Erythrocyte Pyruvate Kinase Research Articles

Interconvertible Kinetic and Physical Forms of Human Erythrocyte Pyruvate Kinase

Abstract Human erythrocyte pyruvate kinase (EC 2.7.1.40) was isolated in forms with K0.5 s values for phosphoenolpyruvate which range from 3 x 10-5 m to greater than 10-3 m. Several activity peaks also were obtained when samples were subjected to Sephadex G-200 chromatography, sucrose gradient sedimentation, or starch gel electrophoresis. The most prevalent peak had a molecular weight of 237,000 ± 1,680 S.E. when analyzed by gel filtration chromatography and 214,000 ± 2,700 S.E. when subjected to sedimentation studies. Assuming this to be a tetrameric form, evidence was also obtained for possible monomeric, dimeric, trimeric, and pentameric forms. Enzyme maintained at pH values below 7.0 had K0.5 s values for phosphoenolpyruvate of less than 8 x 10-5 m, gave hyperbolic rate curves, and was 80% or better apparent tetramer. This form could be reversibly converted to a form which had K0.5 s values for phosphoenolpyruvate of about 2.0 x 10-4 m by aging in alkaline pH or purifying on DEAE-cellulose columns. Such preparations appeared to consist primarily of apparent trimer and could be converted back to the lower K0.5 s apparent tetrameric form by aging in solutions with pH values less than 7.0 or by incubating with fructose 1,6-diphosphate. In addition to this apparent R ⇌ T conformational pair, a form of the enzyme with a K0.5 s value for phosphoenolpyruvate greater than 7 x 10-4 m was also obtained from outdated cells. This form of the enzyme could exist in solutions with other forms, but it could not be reconverted to lower K0.5 s forms by exposure to acidic solutions. Another apparently distinct form of enzyme was found in lysates of fresh blood cells. This differed from any of those previously described in that the K0.5 s and nh values (Hill coefficient) obtained with phosphoenolpyruvate as the variable substrate were highly dependent upon the ADP concentration and in that the apparent dimeric form predominated in Sephadex and starch gel studies.

Selective reticulocyte destruction in erythrocyte pyruvate kinase deficiency.

Radioisotope studies of bilirubin turnover, ferrokinetics, and red cell survival ((51)Cr) in a patient with erythrocyte PK deficiency have provided evidence for prompt reticulocyte sequestration and destruction by the reticuloendothelial system. More mature erythrocytes appeared to survive well despite their deficiency of PK. PK-deficient reticulocytes, dependent upon oxidative phosphorylation for ATP production, are exquisitely sensitive to cyanide- or nitrogen-induced mitochondrial inhibition. If oxidative phosphorylation is unavailable, ATP levels decline rapidly, producing alterations in the cell membrane which allow massive losses of potassium and water. The result is a shrunken, spiculated, viscous cell whose rheologic properties would favor its sequestration by the reticuloendothelial system. Those reticulocytes with particularly low levels of PK exhibit very low glycolytic rates and thus are uniquely reliant upon oxidative phosphorylation. Other reticulocytes, better endowed with PK activity, can meet the increased ATP requirements of young erythrocytes. Upon reaching maturity, such cells have diminished ATP needs and can, therefore, survive despite their enzyme deficiency.

Human erythrocyte pyruvate kinase. Its purification and some properties

1. 1. Human erythrocyte pyruvate kinase (EC 2.7.1.40) was purified 30 000-fold by successive (NH 4) 2SO 4 precipitation and column chromatography with blue dextran 2000. The resulting enzyme preparation had a specific activity of 150 μmoles NADH · min−1 · mg−1 at 25°. 2. 2. The molecular weight as determined by gel filtration with Sephadex G-200 was 205 000 ± 5000. 3. 3. With starch gel electrophoresis only one band was observed after-detection of the enzyme activity with the fluorescent technique. 4. 4. The variation of activity of pyruvate kinase at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. 5. 5. The stimulatory effect of Fru-1,6-P 2 as well as the inhibitory effect of ATP on the activity of pyruvate kinase were pH dependent. 6. 6. Phosphorylated hexoses ( Fru-1,6-P 2 and Glc-6-P ), P 1 and the substrate PEP overcame the inhibition of the activity of pyruvate kinase by ATP at physiological pH. 7. 7. Phosphorylated hexoses and P 1 stimulated the activity of pyruvate kinase.

Blood Changes in Calcium and Magnesium Ion Concentrations after Whole-Body X-Irradiation

Following whole-body x-irradiation (600 R) of rabbits, there was an accumulation of calcium and magnesium ions in the whole blood and in the red blood cells. Pyruvate kinase activity and pyruvate concentration were decreased in the erythrocytes from the irradiated animals while the level of phosphoenolpyruvate was increased following x-irradiation. Erythrocytic pyruvate kinase was also found to be inhibited by high calcium ion concentrations. The results showed an accumulation of calcium and magnesium ions in the red blood cells after irradiation which may result in the decrease in pyruvate kinase activity and, hence, in the erythrocyte glycolytic capacity, previously observed following x-irradiation.

Mechanisms of fructose diphosphate activation of a mutant pyruvate kinase from human red cells

1. 1. Normal human erythrocyte pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) and mutant pyruvate kinase from a propositus with congenital, nonspherocytic, hemolytic anemia were isolated by DEAE-cellulose extraction and (NH 4) 2SO 4 precipitation. 2. 2. Fru-1,6-P 2 lowered the apparent Michaelis constant for phosphoenolpyruvate of the normal enzyme but did not substantially change the maximal velocity. This activator greatly elevated both the apparent Michaelis constant for phosphoenolpyruvate and the maximalvelocity of the mutant enzyme. 3. 3. Fru-1,6-P 2 did not affect the Michaelis constant for ADP or the optimal Mg 2+ concentration in either the normal or the mutant enzyme. 4. 4. Fru-1,6-P 2 altered the extremely sensitive interaction of the binding sites for the two substrates on the mutant enzyme. 5. 5. The sensitivity of the normal enzyme to Fru-1,6-P 2 could be increased by either dilution or heating. With heating, dilute solutions of both normal and mutant enzymes decayed in two steps, indicating the presence of multiple conformations in both enzymes. 6. 6. A molecular model is proposed for Fru-1,6-P 2 activation; Fru-1,6-P 2 acts directly on the phosphoenolpyruvate binding site; interaction of the two substrate sites then alters the conformation of the binding site for ADP.

Pyruvate kinase in human erythrocytes.

The importance of efficient separation of erythrocytes from leucocytes when studying haemolysates is shown experimentally, pyruvate kinase determination given as an example. The use of the spiral centrifuge is recommended.

Stabilization, partial purification, and effects of activating cations, ADP, and phosphoenolpyruvate on the reaction rates of an erythrocyte pyruvate kinase

Stability of erythrocyte pyruvate kinase was found to be independently enhanced by inorganic ortho-phosphate, magnesium, and 2-mercaptoethanol. Phosphoenolpyruvate could substitute for and was more effective than phosphate. The enzyme was more stable below pH 7.4 than above. These data were utilized to help achieve a partial purification (<10,000-fold), of the enzyme. The purified enzyme had a pH optimum of 6.8 – 7.1, was activated by potassium (or ammonium), and also required magnesium for activity. The activating effect of magnesium was proportional to the amount of magnesium-ADP chelate formed. Excess magnesium acted as an inhibitor, competing with potassium. The K m value for magnesium-ADP was found to be about 3.3 × 10 −4 m. High levels of ADP were found to inhibit reaction rates. This inhibition was released by addition of more magnesium, suggesting free ADP was the inhibitor. The K m value for phosphoenolpyruvate was found to be about 4.6 × 10 −5 m. Optimal assay conditions (TES buffer) were found to be at about: pH 7.0, 90 m m K; 6.4 m m Mg: 2 m m ADP, and 0.8 m m PEP.

Les anomalies de la glycolyse au cours de l'anémie hémolytique par déficit du globule rouge en pyruvate kinase

In two cases of hemolytic anemia with pyruvate kinase deficiency, we have studied the modifications of glycolysis in red blood cells and the kinetic behaviour of pyruvate kinase. Red cell glycolysis was markedly diminished. This alteration of the metabolic capacity coincides with an accumulation of phosphoenol pyruvate and phospho-glycerates and with a low level of hexose phosphates and triose phosphates. ATP content is very much decreased (35 % of normal values) ; 2,3-diphosphoglycerate is slightly increased. The level of pyridine nucleotides was found to be low, with predominance of reduced forms. These modifications are interpreted in relation to pyruvate kinase deficiency. The kinetic studies of the purified enzyme revealed two forms of pyruvate kinase in normal erythrocytes as well as in pyruvate kinase-deficient cells : — pyruvate kinase-B, with hyperbolic kinetics and K m app. (phosphoenol-pyruvate) = 3.6· 10 −5 M, insensitive to fructose diphosphate. — pyruvate kinase-A, with allosteric kinetics and K m app. (phosphoenol-pyruvate) = 19.3· 10 −5 M. The allosteric form is activated by fructose diphosphate at very low concentrations ( K m app. (fructose diphosphate) = 43·10 −9 M). In activated A-form, K m (phosphoenolpyruvate) is lowered at the same value as the B-form (= 2.8 · 10 −5 M). ADP is inhibitor at concentrations above 30 · 10 −5 M, and this action is overcome by fructose diphosphate. ATP inhibits at physiological concentrations. Pyruvate kinase-A and pyruvate kinase-B have identical V max and are interconvertible. The transition factors are discussed. In this study, no differences appeared in the molecular structure of pyruvate kinase in normal and deficient cells.

An inherited molecular lesion of erythrocyte pyruvate kinase. Identification of a kinetically aberrant isozyme associated with premature hemolysis.

Atypical cases of heritable hemolytic anemia have been noted that conform clinically and biochemically to anemias of the pyruvatekinase (PK)-deficient type, except for the presence of apparently adequate quantities of erythrocyte-PK activity by the usual assay procedure. Investigations of four such anomalous cases, occurring in two unrelated families, are presented. Erythrocytes contained a kinetically aberrant isozyme of pyruvate kinase (PK(2)). Michaelis constants for the pathologic isozyme relative to phosphoenolpyruvate were over 10-fold greater than control values, but no kinetic abnormality was evident for the second substrate, adenosine diphosphate. PK(2) exhibited a pH optimum almost 1 U lower than the wild enzyme form (PK(1)). Significant differences were also evident in the functional stabilities of the isozymes. Leukocytes were unaffected. Family studies revealed paternal heterozygosity for quantitative PK deficiency of the usual type. Clinically normal maternal relatives and some siblings demonstrated intermediate deviations in erythrocyte-PK kinetics and reaction characteristics compatible with coexistence of normal PK(1) and kinetically abnormal PK(2). Hemolytic anemia in the propositi appeared to require simultaneous inheritance of the gene governing PK(2) production and its presumed allele resulting in quantitative PK deficiency. Both genetic defects were traced through three generations, the defective gene in both instances apparently resident on autosomes.A revision of the PK assay technique is suggested, since catalytic inefficiency of PK(2) was manifested only at low substrate concentrations and was therefore undetectable at the relatively high phosphoenolpyruvate levels employed in the conventional assay.

Erythrocyte Pyruvate Kinase Deficiency in Non-spherocytic Hemolytic Anemia: A System of Multiple Genetic Markers?

Abstract Extreme intrafamilial differences between PK-deficient phenotypes regarding hemolysis, ATP stability, and glucose consumption were observed in two pedigrees in which the index cases had severe nonspherocytic hemolytic anemia. Genetic analysis was consistent with heterozygosity for two distinct interacting mutants in minimally affected relatives of severely anemic homozygotes. Neither the mature erythrocytes of the former nor the reticulocyte-rich cell populations of the latter showed accumulation of glycolytic intermediates, but 2,3-DPG was elevated in both. Despite severe PK deficiency, red cell survival in the minimal type was near normal, glucose consumption was unaffected in three of four subjects tested, and ATP maintenance in vitro was adequate, in contrast to the severe type in which these parameters were grossly depressed. The genetic and pathophysiologic implications of these findings are discussed. The possibility is considered that defective glycolysis may play a subordinate role in the hemolytic process associated with PK deficiency and that the enzyme defect may be a genetic marker for as yet unknown erythrocytic abnormalities involving an increase of 2,3-DPG and possibly primary membrane lesions creating excessive demands on the energy metabolism of the erythrocytes. Regardless of the mode of gene action, it is concluded that the nonspherocytic hemolytic anemias associated with PK deficiency are genetically and phenotypically heterogeneous, and that the genetic diagnosis cannot rest on PK assay alone.

Life-span and organ sequestration of the red cells in pyruvate kinase deficiency.

Abstract To define the role of the spleen in the hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency, removal rates and patterns or organ sequestration of 51Cr-labeled PK erythrocytes were studied. Recipients were the PK patients themselves, normal subjects and persons splenectomized for diverse causes. In one study the 51Cr was attached to young and old populations of PK erythrocytes before their injection; in another the young population was differentially labeled with glycine-2–14C. The results show that the PK-deficient cell is heterogeneously affected by whatever mechanisms induce its destruction. Therefore, a population of young PK cells contains more seriously damaged cells than a population of older cells, the latter living to an older age because of their improved initial outlook. Younger cells, if they pass through the spleen, are subsequently destroyed in the liver. Removal of the spleen improves the survival of the younger cells, for they are then destroyed muc...

Use of the autoanalyzer for determination of erythrocyte pyruvate kinase, glucose-6-phosphate dehydrogenase and cholinesterase.

Use of the autoanalyzer for determination of erythrocyte pyruvate kinase, glucose-6-phosphate dehydrogenase and cholinesterase.

Purification of human erythrocyte pyruvate kinase

Purification of human erythrocyte pyruvate kinase

A nomogram relating pO2, pH, temperature, and hemoglobin saturation in the dog.

Abstract : The data from 591 dog blood samples on which observations were made of Po sub 2, hemoglobin saturation, Pco sub 2, pH, and temperature were analyzed by means of a digital computer. The Hill equation was found to fit the data well in the range from 20% to 98% saturation. Linear terms for pH and temperature effects were found to improve appreciably the prediction ability of the equation. A similar term for Pco sub 2, although statistically significant, did not contribute enough additional precision in prediction to justify its inclusion in the final equations. The equations derived are: log (S/1-S) = 2.5198 log Po sub 2 + 1.1804 (pH-7.0) - .047234T - 2.3621 and log Po sub 2 = .37143 log (S/1-S) - .48047 (pH-7.0) + .019518T + .92247. A nomogram relating these variables is also presented. (Author)

HEREDITARY HEMOLYTIC ANEMIA WITH TRIOSEPHOSPHATE ISOMERASE DEFICIENCY.

THE demonstration of the association of inherited deficiencies of erythrocyte enzymes with certain congenital hemolytic anemias has aided a greater understanding of the genetic and pathological manifestations of these disorders, and has resulted in a more definitive classification of these heterogeneous conditions. In 1954 Selwyn and Dacie1 separated the congenital nonspherocytic hemolytic anemias into two classes, Types 1 and 2, on the basis of differences in the spontaneous autohemolysis of sterile defibrinated blood. Valentine, Tanaka and Miwa,2 , 3 in 1961, described the occurrence of a marked deficiency of erythrocyte pyruvate kinase in association with hemolytic disease classifiable as Type 2 on . . .

EXTREME HEMOLYSIS AND RED-CELL DISTORTION IN ERYTHROCYTE PYRUVATE KINASE DEFICIENCY. II. MEASUREMENTS OF ERYTHROCYTE GLUCOSE CONSUMPTION, POTASSIUM FLUX AND ADENOSINE TRIPHOSPHATE STABILITY.

EXTREME HEMOLYSIS AND RED-CELL DISTORTION IN ERYTHROCYTE PYRUVATE KINASE DEFICIENCY. II. MEASUREMENTS OF ERYTHROCYTE GLUCOSE CONSUMPTION, POTASSIUM FLUX AND ADENOSINE TRIPHOSPHATE STABILITY.

EXTREME HEMOLYSIS AND RED-CELL DISTORTION IN ERYTHROCYTE PYRUVATE KINASE DEFICIENCY. I. MORPHOLOGY, ERYTHROKINETICS AND FAMILY ENZYME STUDIES.

BIZARRE morphologic abnormalities of the erythrocytes are generally not considered a prominent feature of congenital nonspherocytic hemolytic anemias.1 , 2 We have observed a child with a severe congenital nonspherocytic hemolytic anemia associated with a deficiency of erythrocyte pyruvate kinase in whom morphologic abnormalities of the red cells were profound. The bizarre red-cell abnormality was in some respects similar to that observed in the syndrome of acanthocytosis associated with absence of serum beta-lipoprotein.3 Of additional interest was the finding that the mother of the patient was not only a heterozygote for the pyruvate kinase (PK) deficiency but was also a carrier of . . .

A SCREENING METHOD FOR THE DETECTION OF ERYTHROCYTE PYRUVATE KINASE DEFICIENCY.

A screening test is described for the evaluation of erythrocyte pyruvate kinase activity. The method is based on the use of an indicator sensitive to the variations of pH induced by the enzyme reaction. The reliability and reproducibility of the test have been compared with the spectrophotometric assay, in normal subjects, in two patients affected by hemolytic anaemia due to pyruvate kinase deficiency, and in a group of heterozygous subjects with intermediate values of pyruvate kinase activity.

Erythrocyte Pyruvate Kinase Deficiency Resulting in Congenital Nonspherocytic Hemolytic Anemia

Erythrocyte Pyruvate Kinase Deficiency Resulting in Congenital Nonspherocytic Hemolytic Anemia

ERYTHROCYTE PYRUVATE KINASE DEFICIENCY RESULTING IN CONGENITAL NONSPHEROCYTIC HEMOLYTIC ANEMIA.

HEREDITARY spherocytosis with its associated hemolytic anemia is generally easily recognized and satisfactorily treated with splenectomy. A rarer group of hemolytic anemias, however, is sometimes confused with hereditary spherocytosis because of the occasional presence of small numbers of spherocytes in association with varying degrees of abnormality in the incubated osmotic fragility test. In this later group of congenital nonspherocytic hemolytic anemias splenectomy has often been performed but usually does not produce a clinical cure. Because splenectomy is generally of inconsistent value, this type of anemia must be accurately differentiated for the sake of proper treatment and accurate prognosis. . . .