Erythrocyte Pyruvate Kinase Research Articles

Proteolytic processing of human erythrocyte pyruvate kinase: Study of normal and deficient enzymes

The L' subunits, phosphorylatable precursors of L-type pyruvate kinase, can be proteolyzed in vivo and in vitro, and transformed into several molecular species. Trypsin induces the appearance of a phosphorylatable form similar to liver L-type enzyme. Subtilisin splits the phosphorylatable site and induces the appearance of a form similar to the minor form occuring with red cell aging. Sensitivity to proteolysis of some pyruvate kinase variants is modified.

Phosphorylation and inhibition of human erythrocyte pyruvate kinase by erythrocyte membranes

The enzymatic activity of human erythrocyte pyruvate kinase was found to decrease on incubation of the purified enzyme with red blood cell ghosts, ATP and cAMP. If [ 32P]γATP was used radioactivity was found associated with the protein after gel electrophoresis. Various effectors protected the enzyme against phosphorylation. Treatment of the modified enzyme with a protein phosphatase restored enzymatic activity and also caused the loss of the radioactive label. Modification of the pyruvate kinase in this way altered the affinity of the enzyme for one of its substrates (phosphoenolpyruvate), but the binding of the other substrate (ADP) was unaffected.

Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase

1. 1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Trismaleate shows a loss of positive homotropic interactions, as compared to the same preparation in Tris-HCl. Hill coefficients ( n) of n = 1.0−1.2 and n = 1,6−1.8 are obtained in Tris-maleate and Tris-HCl respectively. Half saturation [ S] 0.5 and V max remain unchanged. Pyruvate kinase in Tris-maleate is slightly more stable to heating at 60°C than in Tris-HCl. Incubation of the enzyme in Tris-maleate for one h with high concentrations of dithiotreitol restores the positive homotropic interactions. 3. 3. It is proposed, that the abnormalities of the pyruvate kinase of some patients with acquired pyruvate kinase deficiency, obtained from a study in Tris-maleate, may partly be induced by the buffer itself.

Significance of the electrophoretic modifications of defective pyruvate kinase variants. Study of six new observations

Six new defective pyruvate kinase variants have been characterized in patients suffering from chronic hemolysis. Partially purified enzyme variants exhibited various anomalies from immunological, kinetic, stability and electrophoretic points of view. The significance of the electrophoretic anomalies has been interpreted in view of the normal post-synthetic maturation of the precursor enzyme L' 4 into L 2L' 2 and L 4, and the ability of trypsin to induce in vitro the transition L' 4 → L 4 has been tested. One defective enzyme existed in a single L' 4 form and could not be transformed by trypsin into L 4. In three cases slow-moving L' 4 and L 2L' 2 forms were transformed by trypsin into an abnormal slow-moving L 4 form. In the last two observations the L 4' and L 2L' 2 forms exhibited normal mobility and were normally transformed by trypsin into L 4. The relevance of these data to the functional anomalies of the defective variants and to the nature of the primary genetic anomaly giving rise to the congenital defects in erythrocyte pyruvate kinase is discussed.

A Case of Acute Granulocytic Leukemia with Erythrocyte Pyruvate Kinase Deficiency and Bone Marrow Chromosome Aberration

A Case of Acute Granulocytic Leukemia with Erythrocyte Pyruvate Kinase Deficiency and Bone Marrow Chromosome Aberration

Identification of an Erythrocyte Pyruvate Kinase Variant in a Family from Latium with Non-Spherocytic Congenital Haemolytic Anaemia

Erythrocyte PK deficiency was detected in a family from Latium in Italy. This PK variant is characterized by normal or increased activity immediately after blood collection, instability to storage, to heat and to urea. Only in the propositus the mutant enzyme exhibited an increased Michaelis constant for PEP, slightly increased inhibition by ATP and an altered optimum pH value. The kinetic anomaly was only partially corrected by activation with F-1, 6-DP and by addition of 2-ME. From these results it can be concluded that in the family observed two distinct erythrocyte PK alterations were demonstrable: instability in the propositus and his father; low affinity for PEP and altered optimum pH value only in the propositus.

Congenital Non‐spherocytic Haemolytic Anaemia Variants with Primary and Secondary Pyruvate Kinase Deficiency

The patterns of survival of isotope-labelled erythrocytes were examined in patients suffering from two variants of congenital non-spherocytic haemolytic anaemia with decreased erythrocyte pyruvate kinase (PK) activity. In one variant, with primary PK defect (PPKD) random destruction of erythrocytes was predominant in the process of haemolysis. In the second variant, with primary magnesium activated adenosine triphosphatase (ATP-ase) (Mg++) deficiency and a secondary decrease in PK activity, erythrocytes were destroyed by senescence. Two subpopulations of labelled erythrocytes with different destruction rates were observed in all patients examined, except one with the second variant, with very mild haemolysis. Splenectomy, performed on two patient, was successful only in the variant with PPKD.

Laboratory studies of erythrocytic pyruvate kinase deficiency. Pathogenesis of the hemolysis.

Genetic polymorphism exists in erythrocytic pyruvate kinase (PK)-deficient hemolytic anemia, as briefly described. Destruction of erythrocytes varied in extent, but its mechanisms in different PK-deficient polymorphic persons investigated were similar. A paradoxical post-splenectomy reticulocytosis regularly occurred. Qualitative enzymatic differences, the biochemistry, and measurements of erythrocytic destruction were made in several PK variants. 51Cr autologous and cross-transfusions of PK-deficient erythrocytes into volunteers showed multimodel regression lines of several erythrocytic cohorts. 59Felabeled PK-deficient reticulocytes donated by PK-deficient splenectomized subjects were transfused 20 hours before splenectomy into two PK-deficient infants with hemolysis, and into two adult volunteers with autoimmune thrombocytopenia. The highest reticulocyte concentration in any organ initially was within the spleen. Radioiron-labeled erythrocytes and cytologic data showed large splenic reticulocyte pools. Splenic macrophages ingesting reticulocytes and erythrocytes were seen by both light and electron microscopy of the splenic pulp. After cyanide additives inhibiting reticulocyte oxidative phosphorylation, a bizarre erythrocytic cytologic configuration was found by scanning electron microscopy. These studies of PK-mutant subjects with PK-deficient erythrocytic hemolysis showed age dependent destruction of erythrocytes. Bimodal Cr survival data suggested reticuloendothelial removal of short-lived erythrocytes and reticulocytes. The transfusions of radioironlabeled PK reticulocytes and the data obtained by scanning electron microscopy suggested that the spleen was the initial hostile organ destroying a cohort of susceptible erythrocytes, prinicpally reticulocytes.

Lack of phosphorylatable site and some kinetic properties of erythrocyte pyruvate kinase from the rat A modified L-type isozyme?

Lack of phosphorylatable site and some kinetic properties of erythrocyte pyruvate kinase from the rat A modified L-type isozyme?

Hereditary erythrocyte pyruvate kinase deficiency: molecular and functional studies of four mutant PK variants detected in Spain

The mechanisms of the defect in enzyme activity and the functional anomalies have been studied for four different mutant erythrocyte pyruvate kinase (PK) variants found in Spain. In two cases the PK variants were heat stable, and their antigenic concentration was nearly normal in the patients' red cells: the defect in activity was due to decreased catalytic activity of the mutant molecules. In the two other observations the mutant PK variants were heat-labile, and their antigenic concentration was reduced in the red cells: the defect in activity was entirely due to this decrease of the enzyme molecule concentration in one case, and to decrease of the enzyme molecule concentration plus decrease of catalytic activity of these mutant molecules in the other case. Kinetic properties were normal for one variant and drastically modified in the three other variants. These latter variants showed decreased apparent affinity for PEP, which cannot be simply explained by a shift of the R α T allosteric equilibrium towards the T form: the results of the study of ATP inhibition and FDP activation, indeed, did not correspond to the expected results for a PK enzyme present in a predominant T form. We point out the heterogeneity and the complexity of the anomalies of the mutant PK variant, most likely reflecting the diversity of the possible punctual mutations of the PK structural gene.

Erythrocyte pyruvate kinase deficiency in a beagle dog.

A macrocytic hypochromic anemia with marked reticulocytosis was observed at each of 10 examinations during a 4 month period of a male Beagle dog that was 11 months old when first examined. The owner had noticed pale mucous membranes from the time the dog was purchased at 7 weeks of age. Based on the clinical history and negative LE, ANA and Coombs tests, an intrinsic erythrocyte defect was suspected. Erythrocyte osmotic fragility and hemoglobin electrophoresis were normal. Autohemolysis of incubated erythrocytes was marked. Hemolysis was uncorrected by glucose but partially corrected by ATP. With the exception of pyruvate kinase (PK), all of the erythrocyte enzymes measured had greater activities than those of control Beagle erythrocytes. In comparison to moderately reticulocyte-rich controls. PK activity was low in the affected Beagle. Erythrocyte phosphoenolpyruvate content was increased significantly as expected in PK deficiency. The kinetic constant (Km) for phosphoenolpyruvate was lower than that of normal control dogs, a finding in agreement with studies of PK from deficient Basenjis. On these bases a diagnosis of PK deficiency was made.

Erythrocyte pyruvate kinase deficiency in basenji dogs

Erythrocyte pyruvate kinase deficiency in basenji dogs

Heterogeneity of erythrocyte pyruvate kinase deficiency and related metabolic disorders in patients with hematological diseases

In several patients suffering from congenital non-spherocytic hemolytic anemia or from malignant hematological disorder associated with erythrocyte pyruvate kinase (PK) deficiency, a metabolic study has been carried out involving the following biochemical determinations: assay of red cell enzyme activities; estimation of glucose consumption; measurement of the rate of glycolytic intermediates; and, in some cases, enzyme purification and characterization of the PK variant. Metabolic equilibrium most probably does not depend on kinetic characteristics of PK molecules. Furthermore, the data obtained allow separation of cases with congenital non-spherocytic hemolytic anemia (hereditary defect) and acquired PK deficiencies.

A deficient pyruvate kinase with an electrophoretically slow-moving component.

A deficient erythrocyte pyruvate kinase observed in a patient with congenital non-spherocytic anaemia was characterized by the following properties: very low activity in haemolysates, decreased thermal stability, slightly increased urea denaturation, high affinity for PEP, poor FDP activation, normal ATP inhibition, decreased affinity for ADP, normal pH of optimal activity, and presence of an abnormal slow-moving component in this layer polyacrylamide gel electrophoresis. The patient was probably double heterozygous for two different deficient mutants of erythrocyte pyruvate kinase.

Activation of human erythrocyte pyruvate kinase by hexose-bisphosphates

Summary Human erythrocyte pyruvate kinase (E.C. 2.7.1.40, ATP-pyruvate phosphotransferase) is activated by hexose-1,6-bisphosphates in the order: fructose- > mannose- > glucose-bisphosphate. These activators promote substantial reductions (7 to 10 fold) in the K M for the substrate phosphoenolpyruvate but do not effect the maximum velocity. The ability of mannose- and glucose-1,6-bisphosphate to function as activators of both human erythrocyte phosphofructokinase (Rose, I.A. and Warms, J.V.B. (1974), Biochem. Biophys. Acta 156, 231–239) and pyruvate kinase may account for the presence of these metabolites in red cells at relatively high levels. Fructose-1,6-bisphosphate also induces slight increases in the K M values for the active substrate, Mg·ADP, at sub-saturating levels of phosphoenolpyruvate but is without effect at saturating levels of phosphoenolpyruvate. A possible role for these activators in maintaining a balance in the flux through the pyruvate kinase reaction is proposed.

Genetic distance between the Australian Merino and the Poll Dorset Sheep

SUMMARYMerinos (189) and Poll Dorsets (106) were compared for genetic variation at 30 loci for blood proteins. ‘Malic enzyme’ and NADH diaphorase I polymorphism occur in Merinos but not in Poll Dorsets, whereas both breeds are polymorphic for haemoglobin, erythrocyte X-protein, serum esterase, catalase and transferrin, although the breeds differ in the presence or absence of certain rare transferrin variants. Poll Dorsets but not Merinos have genetic variation of erythrocyte pyruvate kinase at low gene frequencies; Merinos but not Poll Dorsets have genetic variation of two erythrocyte Gly-Leu peptidases, glucosephosphate isomerase, superoxide dismutase, and NADH diaphorase II, all at low gene frequencies (P< 0·05).Using Masatoshi Nei's (1972, 1976) standard genetic distance, over all 30 loci, to calculate the time of divergence of the two breeds, we obtainedt= 69700 years – whereas sheep are believed to have been domesticated by man for not more than 11000 years, and ancestors of the British breeds and of the Merino have probably not been separated for more than 2000 years. Several possible explanations for this discrepancy are discussed, including the accuracy of the coefficient of codon change (α), changes in population size and structure, selection, and additional hybridization in the ancestry of the Merino.

Evidence for a postsynthetic proteolytic transformation of human erythrocyte pyruvate kinase into L-type enzyme.

Evidence for a postsynthetic proteolytic transformation of human erythrocyte pyruvate kinase into L-type enzyme.

Purification and characterization of human muscle pyruvate kinase

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.

Human erythrocyte pyruvate kinase Total purification and evidence for its antigenic identity with L-type enzyme

Erythrocyte pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase, EC 2.7.1.40) has been purified 40 000 times from human erythrocytes, according to an original method. The whole purification procedure included toluene extraction, ammonium sulphate fractionation, DEAE-Sephadex batchwise chromatography and affinity chromatography on a Dextran Blue-Sepharose column with specific elution by fructose 1,6-diphosphate. The final preparation had specific activity of 290 I.U./mg of proteins and the overall yield was about 30%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. Pure enzyme was injected into rabbits and monospecific antiserum was obtained able to neutralize, per ml, 150 I.U. of erythocyte-type pyruvate kinase as well as of l-type enzyme. l-type and erythrocyte-type pyruvate kinases showed reactions of complete identity when tested in immunodiffusion against anti-erythrocyte type pyruvate kinase sera; in all cases a single precipitation line could be detected. l-type pyruvate kinase when mixed with anti-erythocyte pyruvate kinase serum suppressed all ability of that antiserum to react immunological with erythocyte enzyme. Finally the microcomplement fixation curves using anti-erythrocyte pyrurate kinase serum were identical for erythrocyte and l-type enzymes. From these results it appeared that no antigenic difference between L-type and erythocyte enzyme could be detected. Consequently the most likely hypothesis is that both these enzymes are coded by the same single gene, the slight electrophoretic differences between them being due to post-synthetic tissuespecific changes.

A spanish family with erythrocyte pyruvate kinase deficiency: Contribution of various immunologic methods in the study of the mutant enzyme

Erythrocyte PK deficiency was detected in a 38-year-old man from Catalonia, in Spain. His father and his three children were proven to be heterozygous for the same mutant PK variant. This variant was characterized by low immunologic specific activity, normal (or slightly increased) stability to heat and to urea; normal isoelectric point, increased K 0.5 for phosphoenolpyruvate, increased inhibition by ATP and normal activation by 0.35 mM fructose 1,6-diphosphate. The mutant PK variant was antigenically identical with wild enzyme as tested against anti wild erythrocyte PK serum by double immunodiffusion and micro complement fixation. The utility and the significance of the immunologic methods to be used in the study of mutant PK variants are discussed.