and 6 (Delta5,6). Subcellular localization of transcripts suggested that no functional protein was produced by the IVS5+1A allele. The unusual Delta5,6 transcript suggests that efficient inclusion of exon 6 in wild-type PKLR mRNA depends on the presence of splice-enhancing elements in exon 5. The c.1436G>A mutation caused skipping of exon 10 but was mainly associated with a severe reduction in transcripts although these were, in general, normally processed. Accordingly, low amounts of PK were detected in nucleated erythroid cells of the patient, thus correlating with the patient's PK-deficient phenotype. Finally, several low-abundant transcripts were detected that represent the first examples of "leaky-splicing" in PKLR.

Adult↗
PubMed1978

Ontogenesis of pyruvate kinase in the brain and liver tissues of the rat.

The ontogenetic study of pyruvate kinase in the brain and liver tissues was performed in different batches of rats, from the foetus at the 13th day of gestation to the adult subject. -- According to the kinetic study, the shape of the curve is transformed from sigmoid to hyperbolic from the 13th day of foetal life to adulthood in the brain. Hill coefficient increases with the age of animal in the liver tissue. -- According to polyacrylamide gel isoelectrofocusing, a family of four, transitory or definite bands are detected in the foetal brain : they are well defined by their pHi : M4, M3, M2, M1 ; at the adult stage, M1 predominates, M2 is minor. Threee principal bands are distinguished in the liver : two are characteristic of foetal life (Lf and M2), one of adulthood (L). -- According to the immunochemical tests, there are antigenic determinants common to M1, M2, M3 and M4. The confrontation of the first two methods prompts the conclusion that the kinetic of the enzyme (and perhaps its function) varies with the animals age and is linked to its molecular structure. With the third method, it allows to stress the precociousness of the appearance of the common antigenic determinants, simultaneously with immature enzymatic forms. The signification of the kinetic modifications as well as the succession of the isozymes of the M type in a determined order are discussed and the in vivo formation of hydbrids is suggested.

Animals↗
PubMed1984

Thermodynamic linkages in rabbit muscle pyruvate kinase: kinetic, equilibrium, and structural studies.

The mechanism of allosteric regulation of rabbit muscle pyruvate kinase (PK) was examined in the presence of the allosteric inhibitor phenylalanine (Phe). Steady-state kinetic, equilibrium binding, and structural studies were conducted to provide a broad data base to establish a reasonable model for the interactions. Phe was shown to induce apparent cooperativity in the steady-state kinetic measurements at pH 7.5 and 23 degrees C. The apparent Km for phosphoenolpyruvate was shown to increase with increasing Phe concentrations. These results imply that Phe reduces the affinity of PK for phosphoenolpyruvate. This conclusion was substantiated by equilibrium binding studies which yielded association constants of phosphoenolpyruvate as a function of Phe concentration. The binding constant of Phe was also determined at pH 7.0 and 23 degrees C. The effect of ligands on the hydrodynamic properties of PK was monitored by difference sedimentation velocity, sedimentation velocity, and equilibrium experiments. The results showed that PK remains tetrameric both in the presence and in the absence of Phe. However, Phe induces a small decrease in the sedimentation coefficient of the enzyme; hence, it suggests a loosening of the protein structure. The accessibility of the sulfhydryl residues of the enzyme also increases in the presence of Phe. Furthermore, the Phe-induced conformational change was approximately 90% complete when only 25% of the binding sites were saturated. This result suggested that the regulatory behavior of PK might satisfactorily be described by the two-state model of Monod-Wyman-Changeux [Monod, J., Wyman, J., & Changeux, J.-P. (1965) J. Mol. Biol. 12, 88-118].

Allosteric Site↗