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PubMed1983

Modulation of pyruvate kinase phosphatase activity in hepatocyte extracts by pyruvate kinase-L ligands.

Hepatic pyruvate kinase phosphatase activity has been assayed in native conditions, in Sephadex G-25 filtered extracts of rat hepatocytes, by measuring the reactivation rate of glucagon-inactivated pyruvate kinase-L. The ionic requirements for this reaction, as well as the possible regulatory role of some pyruvate kinase ligands, have been investigated. Pyruvate kinase phosphatase activity was dependent on divalent cations (Mg2+, Mn2+ or Co2+). Mg2+ ions highly enhanced the reactivation rate of pyruvate kinase, while the presence of 100 mM KF inhibited this process. Physiological concentrations of phosphoenolpyruvate or fructose 1,6-bisphosphate inhibited pyruvate kinase phosphatase activity. These inhibitory effects were partially antagonized by the presence of L-alanine. Our results suggest that ligands of pyruvate kinase could play a role in the control of pyruvate kinase phosphatase activity(ies), possibly by modifying the conformational state of the substrate protein.

Animals↗
PubMed1983

A direct radioassay for pyruvate kinase activity.

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.

Animals↗
PubMed1976

Pyruvate kinase isozymes in various tissues of rat, and increase of spleen-type pyruvate kinase in liver by injecting chromatins from spleen and tumor.

Pyruvate kinase [EC 2.7.1.40] in various tissues of rats was separable into seven kinds of pI-isozymes by isoelectric separation with Ampholine carrier ampholytes; pI 5.4-isozyme, pI 5.6-isozyme, pI 6.2-isozyme (2 kinds), pI 6.6-isozyme, pI 7.4-isozyme, and pI 7.8-isozyme. Some of these pI-isozymes contained bound fructose 1,6-diphosphate (FDP). The bound FDP was completely dissociated when the pI-isozymes were salted out with ammonium sulfate. In the FDP-free form, pyruvate kinase was classified into three types, liver-type (type L) of pI 6.2, muscle-type (type M) of pI 7.4, and spleen-type (type M2) of pI 7.8. The liver-type isoenzyme had two kinds of FDP-binding sites; the pI 5.6-isozyme and pI 5.4-isozyme were obtained when one and two kinds of sites were bound with FDP, respectively. The association and dissociation of FDP at both sites were reversible in the presence and absence of 0.15 M KC1 (high ionic strength). The muscle-type isoenzyme had no FDP-binding site. The spleen-type isoenzyme had two kinds of FDP-binding sites, like the liver-type isoenzyme. When the ionic strength of solutions containing the enzyme and FDP was sufficiently low, one and two kinds of the sites could bind with FDP, converting the enzyme into pI 6.6-isozyme and pI 6.2-isozyme, respectively. FDP bound with one kind of site (the 2nd site) was easily dissociable, but FDP bound with the other kind of site (the 1st site) was not. Provided that the 1st site carried bound FDP, the 2nd site was associable at high ionic strength. The liver-type isoenzyme free of FDP and the spleen-type isoenzyme bound with FDP at both sites had similar pI values of 6.2 and were not separable by isoelectric separation. Some properties of these pI-isozymes were compared. When Rhodamine sarcoma was transplanted in rats, the content of spleen-type isoenzyme in the livers increased. When rats were injected with chromatin prepared from either Rhodamine sarcoma or spleen, the content of spleen-type isoenzyme in the livers again increased. This was not observed on the injection of chromatin prepared from liver, indicating that the factor capable of controlling the gene expression was present in chromatins of sarcoma and spleen but barely or not at all in chromatin of liver.

Animals↗
PubMed1991

Point mutations in the L-type pyruvate kinase gene of two children with hemolytic anemia caused by pyruvate kinase deficiency.

The molecular alterations responsible for the characteristic enzyme abnormalities in pyruvate kinase (PK) deficiency were investigated in two unrelated children homozygous for PK deficiency. Both variant enzymes were characterized according to the recommendations of the International Committee for Standardization in Haematology. Genomic DNA was specifically amplified by the polymerase chain reaction. Normal and mutant alleles of the L-type PK gene were analyzed by nucleotide sequencing. Heterozygosity of the parents was confirmed by allele-specific oligonucleotide hybridization. In PK Linz a C to T base exchange at position 394 of the L-type PK gene was found. As a result, the 132nd amino acid of the mutant enzyme, arginine (CGC), is replaced by cysteine (TGC). The affected amino acid residue is located within the deduced active site of the protein and the enzyme variant shows strongly altered allosteric properties. PK Beirut shows a C for T substitution at position 1058, changing the 353 amino acid from threonine (ACG) to methionine (ATG). In contrast to PK Linz, this amino acid lies outside the deduced substrate binding site and kinetic parameters of PK Beirut are close to normal. Both enzyme variants show a markedly reduced specific activity and thermolability.

Anemia, Hemolytic↗
PubMed1976

Chronic haemolytic anaemia in two patients heterozygous for erythrocyte pyruvate kinase deficiency. Electrofocusing and immunological studies of erythrocyte and liver pyruvate kinase.

Two patients with mild chronic haemolytic anaemia, a mother and her son, were found to be heterozygous for erythrocyte pyruvate kinase deficiency. In the red blood cells the enzymatic activity was reduced by about 50% and the residual PK had normal kinetic properties, stability and electrofocusing pattern. The PK antigen concentration was also decreased by half, so that the ratio of the enzymatic activity to the immunological reactivity (i.e. the molecular specific activity) was normal. In the son's liver PK enzymatic activity was slightly reduced and, above all, an abnormal active form, more anodic than normal PK, was detected by electrofocusing. The propositus's liver PK was also slightly thermo-unstable. It is suggested that the patients were heterozygous for an unstable PK variant which is found in liver, nucleated tissue actively synthesizing proteins, but which disappeared from the erythrocytes because of its unstability.

Adult↗
PubMed1989

Comparative study of human M2-type pyruvate kinases isolated from human leukocytes and erythrocytes of a patient with red cell pyruvate kinase hyperactivity.

M2-type pyruvate kinases (M2-PK) have been isolated from human leukocytes and from the erythrocytes of a patient with erythrocyte PK hyperactivity. The kinetic characteristics of the patient erythrocyte M2-PK were similar to those of leukocyte M2-PK except for the Hill coefficient of phosphoenol pyruvate kinetics that showed little difference in the values. The patient erythrocyte M2-PK displayed complete immunological identity with leukocyte M2-PK in immunodiffusion, immunoblotting and immunoneutralization. The sensitivity to proteolysis by trypsin and the electrophoretic migration in different conditions were similar for the M2-PK of both origins. These results suggest an identity between this M2-PK abnormally present in erythrocytes and the M2-PK from leukocytes.

Chromatography, Ion Exchange↗
PubMed1995

Case report: pyruvate kinase deficiency.

Pyruvate kinase deficiency is a rare cause of congenital hemolytic anemia. Despite a paucity of reports, splenectomy resulted in successful outcomes for two siblings with this disorder. The sisters were diagnosed at birth with profound jaundice and congenital nonspherocytic hemolytic anemia.

Adolescent↗
PubMed1986

Determination of the mean cell age of erythrocytes from diabetic subjects with pyruvate kinase.

The pyruvate kinase activity of erythrocytes from normal and diabetic subjects was examined in order to establish this enzyme as a valid indicator of mean cell age in the studies of age-dependent erythrocyte functions. This study reveals that the enzyme activity in the erythrocytes was not affected by the condition of diabetes and suggests that it may provide a simple means for the determination of cell age in erythrocyte insulin binding studies. Present data further indicate that the mean cell age of the erythrocytes from diabetic patients was not significantly different from normal although insulin binding to erythrocytes was markedly reduced when compared with that in the normal subjects.

Diabetes Mellitus↗
PubMed1990

Pyruvate kinase deficiency.

Pyruvate kinase (PK) deficiency was initially described by Valentine et al. in 1961. Since then, more than 300 cases have been described, including 65 in Japan. PK deficiency is the most common hereditary nonspherocytic hemolytic anemia among several red cell enzyme defects of the Embden-Meyerhof glycolytic pathway. The clinical manifestations are highly variable. Splenectomy usually increases the hemoglobin level by about 2 g/100 mL. Standardization of methods for characterization of PK variants was achieved in 1979. There are four PK isozymes, M1, M2, L and R, in mammalian tissues. We have clarified the switch from M2-type to L-type PK during maturation of erythroid precursor cells. Recently we cloned and sequenced a full length human L-type PK cDNA. It will be useful to clarify the molecular basis of PK deficiency.

Anemia, Hemolytic, Congenital↗
PubMed1994

Biochemical characterization of four new erythrocyte pyruvate kinase variants.

Pyruvate kinase (PK) from four patients with moderate to severe congenital non-spherocytic haemolytic anaemia was characterized by methods recommended by the ICSH. The possibility that two of the patients are true homozygotes cannot be ruled out, while the other two apparently represent double heterozygotes. All but one had levels of PK activity between 44 and 65% of normal. The variant enzymes were designed 'PK Pontos', 'PK Macedonia', 'PK Athens' and 'PK Larisa'. Multiple physicochemical as well as kinetic aberrations were detected in the above variants. Their altered kinetic behaviour is discussed in terms of the concerted transition model for allosteric enzymes and their abnormal properties are compared with other known variants, while it is also attempted to correlate them with possible mechanisms resulting in chronic haemolytic anaemia.

Adolescent↗
PubMed1981

Molecular lesion affecting the ADP-combining site in a mutant isozyme of erythrocyte pyruvate kinase.

Erythrocyte pyruvate kinase (PK) from a patient with PK deficiency was characterized according to internationally standardized procedures. In addition to low activity, the mutant isozyme displayed impaired kinetics specifically affecting the ADP-combining site:Km (ADP) was 3-5 times greater than normal when determined at three different concentrations of phosphoenolpyruvate (P-enolpyruvate). Maximum reaction velocities were not achieved until ADP was 10 times the concentration normally required for control PK. Substrate inhibition by high concentrations of ADP and competitive inhibition by ATP were markedly diminished. Other nucleoside diphosphates normally capable of replacing ADP in the PK reaction were less effective with the mutant isozyme than with PK from controls or from subjects with other forms of PK deficiency, and Michaelis--Menten constants for several of these (UDP, GDP, CDP) were significantly elevated. Whereas all previously known PK kinetic defects have involved the substrate P-enolpyruvate, the half-saturation constant K0.5s (P-enolpyruvate) for this mutant isozyme was normal, as was its response to fructose-1,6-bisphosphate activation.

Adenosine Diphosphate↗
PubMedDate not supplied

Acquired pyruvate kinase deficiency.

Pyruvate kinase (PK) is an enzyme of critical importance in the glycolytic pathway of the red cells, deficiency of which, whether congenital or acquired, results in a hemolytic anemia. Measurement of the enzyme is now rapid and simple utilizing preprepared substrate and reagents. Some caution must be taken, however, in both measurement and interpretation. Red cells must be separated from other blood cells, particularly granulocytes, since the PK of white cells is different from that of red cells and is present in much higher activity per cell. Sometimes the use of special techniques of measurement [low substrate concentration (phospho-enolpyruvate, known as PEP, 0.4 mM in place of 2 mM) or heating at 53 degrees C for 60 minutes, (to test thermostability)] are necessary to detect abnormal molecular characteristics of PK, particularly in the acquired form of deficiency. Evidence recently presented suggests that the acquired form of PK deficiency may sometimes be due to inhibitors which oxidize sulfhydril bonds in the enzyme or alter its substrate binding properties or other molecular characteristics.

Adenosine Triphosphate↗
PubMed1989

Effects of primary sequence differences on the global structure and function of an enzyme: a study of pyruvate kinase isozymes.

Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Secondary kinase reactions catalyzed by yeast pyruvate kinase.

1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.

Adenosine Triphosphate↗
PubMed2001

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

Adenosine Triphosphate↗
PubMed2001

Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase.

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Allosteric Regulation↗