Pyruvate Kinase Variants Research Articles

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.

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.

Pyruvate kinase deficiency and G6PD deficiency

G6PD deficiency was discovered in 1956. It causes drug-induced acute hemolysis and is inherited by a sex-linked recessive trait. While the disorder is frequently seen in Negroes and inhabitants around the Mediterranean basin, it is rare in Japanese, the frequency of which is about 0.1%. As to the G6PD genetic variants, about 120 variants have been discribed so far in the world and 10 variants were found in Japan. Pyruvate kinase (PK) deficiency was first discovered in 1961. The disorder is inherited by an autosomal recessive trait and causes chronic hemolytic anemia. 29 cases were found in Japan. Using paremeters such as enzyme activity, substrate affinity, velocity maximum, urea stability as well as electrophoretic mobility, we have been able to differentiate several PK variants. The procedures for the characterization of PK variants have not standardized as yet but there is a treand to do so and hopefully this can be accomplished fairly soon. In both G6PD deficiency and PK deficiency, there is a certain correlation between the characteristics of abnormal enzymes and clinical manifestations.

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.

Carrier detection in X-linked recessive (Duchenne) muscular dystrophy: Pyruvate kinase isoenzymes and creatine phosphokinase in serum and blood cells

Allosterism allows individual assay of both isoenzymes, one abundant in muscle, of pyruvate kinase (PK), recently reported superior to serum creatine phosphokinase (CPK) in detecting patients with and female carriers of X-linked recessive (Duchenne) muscular dystrophy (DMD). Extensive comparative studies did not support these findings, and confirmed the marked superiority of CPK over variants of PK or other enzymes in sensitivity, stability and convenience. Deducting the adenylate kinase increment (AKI) further refined the CPK assay, eliminating the effect of haemolysis in diagnosis and enabling studies of blood cell content. Both leucocytes and erythrocytes liberated PK and lactate dehydrogenase (LDH) after brief chilling or disruption. Only erythrocytes showed a CPK content, however, constantly adjusted to match that of serum as if by free cell membrane passage, but less accomodating to a sudden large influx of CPK than of LDH, where an apparent buffering effect could account for differences in clinical response.

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.

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°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-poly-acrylamide gel electrophoresis.

Effects of Sulphydryl Compounds on Abnormal Red Cell Pyruvate Kinase

The effect of some sulphydryl compounds on two new variants of red cell pyruvate kinase (ATP: pyruvate phosphotransferase, PK) is reported. In vitro a striking correction has been obtained of both the qualitative and, in one case, the quantitative defect of red cell PK. In vivo, a correction of the qualitative and quantitative abnormalities has been produced in both patients, with clinical improvement of one of them. These findings, together with the unexpected results in respect to the functional properties of PK found in the affected members of the two families studied, suggest that the PK abnormality is not the cause of the haemolytic anaemia, but an epiphenomenon of a primary unknown defect that apparently involves the red cell thiol groups.

Molecular mechanism of erythrocyte pyruvate kinase deficiency.

Erythrocyte pyruvate kinase (PK) from 5 patients with cogenital non-spherocytic hemolytic and erythrocyte PK deficiency have been studied by immunological methods and electrofucusing. L type immunologically related PK was titrated in crude hemolysate with anti human liver L type PK rabbit serum and M2 type immunologically related PK with anti human leukocyte M2 type PK serum. After partial purification, molecular specific activity of erythrocyte PK was measured by immunoinactivation and electroimmunodiffusion and anti L type PK serum. Partially purified erythrocyte PK was focused on continuous sucrose gradient with 2% ampholines covering the pH range 5--8. PK enzymatic deficiency was due two times to a lowered molecular specific activity of the PK variants, the concentration of PK antigen being in the normal range. In the 3 other cases enzyme activity and immunological reactivity were likewise lowered. In the 2 patients with the most marked erythrocyte PK deficiency about 50% of the residual activity in crude hemolysate were non inhibited by anti L type PK serum, but were inhibited by anti M2 type PK serum. In 3 patients, the electrofocusing pattern of partially purified PK was significantly different from than of normal controls. In conclusion, the heterogeneity of the molecular mechanisms of the deficiency on the one hand, and the abnormalities of electrofucusing patterns on the other hand, seem to indicate that erythrocyte PK deficiency is due to the synthesis by muted structural genes of various abnormal PK molecules.

Purification and properties of pyruvate kinase in normal and in pyruvate kinase deficient human red blood cells

1. 1. Pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) from normal and enzyme-dificient human red blood cells was purified 10 000-fold with an overall yield of 10%. 2. 2. The molecular weight of the different variants of pyruvate kinase was determined by dextran gel filtration by Sephadex G-200 and was found to be 195 000 ± 6000. 3. 3. In high-voltage electrophoresis the pattern of normal red blood cell pyruvate kinase shows three fractions, two being interconvertible. In enzyme-different material one of the three fractions was not detectable because of its pronounced instability. 4. 4. A Stepwise purification of the three electrophoretic fractions can be achieved by (NH 4) 2SO 4 precipitation. The kinetic constants of the fractions from all phenotypes do not differ significantly with respect to ADP and phosphoenolpyruvate.A striking difference was found in enzymic activation by the allosteric effector fructose 1,6-diphosphate.

Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties.

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the K(m) for phosphoenolpyruvate. 2. A ;cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal K(m) for substrate at 5 degrees . At physiological concentrations of phosphoenolpyruvate the ;cold' enzyme is active only below 10 degrees . A ;warm' pyruvate kinase has a minimal K(m) for substrate at about 12 degrees . This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9 degrees . 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4-12 degrees . 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the ;warm' pyruvate kinase and the ;cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the ;warm' enzyme is converted into the ;cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.