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PubMed2006

Red cell pyruvate kinase deficiency in neonatal jaundice cases in India.

OBJECTIVE: Pyruvate Kinase (PK) deficiency is the most common enzymopathy of the glycolytic pathway in erythrocytes. It constitutes one of the common causes of hereditary non-spherocytic hemolytic anemia. The aim of this study was to screen newborns in India for pyruvate kinase (PK) deficiency in relation to unconjugated hyperbilirubinemia. METHODS: Laboratory investigations done included complete blood counts, reticulocyte counts, direct and indirect bilirubin, assay of G6PD and PK activity, ATP and 2,3 DPG levels. All variables were studied in 50-cord blood samples from normal deliveries and 218 neonates with hyperbilirubinemia. RESULTS: 7 of the 218 cases of neonatal jaundice were PK deficient with 30-40% reduction in PK activity. These cases also had a 3-4-fold increase in 2,3 DPG:ATP ratios, which is one of the additional indicators for PK deficiency. Six of the 7 infants had a severe clinical course. CONCLUSION: This study shows that the prevalence of PK deficiency in Indian neonatal jaundice cases is 3.21%, which is relatively high. This emphasizes the need for screening neonatal hyperbilirubinemia cases in India for PK deficiency.

Erythrocytes↗
PubMed1983

Red blood cell age, pyruvate kinase activity, and insulin receptors. Evidence that monocytes and RBCs may behave differently.

Data emerging from insulin receptor studies performed on red blood cells (RBCs) and monocytes from the same subject are not always in agreement; dichotomy might occur since variations in mean RBC age are not taken into account or because insulin receptors on the two cell types behave differently. In the present investigation RBCs from normal male subjects were separated into five populations of different mean age by means of centrifugation of RBCs on a discontinuous gradient of buffered Percoll for 10 min at 1000 X g. Insulin binding varied significantly depending upon the RBC population tested and was closely correlated to the activity of pyruvate kinase (r2 = 0.86), a well-known marker of RBC age. These data suggested that pyruvate kinase assay might be helpful in studies of RBCs. To confirm this hypothesis, RBCs from 10 normal male subjects and 13 male patients with hemolytic anemia were studied; insulin binding was correlated to pyruvate kinase activity. By adjusting insulin binding to 2 X 10(9) RBCs/ml the range of data was abnormally high, but it became acceptable after adjusting insulin binding to pyruvate kinase activity (0.75 U/2 X 10(9) RBCs). The overall data indicated that insulin binding was highly correlated to pyruvate kinase activity (r2 = 0.82) but only slightly to reticulocyte number (r2 = 0.56) since not only reticulocytes but also erythrocytes lose receptors during maturation. Pyruvate kinase activity was measured in RBCs from normal men and from normally menstruating women at the seventh and twenty-fourth days of the cycle; results demonstrated that adjustment of data, according to mean RBC age, broadens dichotomy of monocyte and RBC data.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1978

Deviation of isozyme pattern of pyruvate kinase in the liver of nude mice bearing allogeneic or xenogeneic tumor.

Isozyme pattern of pyruvate kinase in the liver of nude mice bearing Ehrlich ascites tumor changed markedly during tumor growth. The change in isozyme pattern of pyruvate kinase was not due to infiltration or metastasis of tumor cells in the liver. Such a change in isozyme pattern of pyruvate kinase was also found in the liver of nude mice bearing canine gastric leiomyosarcoma induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not in the liver of nude mice bearing MNNG-induced canine gastric adenocarcinoma or human gastric adenocarcinoma.

Animals↗
PubMed1988

Regulation of pyruvate kinase in Reuber H35 hepatoma cells by insulin and fructose.

1. Kinetic and immunological studies as well as electrophoretic behaviour indicated that pyruvate kinase in Reuber H35 hepatoma cells is of the M2-type. 2. Addition of 0.1 microM insulin or 2 mM fructose to the incubation medium for 72 hr increased the activity of the M2-type pyruvate kinase in Reuber H35 hepatoma cells by 103 and 25% respectively. 3. Incorporation studies with [3H]leucine followed by immunoprecipitation showed that the apparent rate of synthesis of the M2-type pyruvate kinase was increased by both insulin and fructose. 4. Degradation studies indicated that the addition of insulin and fructose to the incubation medium increased the half-life of the M2-type pyruvate kinase from 4.8 to 8.6 and 6.8 hr respectively.

Animals↗
PubMed1984

Regulation of L-pyruvate kinase activity by insulin and glycolytic intermediates.

The presence of insulin and various carbohydrates in long term cultures of hepatocytes was studied to examine the mechanism by which insulin and glycolytic metabolites regulate L-pyruvate kinase activity. When hepatocytes were isolated from a control rat and cultured in the presence of insulin, a constant level of enzyme activity (16 EU/mg DNA) was maintained for 12 days. The L-pyruvate kinase activity in hepatocytes from refed rats initially was elevated (45 EU/mg DNA) and decreased to control values by the 5th day in culture. Cells isolated from a fasted rat initially contained a low level of L-pyruvate kinase activity (5 EU/mg DNA) which increased to control values by the 8th day in culture. The enzyme activity was 5 EU/mg DNA when control cells were cultured for 4 days in medium containing either glucose, glycerol or fructose without insulin; 10 EU/mg DNA in medium containing galactose and insulin but without glucose, glycerol or fructose; and 20 EU/mg DNA in medium containing both insulin and either glucose, glycerol or fructose. It is suggested that insulin is essential for the induction and maintainance of L-pyruvate kinase activity, that carbohydrates in the absence of insulin are unable to maintain the enzyme activity in cultures of hepatocytes, and that insulin and glycolytic metabolites may act synergistically to increase the activity of L-pyruvate kinase by increasing the synthesis of the enzyme.

Animals↗
PubMed1985

Stimulation of Trypanosoma brucei pyruvate kinase by fructose 2,6-bisphosphate.

The activity of pyruvate kinase present in a crude extract of the bloodstream form of Trypanosoma brucei was greatly increased by fructose 2,6-bisphosphate, which converted the saturation curve for phosphoenolpyruvate from a sigmoid into a hyperbola with no change in V. Phosphate and arsenate had an effect opposite to that of fructose 2,6-bisphosphate and the apparent Ka for fructose 2,6-bisphosphate was shifted from 75 nM to 1.5 microM by the presence of 5 mM phosphate. Fructose 1,6-bisphosphate had effects similar to those of fructose 2,6-bisphosphate but at approximately 4000-fold higher concentrations. Pyruvate kinases of Crithidia luciliae and of Leishmania major, two trypanosomatids which are like T. brucei in containing glycosomes, were also stimulated by fructose 2,6-bisphosphate and inhibited by phosphate.

Animals↗
PubMed1979

Modification of pyruvate kinase activity by proteins from chicken liver.

The partial purification of a protein fraction inhibiting pyruvate kinase isoenzymes is described. The fraction was isolated from the (NH4)2SO4 step of the purification procedure for pyruvate kinase isoenzymes from chicken liver (Eigenbrodt, E. & Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1033-1046) by extraction with 1N NaOH, acidification to pH 3, ethanol precipitation and chromatography of the supernatant on DEAE-cellulose. The inhibitor fraction was further purified by disc gel electrophoresis using a gel gradient from 10 to 25%; this procedure separated activating proteins from the inhibitor fraction. The inhibitor fraction inhibited the pyruvate kinase isoenzymes from chicken in the sequence of decreasing effect: M2 greater than L greater than M1. The inhibition was due to a decrease in the affinity for phosphoenolpyruvate. The inhibitor is stable against heating for 5 min in 1% sodium dodecyl sulfate at 100 degrees C; it is destroyed by pepsin digestion. The inhibitor fraction could be purified further only by dodecyl sulfate gel electrophoresis. This resulted in the separation of 2 inhibitors (Mr = 33,500 +/- 8500 and ca. 5000), an activator (Mr = 15,100 +/- 5200), and an unidentified protein (Mr = 27,000).

Animals↗
PubMed1975

Spectral properties of Co(II)- and Ni(II)-activated rabbit muscle pyruvate kinase.

Stoichiometry, kinetics, and optical properties of rabbit muscle pyruvate kinase activated with Co(II), Ni(II), Mg(II), and Mn(II) were studied. The stoichiometry of metal binding to enzyme was found to be 4 metal ions per tetrameric enzyme for Co(II) and Ni(II) by carrying out circular dichroic titrations. Cu(II) and Fe(II) were inactive. Ca(II) and Zn(II) were not activating, and were inhibitory with respect to all of the active cations. The temperature dependence of the optimal velocity is similar for all activating metals. The pH rate profiles suggest that there are two classes of enzyme activation by metal ions. Mg(II) and Mn(II) are quite similar to each other while Co(II) and Ni(II) are different from them but similar to each other. Absorption, natural, and magnetic CD in the visible region were used to probe the environment of the activating divalent cation in Ni(II)- and Co(II)-activated pyruvate kinase and their complexes with substrates and inhibitors...

Animals↗
PubMed1993

Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes.

Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10:1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis.

Adenosine Diphosphate↗
PubMed1995

Affinity labelling of the catalytic and allosteric ATP binding sites on pyruvate kinase type I from Escherichia coli.

The allosterically regulated pyruvate kinase type I (PKI) from E. coli was inactivated by the ATP analog 2',3'-dialdehyde ATP (o-ATP) with a Ki of 3.6 mM. ATP and phosphoenolpyruvate protected the enzyme activity while the allosteric activator fructose 1,6-bisphosphate enhanced the rate of inactivation. Incubation with o-ATP, followed by reduction of the formed Schiff bases with radioactive sodium borohydride, was employed to determine the ATP binding sites of PKI. After tryptic digestion, the purification of the labelled peptides and the sequence analysis allowed to identify four modified lysyl residues, namely Lys173, Lys175, Lys272, and Lys317 of the known DNA-deduced sequence of PKI. The close lysines 173 and 175 reacted with o-ATP in a mutually exclusive way and accounted together for 53% of the recovered radioactivity, the rest being distributed on Lys272 (31%) and Lys317 (16%). When fitted on the available three-dimensional structure of muscle pyruvate kinase, the position of the modified lysines defines both the catalytic and the allosteric ATP binding sites on PKI.

Adenosine Triphosphate↗
PubMed1981

Reversible solvent denaturation of rabbit muscle pyruvate kinase.

The structural transitions of the tetrameric rabbit muscle pyruvate kinase induced by guanidine hydrochloride and urea are characterized by elastic and quasi-elastic light-scattering, sedimentation velocity, and intrinsic viscosity experiments as well as by protein fluorescence, circular dichroism, and enzymic activity measurements. The transition curves are shown to be reversible. We find a new pathway of unfolding which is different from that described in the literature: The first intermediate with increasing concentration of denaturant is a less compact and inactive tetramer which can be renatured if substrates are added. Dissociation of the tetramer results in an expanded dimer with a partial loss of the secondary structure. The final state is a completely disordered monomer. These intermediates are consistent with a domain structure of pyruvate kinase, as it was suggested by Stammers & Muirhead [Stammers, D. K., & Muirhead, H. (1975) J. Mol. Biol. 95, 213--225] on the basis of their X-ray data. Using Schellman's solvent denaturation model [Schellman, J. A. (1978) Biopolymers 17, 1305--1322], we calculate the free energies of stabilization of the folding--unfolding equilibrium.

Animals↗
PubMedDate not supplied

[Effect of insulin and hydrocortisone on pyruvate kinase from the medullar and cortical layers of rabbit kidney].

Activity of M2-pyruvate kinase from medullar layer of rabbit kidney was studied in diabetes, in starvation within 1 day and 10-16 days and in long-term starvation of rabbits after administration of glucose or hydrocortisone and protamine-Zn-insulin. The enzymatic activity was increased in diabetes and decreased in long-term starvation and after administration of insulin. A correlation was observed between low activity of pyruvate kinase in kidney medulla under conditions of long-term starvation of rabbits and deficiency of the enzyme substrate. The data, obtained after study of the enzymatic activity in kidney medulla as compared with that of rabbit kidney cortex, demonstrate various adaptability of cells from these kidney layers to regulatory effects of hormones on the pyruvate kinase activity.

Animals↗
PubMed1985

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. A mathematical model of glycolysis in normal and pyruvate-kinase-deficient red blood cells.

A mathematical model of glycolysis in human erythrocytes is proposed to study the influence of a pyruvate kinase deficiency on the energy metabolism. The model takes into account the main regulatory properties of the non-equilibrium enzymes and the magnesium-complex formation by the adenine nucleotides and by 2,3-bisphosphoglycerate. In the normal case (no enzyme defect) the calculated flux rates and metabolite concentrations are in a good agreement with experimental data. It is shown that a severe pyruvate kinase deficiency manifested in a tenfold diminished activity of that enzyme leads to a remarkable decrease of the glycolytic flux and the ATP concentration of about 50% of the normal values. On the other hand a lowering of the pyruvate kinase activity to half of the normal value, characteristic for the heterozygotes, gives no significant alterations of the metabolite concentrations and the flux rates compared with the normal case which is in accordance with the lack of clinical symptoms for a metabolic disease of these probands. For three patients with known alterations of their pyruvate kinase mutants the calculated metabolite concentrations and the control characteristics permit estimation of the degree of disorder of the glycolytic pathway. The resulting classification corresponds well to other independent experimental and clinical findings. In particular, the calculation demonstrates that there is no simple correlation between the lowered enzyme activity and the reduced flux rate through the affected pathway.

Adenosine Triphosphate↗
PubMed1974

Some Properties of Partially Purified Pyruvate Kinase from Euglena gracilis Klebs var. bacillaris.

A method of purification of pyruvate kinase (EC 2.7.1.40) from light-grown Euglena gracilis var. bacillaris was developed which yielded an enzyme preparation purified 115-fold over crude extracts. During organelle formation, levels of pyruvate kinase in extracts prepared from cells engaged in light-induced chloroplast development do not change significantly. The enzyme has a molecular weight of approximately 240,000 and a requirement for both K(+) and Mg(2+). Fructose 1,6-diphosphate activates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentration of ADP is limiting. ATP, citrate, and Ca(2+) are inhibitors of the enzyme and inhibit the fructose 1,6-diphosphate stimulation of the enzyme activity. ATP inhibition is only partially reversed by high concentrations of fructose 1,6-diphosphate. Further reversal of inhibition can be achieved by dialysis. Ca(2+)-dependent inhibition can be reversed by a chelating agent but not by increased concentrations of Mg(2+).The significance of the properties of pyruvate kinase in the regulation of photosynthetic carbohydrate metabolism, especially in connection with the inability of fructose 1,6-diphosphate to reverse Ca(2+) and ATP inhibitions, is emphasized.

Journal Article↗
PubMed1987

Interactions of insulin and dexamethasone in the control of pyruvate kinase activity and glucose metabolism in sheep adipose tissue.

1. The Vmax. activity of pyruvate kinase of sheep adipose tissue increased during tissue culture up to 48 h; the increase was blocked by actinomycin D (an inhibitor of transcription) and was promoted by insulin and antagonized by dexamethasone. 2. In contrast with their effects on pyruvate kinase, insulin and dexamethasone acted synergistically to increase the activity of glucose-6-phosphate dehydrogenase of sheep adipose tissue maintained in culture. 3. Insulin stimulated, whereas dexamethasone inhibited, glucose utilization by sheep adipose tissue maintained in culture; the two agents were mutually antagonistic, and their effects were prevented by actinomycin D. 4. Antimycin A (an inhibitor of the electron-transport chain) stimulated glucose uptake and lactate output by sheep adipose tissue in the presence of dexamethasone and insulin, suggesting that the effects of dexamethasone on glucose utilization by sheep adipose tissue were not due to an inhibition of glucose transport. 5. Comparison of these findings with previous studies on the endocrine control of hepatic pyruvate kinase shows that there are major differences in the control of these Vmax. activities in liver and adipose tissue. 6. Although glucocorticoid hormones inhibit glucose utilization themselves and can antagonize the stimulatory effects of insulin on glucose utilization in adipose tissue from both sheep and rats, there appear to be major differences in the sites of action of these hormones in the two species.

Adipose Tissue↗
PubMed1981

Metabolic consequences of pyruvate kinase inhibition by oxalate in intact rat hepatocytes.

The effects of oxalate on glycolysis and glucose production from trioses were studied in hepatocytes isolated from fed and fasted rats. 1--In cells from fed rats oxalate inhibited glycolysis at the pyruvate kinase step, as shown by an increased phosphoenolpyruvate concentration, a decreased lactate and pyruvate production and a reduction of the glycolytic flux estimated by the rate of detritiation of [6-3H] glucose. The plot of 1/lactate production versus oxalate concentration showed that pyruvate kinase is a limiting step of glycolysis and allowed to determine the apparent inhibition constant for oxalate: about 3035 microM which is near the physiological concentration of blood oxalate. Under conditions where both pyruvate kinase and glycolytic flux are inhibited, oxalate had no effect on the synthesis of [14C] glucose from [14C] triose. 2--In hepatocytes prepared from fasted rats and incubated with lactate and pyruvate, oxalate decreased gluconeogenesis. In cells isolated from fasted rats and incubated with dihydroxyacetone, oxalate decreased lactate and pyruvate production whereas glucose synthesis remained unchanged. It is concluded that the inhibition of pyruvate kinase cannot by itself increase the gluconeogenic flux from triose.

Animals↗
PubMed1989

Erythrocyte enzyme activities in myelodysplastic syndromes: elevated pyruvate kinase activity.

The erythrocyte enzyme activities in twenty-six cases of myelodysplastic syndromes were determined. There were remarkably abnormal levels in seven cases; namely, four cases showed increased hexokinase activity, three cases showed increased pyruvate kinase activity, and two cases showed increased adenosine deaminase activity. Among these, one case with elevated pyruvate kinase activity showed the novel expression of M2-type pyruvate kinase activity, in addition to the R-type pyruvate kinase activity normally found in erythrocytes. Southern blotting of peripheral leucocyte DNA revealed only an amplified PK-LR genome, which derived from the chromosomal abnormality of a 1;7 translocation. The mechanism responsible for switching M2-type to R-type during erythroid maturation was considered to be partially disrupted in this case.

Blotting, Southern↗
PubMed1984

Pyruvate kinase in human amniotic fluid--a new indicator of fetal maturity in late pregnancy.

During the second and third trimesters increasing activities of the glycolytic enzyme pyruvate kinase are to be found in human amniotic fluid. At term (37 to 42 weeks), a very steep rise in amniotic fluid pyruvate kinase can be noticed, which may serve as an additional prenatal indicator of fetal maturity. The presumable main sources of the enzyme in amniotic fluid are the epithelium cells of the skin, the respiratory tract and the amnion. In meconium stained amniotic fluid no pyruvate kinase activity is detectable. Addition of meconium to amniotic fluid samples with previously present pyruvate kinase activity leads to a disappearance of the enzyme activity.

Amniotic Fluid↗