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PubMed1981

Inhibitory effect of Zn2+ on rabbit muscle pyruvate kinase and reactivation by histidine.

Histidine was found to be an activator of rabbit muscle pyruvate kinase activity with a K1/2 value of 0.6 mM. Carnosine and anserine are also effective, but only at much higher concentrations. Hyperbolic kinetics with phosphoenolpyruvate of the enzyme were found in either the presence or absence of histidine. Of a number of divalent cations tested, only Zn2+ was found to be an effective inhibitor of enzyme activity at low concentrations. The kinetic data suggested that Zn2+ acted as inhibitor as well as activator for the enzyme activity; a high affinity binding site was associated with Ki of approximately 4.8 microM Zn2+ and a catalytic site was associated with Km of approximately 80 microM Zn2+ Zn2+, which is associated to a high affinity binding site of the enzyme, was removed by the addition of histidine with a K1/2 of approximately 0.6 mM. From these findings, histidine including anserine and carnosine in muscle may act as a chelating agent for the enzyme activity.

Animals↗
PubMed1987

Human erythrocyte pyruvate kinase (L'/R-PK): production and characterization of a monoclonal antibody.

Two preparations of L'/R-type pyruvate kinase from human erythrocytes characterized by SDS-PAGE were used for immunization of BALB/c mice. Their spleen cells were fused with mouse myeloma cells by polyethylene glycol according to standard techniques. Supernatants of hybridomas resulting from two separate experiments were assayed by ELISA and further characterized by immunoblotting. Using those monoclonal antibodies reacting with L'/R-PK in immunoblotting, a major band of 62 KDa MW was recognized in both preparations employed for immunization. Additionally, four smaller bands were detected. Furthermore, the monoclonal antibodies also detected a single band of 62 KDa MW in a highly purified L-PK prepared from human liver. In contrast, they showed no reaction with muscle and brain tissues containing M1-type PK. However, they reacted strongly with a single band of approximately 62 KDa in liver and kidney homogenates which is in line with immunocytochemical studies showing immunoreactive material in hepatocytes and proximal tubules of the kidney.

Antibodies, Monoclonal↗
PubMed1976

Isoelectric focussing of normal human erythrocyte pyruvate kinase (PK) and PK variants with abnormal electrophoretic patterns.

Isoelectric points of normal human erythrocyte pyruvate kinase (PK) and PK variants with abnormal electrophoretic patterns, were obtained by means of isoelectric focussing. The obtained isoelectric points of normal, PK Nagasaki, PK Tokyo II, PK Ube, and case T.S. were 7.36, 7.38, 7.05, 6.90, and 7.04, respectively, at 6 degrees C. All of the PKs collected after isoelectric focussing were shown to be not of the L-type PK but to be the erythrocyte PK (by means of thin-layer-polyacrylamide gel electrophoresis). The PKs with fast migration had isoelectric points that were shifted to the acidic side as compared with those of the normal subjects. The PK with slow migration had an isoelectric point that was shifted to the alkaline side, demonstrating that the isoelectric points obtained by isoelectric focussing were consistent with the electrophoretic patterns of the crude hemolysates. Isoelectric focussing also revealed the existence of PK variants with abnormal electric net charge, just as well as thin-layer-polyacrylamide gel electrophoresis.

Erythrocytes↗
PubMed1996

PK Mondor: prenatal diagnosis of a frameshift mutation in the LR pyruvate kinase gene associated with severe hereditary non-spherocytic haemolytic anaemia.

A mutant form of pyruvate kinase (PK) from the red blood cells of a consanguineous family with severe non-spherocytic haemolytic anaemia has been characterized by polymerase chain reaction (PCR) amplification and sequencing. The variant enzyme was named PK Mondor, according to the recommendations of the International Committee for Standardisation in Haematology. The propositus lacked PK activity and the low level of PK activity found resulted more likely from PK-M2 (fetal isozyme) expression in the red blood cells of the propositus. PK Mondor corresponds to a frameshift mutation with deletion of one G in a repetition of four Gs in positions 1231-1234. This family, whose first child was stillborn and whose second was homozygous for the frameshift mutation, requested prenatal diagnosis during the third pregnancy. Diagnosis was made after chorionic biopsy by a specific approach combining PCR amplification and restriction enzyme digestion.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1992

Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells.

The human pyruvate kinase (PK) L-gene is organized in 12 exons over 9.5 kilobases, and the first and second exons are specifically transcribed to the R- and L-type PK mRNA. The 5'-flanking region upstream the first exon has two CAC boxes and four GATA motifs within 250 bp from the translational initiation codon. Comparison with the rat L-gene revealed four well-conserved elements in the region. The transient transfection demonstrated that the 270-bp upstream region was a powerful promoter in K562 cells, whereas deletion of the distal 150-bp sequences, which included three GATA motifs, resulted in drastic reduction of the activity. When the hypersensitive site 2 of the human beta-globin gene locus was joined to the promoter, the activity of the proximal 120-bp region was enhanced. We concluded that the proximal 120-bp region had a basal promoter activity and that the distal 150-bp region acted as an enhancer in erythroid cells.

Animals↗
PubMed1982

Molecular organization of human L' and L pyruvate kinases.

Extremities, peptide maps and phosphorylatable site localization of human erythrocyte L' and liver L pyruvate kinases (EC 2.7.1.40) were investigated. L' and L subunits seemed to have similar, blocked NH2 termini and differ in their sensitivity to carboxypeptidase A, that is to say in their C-terminal ends. After digestion by Staphylococcus aureus V8 protease, the phosphorylated sites of both L' and L subunits were located on those peptides which were different in L' and L, that is to say on the C-terminal sides. A mild proteolytic attack of the native tetrameric enzymes by trypsin partially degraded the phosphorylatable peptides without removing the phosphoserine residue; in the same conditions, chymotrypsin split off this phosphorylated residue and subtilisin totally degraded the phosphorylated peptides. From these results it appears, therefore, that age-dependent proteolytic degradation of L' subunits in old red cells involves the C-terminal side of the molecules, ultimately resulting in cleavage of the phosphorylated site. Since erythrocyte L' and liver L subunits are encoded by different species of messenger RNAs, our results indicate, in addition, that these messenger RNA species should differ by their 3' coding sequences.

Carboxypeptidases↗
PubMed1995

Study of the molecular defects in pyruvate kinase deficient patients affected by nonspherocytic hemolytic anemia.

We have examined DNA from fifteen unrelated pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia (HNSHA) for the molecular alterations responsible for the enzyme deficiency. All but 3 of the 30 putative mutations were identified. Fourteen different mutations were found. Nine were missense mutations: 320 T-->C, 823 G-->C, 1276 C-->T, 1378 G-->A, 1484 C-->T, 1529 G-->A, 1654 G-->A, 1675 C-->G; three were nonsense mutations: 603 G-->A, 721 G-->T, 1501 C-->T; one was an insertion at 1574 GGG-->GGGG and the other a three nucleotide in-frame deletion 391-392-393 ATC. Eight of these mutations have not been previously described. We also investigated all of the patients for the C/A polymorphism at nt 1705 and the microsatellite ATT repeat in intron 11. All of the mutations that had previously been reported by us (391-393del, 721T, 1484T, 1529A) were found in the context of the same haplotype as the earlier cases, supporting the concept that each may have a single origin.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1976

Respiratory characteristics of blood from Basenji dogs with classical erythrocyte pyruvate kinase deficiency.

The oxygen affinities of blood from eight Basenji dogs homozygous for classical erythrocyte pyruvate kinase deficiency and four dogs heterozygous for the defect were compared with blood from 14 Labrador retrievers and two normal Basenji dogs. The homozygous dogs showed significant anemia compared to heterozygous and normal dogs (P is less than 0.01). The average blood P50 value (at 38C and plasma pH of 7.40) for both homozygous and heterozygous dogs was significantly higher (P is less than 0.01) than for normal dogs (33.6+/-0.4 and 31.8+/-0.7 vs 30.8+/-0.6 mm Hg). The concentrations of 2,3-DPG in the blood of both heterozygous and homozygous dogs were significantly higher than normal values. Four months after splenectomy P50 values declined to normal in four homozygous Basenji dogs without any change in the degree of anemia or blood 2,3-DPG concentrations. Iron kinetic studies showed a shorter plasma clearance time in a homozygous than in a normal dog with the heterozygote falling midway between. The red cell life span in the normal and heterozygous dogs was approximately 120 days. The 59Fe studies on the homozygous dog indicate markedly different survival characteristics which can be attributed to the existence of three populations of red cells differing in their life spans.

Animals↗
PubMed1991

In vivo regulation of monomer-tetramer conversion of pyruvate kinase subtype M2 by glucose is mediated via fructose 1,6-bisphosphate.

The activity of pyruvate kinase, subtype M2 (PKM2), is known to be increased by fructose 1,6-bisphosphate (Fru-1,6-P2), one of the metabolites in the glycolytic pathway. Recently, we have shown that in vitro, Fru-1,6-P2 activated the association of monomer to form the tetrameric PKM2. To ascertain whether this mode of regulation also occurs in vivo, we prepared monomer-specific monoclonal antibody and quantified the monomer formation in situ in cultured cells by immunocytochemistry. The intracellular Fru-1,6-P2 was manipulated by the glucose concentration in the media. At the physiological concentration of glucose (4-6 mM), 30-35% of PK existed as a monomer. However, PKM2 was dissociated into monomer within minutes after cells were deprived of glucose. The maximal level of monomer was detected after 1 h at 37 degrees C. Monomer was rapidly (within minutes) converted to tetramer after addition of glucose. Furthermore, when cells cultured in 10 mM of glucose were treated with cytochalasin B, an inhibitor of the glucose transporter, a maximal level of monomer was detected within 20-30 min. Determination of Fru-1,6-P2 indicated that its intracellular concentration decreased concomitantly with the reduction in glucose concentration in the medium. These results indicate that monomer-tetramer inter-conversion is a major in vivo cellular regulatory mechanism in response to changes in the extracellular glucose concentration via Fru-1,6-P2.

Animals↗
PubMed2003

Monitoring active site alterations upon mutation of yeast pyruvate kinase using 205Tl+ NMR.

The interaction of the monovalent cation with wild type (WT) yeast pyruvate kinase (YPK) and with the T298S, T298C, and T298A mutants was investigated by 205Tl+ NMR to monitor possible structural alterations at the active site by Thr-298 mutation. TlNO3 activates WT YPK with a kcat value similar to that obtained with KCl and an apparent Ka of 0.96 +/- 0.07 mm in the presence of Mn2+ and fructose 1,6-bisphosphate. With the three mutants, Tl+ is a better activator than is K+ based on kcat values. Tl+ activation and inhibition of YPK is affected by mutation of the active site Thr-298. The effect of Mn2+ on the 1/T value of 205Tl+1 in the presence of the WT and mutant YPK complexes was determined at 173 MHz (300 MHz, 1H) and 346 MHz (600 MHz, 1H). For each complex studied, 1/pT2p >> 1/pT1p and 1/pT1p is frequency-dependent suggesting fast exchange conditions. The values of 1/pT1p differ for each mutant. A correlation time of 0.65 +/- 0.35 ns was estimated for the Mn2+-205Tl+ interaction. The Tl+-Mn2+ distances at the active site of YPK were calculated from the paramagnetic contribution of Mn2+ to 1/T1M of YPK-bound 205Tl+. The calculated Tl+-Mn2+ distance for the Thr-298 mutants is decreased by about 1 A from 6.0 +/- 0.2 A observed with WT. The results suggest conformational alterations at the active site of YPK where phosphoryl transfer occurs upon mutation of Thr-298. These conformational changes may, in part, explain the alteration in kcat and kcat/Km,PEP observed with the Thr-298 mutants.

Animals↗
PubMed2002

Erythrocyte pyruvate kinase activity during chemotherapy in children with leukemia and lymphoma.

OBJECTIVES: To determine both erythrocyte pyruvate kinase activity (ePKA) at the time of diagnosis of patients with acute leukemia or lymphoma and the differences in the ePKA profiles during the malignant disease and its chemotherapy. METHODS: A prospective, longitudinal clinical study was performed involving 57 patients, 10 were the ones with relapse of acute lymphoblastic leukemia, 32 were the ones with acute lymphoblastic leukemia (ALL) and 15 were the ones with lymphoma. None of the subjects in this study group received treatment or blood transfusion before the study, except the ones diagnosed with relapse of ALL. Forty two healthy children were also selected to form the control group. In order to measure ePKA, blood samples were taken for five times, with 1.5 months apart between each other during the study. Statistical analysis were done by using Wilcoxon's signed rank test, Kruskall-Wallis with Mann-Whitney U Test and Spearman rank correlation coefficient test. RESULTS: The ePKA of the patients with relapse of ALL, and ALL, but not the patients with lymphoma, at the time of diagnosis were found to be lower compared to the one's in the control group (respectively p = 0.001, p = 0.003). The comparison between the first ePKA samples and the third ePKA samples of the patients with both ALL and lymphoma showed a significant increase (respectively p = 0.006, and p = 0.047). CONCLUSION: The measurement of ePKA can be considered for follow-up the neoplastic treatment due to the fact that it is detected to be low in leukemia and relapse of ALL and in normal values after chemotherapy. However, more long-term studies, including more number of cases, are required to be carried out in order to prove the accuracy of this hypothesis. (Tab. 2, Fig. 1, Ref. 28.)

Adolescent↗
PubMed2004

Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis.

Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the P(nisA)-pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain. In vivo (13)C- and (31)P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells. A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD(+) and NADH was obtained. A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced. Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD(+)/NADH ratio during glucose catabolism. In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels. Furthermore, the level of NAD(+) decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020). The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions. Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert. However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.

Adenosine Triphosphate↗
PubMed2004

Cadmium induces both pyruvate kinase and Na+/H+ exchanger activity through protein kinase C mediated signal transduction, in isolated digestive gland cells of Mytilus galloprovincialis (L.).

The present study investigates the transduction pathway mediated by cadmium in isolated digestive gland cells of mussel Mytilus galloprovincialis. The effects of cadmium treatment on a key glycolytic enzyme, pyruvate kinase (PK), and on Na(+)/H(+) exchanger activity were examined. Cadmium (50 micro mol l(-1)) caused a significant elevation of intracellular pH (pHi) and a rise (176%) of Na influx relative to control values. The amiloride analogue, EIPA (20 nmol l(-1)), a Na(+)/H(+) exchanger blocker, together with cadmium, significantly reduced the effect of treatment by cadmium alone on both Na(+) influx and pHi. In addition, PK activity was significantly increased after treatment with cadmium. PK activity was inhibited after treatment of cells with amiloride or EIPA together with cadmium. Moreover, phorbol-ester (PMA), a potent activator of protein kinase C (PKC), caused a significant rise in both pHi and PK activity, while staurosporine or calphostin C reversed both events. Adrenaline, isoprenaline and phenylephrine alone or together with cadmium also significantly increased the pHi and PK activity of isolated digestive gland cells. The latter effectors in combination with cadmium showed a synergistic effect on pHi and PK. These responses seem to be blocked by propranolol, metoprolol and prazosin. Our findings suggest a hormone-like effect of cadmium on digestive gland cells. The signal transduction pathway induced by cadmium involves the stimulation of PK, PKC and Na(+)/H(+) exchanger in isolated digestive gland cells of Mytilus galloprovincialis.

Amiloride↗
PubMed1985

Purification and properties of aerobic and anoxic forms of pyruvate kinase from the hepatopancreas of the channelled whelk, Busycotypus canaliculatum.

Aerobic and anoxic variants of pyruvate kinase (termed PK-aer and PK-anx) from the hepatopancreas of the gastropod mollusc, Busycotypus canaliculatum, were purified to apparent homogeneity with final specific activities of 14 and 2.3 units/mg protein, respectively. Both enzymes were homotetramers of the same molecular weight. The enzymes also showed equivalent affinities for ADP (0.22 mM) and very similar affinities for Mg2+, Mn2+, K+, and NH4+. PK-aer and PK-anx differed strongly, however, in maximal enzyme velocity (Vmax 9-fold higher for PK-aer), in affinity for P-enolpyruvate (PEP0.5 = 0.38 mM for PK-aer and 1.1 mM for PK-anx), and in the effects of activators and inhibitors on the enzymes. PK-aer was much more strongly stimulated by fructose-1,6-P2 and aspartate as activators (a 19- and 32-fold activation of enzyme velocity at subsaturating PEP levels versus only 4.1- and 2.6-fold activation for PK-anx, respectively). K alpha for fructose-1,6-P2 was 3-fold lower (0.16 microM) for PK-aer than for PK-anx (0.48 microM), but K alpha for aspartate was the same for both enzymes (1.5 mM). Activators decreased the PEP0.5 (to 0.05 mM for PK-aer and 0.07 mM for PK-anx), relieved inhibitions by alanine, Mg ATP, ADP, and Pi, and, when added together, showed a strong synergistic activation of PK-aer (but not PK-anx). The kinetic differences between PK-aer and PK-anx are similar to those of the dephosphorylated versus phosphorylated forms of PK from other sources, including those of red muscle PK of B. canaliculatum, and indicate that the change in enzyme form brought about during anaerobiosis may be due to enzyme phosphorylation. The powerful activation of hepatopancreas PK by aspartate is a novel regulatory control of the enzyme. Aspartate is one of the substrates of anaerobic energy production in marine molluscs and its effects on the enzyme may be important in a tissue where inactivation of PK can occur for one of two reasons: anaerobiosis or gluconeogenesis.

Adenosine Diphosphate↗
PubMed1980

Studies of the structure-function relationships of Neurospora pyruvate kinase: denaturation by urea and the effect of ligands on the refolding and renaturation process.

1. The renaturation of Neurospora pyruvate kinase following denaturation with urea was investigated. 2. The substrates, phosphoenol pyruvate and adenosine diphosphate, were observed to stabilize the enzyme against urea-induced structural disorganization. 3. It was demonstrated that for refolding/reactivation of the denatured protein the allosteric activator, fructose-1,6-diphosphate and a sulphydryl protectant are required. 4. The enzyme recovered following renaturation showed complete immunological identity with the native enzyme in Ouchterlony double diffusion tests.

Adenosine Diphosphate↗
PubMed1992

Effect of tolbutamide and glyburide on pyruvate kinase flux in isolated rat hepatocytes.

The effects of two representative sulfonylureas, tolbutamide and glyburide, on pyruvate kinase (PK) flux were examined in fasted rat hepatocytes. PK flux was estimated by trapping 14C from NaH14CO3 in a 2 mM lactate pool, accounting for any incomplete trapping by parallel incubations with L-[1-14C]alanine. Glyburide (20 microM) and tolbutamide (1 mM) decreased glucose formation by 34.9% and 54.8%, respectively, from 2 mM lactate. This decrease in glucose formation was associated with a proportional decrease in pyruvate carboxylase (PCOX) flux (32.7% and 50.5%, respectively). Under these conditions, no net change in PK flux was observed. When hepatocytes were preincubated with lactate and/or sulfonylurea addition for 30 min prior to radiolabeling with NaH14CO3, the metabolic state of the cells changed markedly. Glyburide produced a 34.6% decrease in glucose formation and a 31.3% decrease in PCOX flux, but no change in PK flux. In contrast, tolbutamide decreased glucose formation by 12.5% and increased PK flux by 53.2%, but no change in PCOX flux was observed. Such an increase in PK flux may be linked to tolbutamide-mediated increases in fructose-1,6-bisphosphate (F16P) via fructose-2,6-bisphosphate (F26P). These findings demonstrate that tolbutamide and glyburide decrease hepatic glucose production through various alterations in carbohydrate metabolism, depending upon the metabolic state of the cell. In addition, F26P may play a larger role in the hypoglycemic mechanism of action of tolbutamide than glyburide, since pyruvate carboxylase accounted for most of the decrease in glucose formation observed with glyburide and because preincubation with tolbutamide resulted in an activation of PK.

Animals↗
PubMed2004

Coexistence of congenital red cell pyruvate kinase and band 3 deficiency.

The authors report the case of a 9-year-old Caucasian girl, born in northern Portugal, with chronic nonspherocytic haemolytic anaemia and without family history of anaemia. The aethiological study of this anaemia revealed pyruvate kinase deficiency (PKD), because of two previously described mutations (426Arg-->Trp and 510Arg-->Gln). Since the blood smear revealed features not fully compatible with PKD diagnosis, additional tests were performed for the propositus and her parents, namely red blood cell membrane protein analysis. A decrease in proteins band 3 (15%) and 4.2 (18%) was found in the propositus. Her father presented only a decrease in band 3 (11%). Coexistence of PKD and erythrocyte membrane proteins deficiency in the same patient is very uncommon. Our findings suggest that a careful blood smear observation may lead to the identification of a combined deficiency in erythrocyte membrane proteins and enzymopathies.

Anemia, Hemolytic, Congenital↗