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PubMed1990

The theory of diazymes and functional coupling of pyruvate kinase and creatine kinase.

The physical-chemical principles governing the interactions of enzymes having common metabolic products are presented. Methods for comparing the dissociation rates of the metabolic product and the rates of enzyme-enzyme interaction are given. Using muscle pyruvate kinase (PK) and creatine kinase (CK) as an example, it is shown that the probability of forming an enzyme-product-enzyme complex is much greater than the rate of ATP dissociation from either enzyme. Experimental evidence using 31P-NMR demonstrates that in the presence of both pyruvate kinase and creatine kinase, there is exchange of phosphate between phosphocreatine and phosphoenolpyruvate without a change in the intermediate, ATP. This confirms the formation of a PK.ATP.CK complex in an aqueous solution without enzyme attachment to a substructure. Enzymes capable of forming these mobile clusters are defined as diazymes, and the criteria for their formation are given. The metabolic implications of diazymes are discussed.

Adenosine Diphosphate↗

PubMed2001

Allosteric activation of pyruvate kinase via NAD+ in rat liver cells.

In isolated rat hepatocytes, it has previously been reported that a rise in the ATP content induces a proportional increase in cytosolic NAD+ concentration [Devin, A., Guérin, B. & Rigoulet, M. (1997) FEBS Lett. 410, 329-332]. This occurs under physiological conditions such as various substrates or different energetic states. To investigate the effect of a physiological rise in cytosolic [NAD+] per se on glycolysis and gluconeogenesis, an increase in [NAD+] induced by exogenous nicotinamide addition was obtained without a change in redox potential, ATP/ADP ratio and ATP concentration. Using dihydroxyacetone as substrate, we found that an increase in cytosolic [NAD+] decreases gluconeogenesis and enhances glycolysis without significant alteration of dihydroxyacetone consumption rate. These modifications are the consequence of an allosteric activation of pyruvate kinase via cytosolic NAD+ content. Thus, in addition to the well-known thermodynamic control of glycolysis by pyridine-nucleotide redox status, our study points to a new mechanism of glycolytic flux regulation by NAD+ concentration at the level of pyruvate kinase activity.

Adenosine Diphosphate↗

PubMed1986

Sequences complementary to the brain-specific "identifier" sequences exist in L-type pyruvate kinase mRNA (a liver-specific messenger) and in transcripts especially abundant in muscle.

A sequence complementary to the brain-specific identifier sequence has been found in the 3' untranslated extension of the heavy 3.2-kilobase (kb) long liver L-type pyruvate kinase mRNA while it is absent in the other two 2- and 2.2-kb long pyruvate kinase mRNA species. A 53-base fragment corresponding to this identifier sequence was subcloned in both orientations in the single-stranded bacteriophage M13, both strands being used as probes to detect homologous sequence in different tissues. Both strands are transcribed in various tissues and are detected in heterogeneous high molecular weight RNA species which are especially abundant in the adult muscle. In addition, the probe identical to the identifier sequence recognized a discrete 0.6-kb RNA species in the muscle and the probe complementary to the identifier-sequence recognized the expected two small brain-specific identifier BC-1 and BC-2 RNAs described by Sutcliffe et al. (Sutcliffe, J. G., Milner, R. J., Bloom, F. E., and Lerner, R. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4942-4946; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Lerner, R. A. (1984) Nature 308, 237-241; Milner, R. J., Bloom, F. E., Lai, C., Lerner, R. A., and Sutcliffe, J. G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 713-717; Sutcliffe, J. G., Milner, R. J., Gottesfeld, J. M., and Reynolds, W. (1984) Science 225, 1308-1314).

Animals↗

PubMed2006

Pyruvate kinase activation and lipoperoxidation after selective hepatic ischemia in Wistar rats.

PURPOSE: Hepatic ischemia and reperfusion can cause several problems in hepatic surgery. The aim of this study was to determine pyruvate kinase activation and lipid peroxidation after hepatic ischemia. METHODS: Twenty-four Wistar rats were submitted to 90 minutes of selective liver ischemia and 15 minutes of reperfusion. Twelve animals were submitted to selective liver ischemia and reperfusion (Group A) and the other 12 were submitted to sham operation (Group B). After 15 minutes of reperfusion, the following parameters were measured: mean arterial pressure (MAP), alanine aminotransferase (ALT), glycemia (GLY), hepatic glycogen (GH), pyruvate kinase (PK) activation, hepatic glutathione (GSH) and malondialdehyde (MDA). Analysis of the results were made by the Student t-test and has been considered significant difference for p<0.05. RESULTS: A and B were differents for all parameters analized. CONCLUSION: The animals of group A showed reperfusion syndrome with a fall in MAP, activation of glycid metabolism through the glycolitic pathway and presence of lipid peroxidation compared to group B.

Animals↗

PubMed1993

Expression of glucose transporter isoforms (GLUT1, GLUT2) and activities of hexokinase, pyruvate kinase, and malic enzyme in preneoplastic and neoplastic rat renal basophilic cell lesions.

Sequential changes in the expression of two glucose transporter isoforms (GLUT1, GLUT2), and in the activities of hexokinase, pyruvate kinase and malic enzyme during the development of rat renal basophilic cell tumors were studied using histochemical techniques. Early basophilic cell tubules are similar to proximal convoluted tubules (PCT) in their overall histochemical pattern, particularly in the expression of glucose transporters, suggesting that basophilic cell tubules and tumors derived from them arise from PCT. In comparison with PCT, basophilic cell tubules show slightly increased activities of all the enzymes studied. In basophilic cell tumors, markedly elevated hexokinase and pyruvate kinase activities are accompanied by a considerable reduction in the expression of GLUT2. GLUT1 expression is not found in basophilic cell tubules or PCT. Small basophilic cell tumors also do not express GLUT1, but GLUT1 is regularly expressed in several cell layers surrounding necrotic areas within large basophilic cell tumors. Our results indicate that increased glycolytic activity and reduced GLUT2 expression take place during the development of renal basophilic cell tumors.

Animals↗

PubMed1984

Thermodynamic linkages in rabbit muscle pyruvate kinase: analysis of experimental data by a two-state model.

A concerted, allosteric model is developed, and equations are derived for quantitative interpretation of the kinetic and equilibrium binding data of rabbit muscle pyruvate kinase at pH 7.5 and 23 degrees C. The simplest model which seems likely to rationalize the experimental data involves two conformational states. In this model, two simplifying assumptions are made. First, the affinities of pyruvate kinase for both substrate and inhibitor are assumed to depend only upon the conformational state of the tetrameric enzyme. Second, the rate of product formation is also assumed to depend only upon the enzyme conformation. All types of experimental data are analyzed simultaneously to estimate the parameters which best predict the total body of experimental results. The fitted parameters indicate that the intrinsic allosteric equilibrium favors the active (R) state by 11 to 1. The substrate phosphoenolpyruvate binds preferentially, by a factor of 10, to the R state, whereas the inhibitor Phe has about 23 times higher affinity for the inactive (T) state. In all cases tested, the calculated values are in good agreement with the experimental data.

Allosteric Site↗

PubMed1985

Fluorine NMR studies on stereochemical aspects of reactions catalyzed by transcarboxylase, pyruvate kinase, and enzyme I.

The stereochemistry of the transcarboxylase-catalyzed carboxylation of 3-fluoropyruvate has been studied by using fluorine NMR of unpurified reaction mixtures. When the product 3-fluorooxaloacetate was trapped by using malate dehydrogenase, only the 2R,3R diastereomer of 3-fluoromalate was formed. The fluoromethyl group of fluoropyruvate does not take up deuterium label from the solvent during the reaction. These results confirm and extend those obtained previously by Walsh and co-workers [Goldstein, J. A., Cheung, Y. F., Marletta, M. A., & Walsh, C. (1978) Biochemistry 17, 5567-5575] showing that transcarboxylase is specific for one of the two prochiral hydrogens in fluoropyruvate. Transcarboxylase, coupled to malate dehydrogenase, has been used to analyze samples of chiral fluoropyruvate obtained by dephosphorylation of (Z)-fluorophosphoenolpyruvate in D2O in the presence of either pyruvate kinase or enzyme I from the Escherichia coli sugar transport systems. Analysis of the fluoromalate produced showed that fluoroenolpyruvate is deuterated from opposite faces by these two enzymes: enzyme I protonates (deuterates) fluoroenolpyruvate exclusively from the 2-re face and pyruvate kinase does so mainly from the 2-si face. Fluoropyruvate is carboxylated by transcarboxylase with absolute retention of configuration.

Animals↗

PubMed2002

Concomitant laparoscopic splenectomy and cholecystectomy as an effective and minimally invasive treatment of pyruvate kinase deficiency with gallstones.

A 4-year-old girl with pyruvate kinase deficiency underwent partial splenic embolization initially. However, even after this procedure, she still had to be transfused every 2 months and then every month. At 5 years of age, she was admitted to our hospital to undergo splenectomy. She underwent laparoscopic splenectomy and concomitant cholecystectomy for gallstones. The hemogram recovered to the normal range after surgery, and her postoperative course was uneventful. Considering the absence of morbidity, the short hospitalization, the quick return to normal activity, the good cosmetic result, and the improved clinical and hematologic results, we consider that simultaneous laparoscopic splenectomy and cholecystectomy is safe and effective for the management of hemolytic anemia resulting from pyruvatre kinase deficiency and associated with cholelithiasis.

Child, Preschool↗

PubMed1976

The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate.

The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium. The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme. There is one site for each of these ligands per monomer. In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, i.e., all homotropic interactions of the substrate are lost. In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator. The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator. The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer. In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer. Magnesium ions inhibit binding of GDP and GTP. KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc., enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate. The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other.

Allosteric Regulation↗

PubMed1990

Chromosomal localization of the gene for a human cytosolic thyroid hormone binding protein homologous to the subunit of pyruvate kinase, subtype M2.

A cDNA for the gene that encodes a human cytosolic thyroid hormone binding protein (p58) recently has been isolated and sequenced. Analysis of the p58 sequence indicates that it is identical to the subunit of pyruvate kinase, subtype M2. By in situ hybridization, the gene for p58 was mapped to 15q24-25. This localization shows that the p58 gene is not linked to the L-type of pyruvate kinase, which is located on chromosome 1. The p58 gene was found to be activated in several forms of cancer. Current localization will permit us to assess the effect of alterations involving chromosome 15 on the structure and activity of the p58 gene in neoplasms or chromosome syndromes.

Autoradiography↗

PubMed1979

Acute regulation of pyruvate kinase activity in rat epididymal adipose tissue by insulin.

1. Evidence is presented that exposure of epididymal fat-pads from fed rats to insulin leads to a marked diminution in the Km for phosphoenolpyruvate of pyruvate kinase. Effects of insulin may be readily demonstrated in experiments both in vivo and in vitro and are not secondary to the activation by the hormone of glucose transport. No effect of insulin is apparent in tissues from 48 h-starved animals. 2. The mechanism of the effect of insulin on pyruvate kinase was not established. The observed changes in Km do not appear to be the result of alterations in the amounts of bound effectors such as fructose 1,6-bisphosphate and alanine. Rather, as the effect persists in incubated extracts, it appears that a change in the degree of phosphorylation or some other covalent modification of the enzyme may be involved.

Adipose Tissue↗

PubMed1988

The effect of fructose 1,6 diphosphate on pyruvate kinase from the liver of the flounder (Platichthys flesus L.).

1. Pyruvate kinase purified from flounder liver in two forms, i.e. PKI and PKII, is activated by fructose 1,6 diphosphate. 2. Two or more binding sites for FDP are demonstrated for PKII, the binding to which is influenced by the levels of substrates. 3. FDP reduces or abolishes the cooperative effect of PEP. 4. FDP increases the maximal activity. 5. The inhibition observed at higher levels of ADP is not abolished by FDP.

Adenosine Diphosphate↗

PubMed1985

Starvation and feeding with a high-carbohydrate diet induce changes in the specific activity of rat hepatic pyruvate kinase.

A highly specific radiosandwich assay for hepatic type-L pyruvate kinase (PK) determinants was developed. This assay was used to evaluate changes in PK specific activity which occur on starvation or feeding with a high-carbohydrate diet. Despite a large increase in both catalytic activity and immunoreactive protein, the calculated specific activity falls on feeding, and the opposite effect occurs on starvation. These change can be attributed to disproportionate changes in immunoreactive protein compared with changes in catalytic activity.

Animals↗

PubMedDate not supplied

Erythrocyte pyruvate kinase activity in term and preterm infants.

The activity of erythrocyte pyruvate kinase (PK), one of the most important enzymes in the anaerobic glycolytic pathway, was measured in a total of 57 healthy (22 term and 35 preterm) infants. The mean PK activity was 8.98 +/- 3.43 IU/gHb in term and 16.56 +/- 7.26 IU/gHb in preterm infants. The mean PK activity was significantly higher in preterm babies than term infants (16.56 +/- 7.26 IU/gHb and 8.98 +/- 3.43 IU/gHb, respectively) (P < .001). A significant negative correlation was found between gestational age, birth weight, and PK activity (r = 0.40, P < .05; r = -0.37, P < .05). No correlation was found between postnatal age and PK activity in both preterm and term infants. The increased PK activity in preterm babies was thought to be due to increased glycolytic activity and energy production in these infants.

Birth Weight↗

PubMed1983

The regulatory properties of rabbit muscle pyruvate kinase. The influence of substrate concentrations.

The kinetics of rabbit muscle pyruvate kinase were studied in assays at pH 7.4, where the relationships between the initial velocities of the catalysed reaction and the concentrations of substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The data were used to test the applicability of the exponential model for a regulatory enzyme, which has been here extended to describe the behaviour of a three-substrate enzyme. It appears that the data can be represented by the model and as a result permit the conclusion that the substrates influence one another's binding by the same type of charge interactions that are evident in the Michaelis-Menten kinetics of the enzyme observed at pH 6.2. Evidence is also presented indicating that MgADP acts as a dead-end inhibitor of the enzyme at pH 7.4.

Adenosine Diphosphate↗

PubMed1988

Erythrocyte pyruvate kinase deficiency: 11 new cases.

Eleven new cases of red cell pyruvate kinase (PK) deficiency with congenital haemolytic disease from 10 unrelated Italian families were characterized using the methods recommended by the International Committee for Standardization in Haematology (ICSH). All patients were double heterozygotes for the PK gene. The 10 variants were designated PK 'Lecce,' 'Parma,' 'Verona,' 'Milano,' 'Soresina,' 'Macerata,' 'Sassari,' 'Genova,' 'Mantova' and 'Brescia.' PK 'Sassari' was associated with glucose-6-phosphate dehydrogenase deficiency in two siblings. All mutants displayed multiple biochemical abnormalities except for PK 'Lecce' that only showed decreased red cell PK activity. No relation was found between the severity of anaemia and either the residual PK activity or specific biochemical enzyme abnormalities. Increased serum ferritin levels were detected in most of the patients, suggesting the need for systematically monitoring iron status in this disease.

Adolescent↗

PubMedDate not supplied

Monovalent cations requirement of the fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli.

The fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli has been purified by a simplified procedure, which gives a homogeneous enzyme in approximately half the working time required by other methods and is suitable for large scale preparations. The activity of the enzyme is strictly dependent on the presence of monovalent cations. Enzyme activity is elicited by K+ and NH4+, but not by Na+. Homotropic cooperativity is displayed in the activation by K+ and NH4+ and heterotropic effects are reciprocally exerted by monovalent cations and other ligands, such as phosphoenolpyruvate and fructose 1,6-bisphosphate. The allosteric nature of such interactions is suggested by changes in heat stability of the enzyme induced by K+ and fructose 1,6-bisphosphate. NH4+, but not K+, at high concentrations, cause an inhibition of enzyme activity.

Cations, Monovalent↗

PubMed1984

A new pyruvate kinase mutation with hyperactivity in the mouse.

A mouse mutant with pyruvate kinase (PK) hyperactivity has been found in offspring of 1-ethyl-1-nitrosourea (ENU)-treated male mice. The activity alteration was detected in the blood and could also be found in the liver but not in the muscle, kidney, heart, spleen, lung, or brain. Heterozygous mice have erythrocyte PK activity enhanced up to about 160% and homozygotes up to about 240%, compared to homozygous wild types. The mutation is codominantly expressed. The heterozygous and homozygous mutants are viable and fully fertile and do not show symptoms of erythrocytosis. The mutation does not affect the heat stability, the electrophoretic mobility, or the Km (for phosphoenolpyruvate) of the PK molecule. It is suggested that the regulatory locus of PK-1 is affected by this mutation. The observations support also the theory of one structural locus for the erythrocyte and liver isozymes.

Animals↗