The effect of copper on red cell enzyme activities.

Previous studies have shown a marked effect of very high levels of copper on red cell glucose-6-phosphate dehydrogenase and glutathione. When the effect of more nearly physiological levels of copper were studied, red cell hexokinase, phosphofructokinase, phosphoglyceric kinase, pyruvate kinase, and 6-phosphogluconate dehydrogenase were found to be inhibited. Inhibition was observed both when copper was added directly to hemolysates or when hemolysates were prepared from red cells from whole blood which had been incubated with copper and washed. The inhibition of red cell enzymes by copper was completely reversed by the addition of EDTA.

Heterogeneity of human platelets. V. Differences in glycolytic and related enzymes with possible relation to platelet age.

Human platelets were separated by desity-centrifugation into heavy and light populations. Heavy platelets have an average volume approximately twofold greater than light platelets, and have previously been shown to be young platelets. All 11 enzymes of the Embden-Meyerhof pathway plus the five related enzymes: phosphoglucomutase, glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, alpha-glycerol-P dehydrogenase, and glutathione reductase (TPNH) were examined in cell lysates from total, heavy, and light platelet populations. Apparent Km for individual enzymes were measured in a total platelet population. Empirical V(max) of the individual enzymes were measured in total, heavy, and light platelet populations. The three apparent rate-limiting enzymes for glycolysis were hexokinase, phosphofructokinase, and glyceraldehyde-3-P dehydrogenase. Heavy platelets contained approximately twofold greater enzyme activity (per gram wet weight) than light platelets for 7 of the 16 enzymes measured: hexokinase, phosphohexoisomerase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase, phosphoglycerokinase, lactic dehydrogenase, and phosphoglucomutase. Heavy platelets also contained 1.9-fold greater reduced glutathione (GSH), 1.7-fold greater DPNH, and 1.2-fold greater TPNH than light platelets. Heavy platelets contained 1.8-fold less lipid peroxidation products (malonyl aldehyde equivalents) than light platelets and were 2.4-fold more resistant to lipid peroxidation catalyzed by 0.1 mM FeCl(3). Sterile incubation of heavy platelets, in vitro for 17 hr, resulted in a significant loss of enzyme activity for the "elevated" seven enzymes when compared with the remainder. Reducing agents such as GSH (0.1 mM), ascorbic acid (0.1 mM), and dithiothreitol (0.01 mM), when added to the incubation mixture, significantly reduced the in vitro loss of activity. In vitro incubation was also associated with a significant loss of GSH and DPNH and a 1.8-fold increase in lipid peroxidation products.

Human platelet 6-phosphofructokinase. Relation between inhibition by Mg-ATP2-and cooperativity towards fructose 6-phosphate and investigations on the formation of a ternary complex.

Human platelet 6-phosphofructokinase (EC 2.7.1.11) shows cooperativity towards Fru-6-P and is allosterically inhibited by high Mg-ATP2- concentrations. No relation could be demonstrated between the cooperativity towards Fru-6-P and the inhibition by Mg-ATP2-. Increasing the concentrations of Mg-ATP2- only raised the apparent Km values for Fru-6-P, but did not change the Hill constants. A possible formation of a Mg-ATP2--enzyme-Fru-6-P complex during catalysis was investigated. Our calculations suggest that such a ternary complex is indeed formed during the reaction.

A liver-type mutation in a case of pronounced erythrocyte phosphofructokinase deficiency without clinical expression.

A marked erythrocyte phosphofructokinase deficiency was detected in a healthy man. His enzymatic activity was only 25% that of normal controls. His father and his son had erythrocytic phosphofructokinase activities of 50-55% that of normal controls. The chromatographic separation of erythrocytic phosphofructokinase isozymes, as well as immunological studies revealed a decrease in L-type phosphofructokinase activity. The lowered erythrocytic L-type phosphofructokinase activity was not accompanied by a decreased level of L-type phosphofructokinase in proteins. The L/M subunit ratio was similar to that of normal subjects. The defect resulted from the synthesis of stable L-type mutant subunit with high electrophoretic mobility. White blood cells, which synthesize mostly the same isozyme as L-type phosphofructokinase also showed a decreased activity and a high electrophoretic mobility. In spite of this important deficiency, and of significant metabolic alterations (a slight decrease in ATP; 2,3-diphosphoglycerate; triose phosphate), hemolysis did not appear in the propositus.

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect.

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).

Purification of F4 phosphofructokinase from human platelets and comparison with the other phosphofructokinase forms.

The phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) tetramers F4, F3L and F2L2 have been separated from human platelets, and purified to homogeneity by affinity chromatography on Dextran Blue-Sepharose 4B. The F subunits have a molecular weight of 85 000, identical to that of the M subunits. By contrast with L-type phosphofructokinase, the F-type enzyme seems to exist predominantly in a tetrameric form and not to aggregate to high molecular weight polymers. Specific activity of pure F4 phosphofructokinase is about 140 IU/mg of protein. Immunologically, it is easy to distinguish all the basic phosphofructokinase forms (i.e. M, L and F types); nevertheless a slight immunological cross-reactivity seems to exist between all these forms.

Modifications in the allosteric properties of phosphofructokinase in rat erythrocytes and reticulocytes cross-linked with dimethyl suberimidate and 3,3'-dithiobispropionimidate.

The kinetic behaviour of phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been studied in situ, by using rat erythrocytes and reticulocytes treated with dimethyl suberimidate and 3,3'-dithiobispropionimidate as cross-linking reagents and with digitonin as the delipidating agent. Comparison of the ATP and fructose-6-P saturation curves of phosphofructokinase in dimethyl suberimidate-permeabilized cells with those obtained in haemolysates showed the enzyme to have reduced allosteric properties under in situ conditions, although it still responded to cyclic AMP (300 microM) added as allosteric effector. Non-sigmoidal fructose-6-P saturation curves were also observed using 3,3'-dithiobispropionimidate-permeabilized erythrocytes, either in the absence or in the presence of cyclic AMP. A hyperbolic behaviour was shown after cross-linking reversal of 3,3'-dithiobispropionimidate-permeabilized erythrocytes by treatment with dithiothreitol. Specific activity values of phosphofructokinase were always lower in permeabilized cells than in haemolysates. A significant inhibition of phosphofructokinase specific activity, without any effect on its allosteric behaviour, is exerted by reaction of dimethyl suberimidate or 3,3'-dithiobispropionimidate with erythrocyte lysates in the presence of an inhibitory concentration of ATP. These results suggest that penetration of the cross-linking reagent and its subsequent reaction with intracellular phosphofructokinase will have a direct effect upon the results obtained using this in situ approach.U

Reaction mechanism of erythrocyte phosphofructokinase.

The reaction mechanism of erythrocyte phosphofructokinase (PFK) was investigated by the initial velocity and the product inhibition. Intersecting lines obtained with initial velocity studies are consistent with a sequential mechanism and the formation of ternary complex as an intermediate. The product inhibition studies support an ordered Bi Bi mechanism in which fructose 6 phosphate (F6P) is the first substrate binding and adenosine diphosphate (ADP) is dissociated from the enzyme before fructose-1,6-P2 (FDP).

Influence of free Mg2+ on the kinetics of human erythrocyte phosphofructokinase.

The influence of Mg2+ on the reaction catalyzed by human erythrocyte phosphofructokinase has been investigated using kinetic methods. The catalytic activity of PFK is dependent upon the presence of Mg2+ which constitutes with ATP the true Mg-ATP2- substrate. Free Mg2+ has no influence on the affinity of the enzyme for Mg-ATP2- substrate. Erythrocyte PFK is more inhibited by ATP4- and uncomplexed citrate than it is by Mg-ATP2- and Mg-citrate. Free Mg2+ relieves the MgATP2- and Mg-citrate inhibition under conditions where free ATP4-is negligible. We can assume that uncomplexed Mg2+ acts as positive effector by direct binding to the enzyme. These results emphasize the role of Mg2+ in the regulation of PFK activity in the erythrocyte.

Alterations in host regulatory glycolytic enzymes during pathogenesis of primate malaria and following chloroquine therapy.

Hexokinase, phosphofructokinase and pyruvate kinase, the regulatory enzymes of the glycolytic sequence, showed progressive increases in their activities with the rise of parasitaemia in Plasmodium knowlesi-infected rhesus monkey serum and red blood cells. Chloroquine therapy cleared the parasitaemia in 72 h and brought the elevated levels of these enzymes back to almost normal in about 30 days.

Mechanisms of the acquired erythrocyte enzyme deficiencies in blood diseases.

Acquired enzymatic activity defects of erythrocyte pyruvate kinase, glucose phosphate isomerase and phosphofructokinase have been studied in patients with acute myeloid leukemias, sideroblastic refractory anemias and unclassified acquired dyserythropoiesis. 6 patients with acute myeloid leukemia had a lowered erythrocyte pyruvate kinase activity; in 5 of them the concentration of the "pyruvate kinase"-antigen was parallely decreased, in such a manner that the ratio enzyme activity/immunologic reactivity (i.e. the molecular specific activity) was normal. In 1 patient with acute leukemia, 4 with refractory anemia and 1 with acquired dyserythropoiesis the defect of the pyruvate kinase activity was associated with a normal antigen concentration (and, therefore, the molecular specific activity in whole hemolysate was lowered). The enzyme activity was restored by incubation with SH reagents in two cases and by partial purification as often as it was performed. The electrofocusing pattern of erythrocyte pyruvate kinase was normal in both these types of defects. In two patients with so-called "acquired dyserythropoiesis" an erythrocyte glucose phosphate isomerase deficiency has been detected; in both the cases it was associated with a parallel decrease of the antigen concentration. The residual enzyme had a normal electrofocusing and electrophoretic pattern and a normal heat stability; the enzyme activity could not be restored by any treatment. In 1 patient with erythroleukemia and in 1 other with acquired dyserythropoiesis the erythrocyte phosphofructokinase activity was lowered. The enzyme activity was not restored by cross incubation in isologous plasma or by the SH reagents. In one case immunologic study could be performed, indicating that the enzyme defect was mainly due to the decreased ratio of the muscle type subunit of the erythrocyte phosphofructokinase. The electrofocusing pattern of deficient phosphofructokinases was normal. Finally, we point out the probable existence of several direct mechanisms, genetic and post translational, accounting for the acquired enzyme defects of red blood cells in various blood disorders.

Erythrocyte glucose metabolism in Duchenne muscular dystrophy.

We compared glucose metabolism by erythrocytes from patients with Duchenne muscular dystrophy and by erythrocytes from control individuals. There was a significant decrease in the rate of lactate production at pH 7.2 in the dystrophic group. When the pH of the incubation medium was changed to 8.0, we found that the increase in the rate of lactate production for the dystrophic group was significantly larger. We measured the concentrations of key glycolytic metabolites and adenine nucleotides and determined the values of the energy charge during these incubations. We also determined the concentrations of polyol pathway intermediates, the activities of the oxidative portion of the pentose phosphate pathway and the activities and kinetics of phosphofructokinase from both cell groups. There were no significant differences between groups for any of these variables.

Erythrocyte phosphofructokinase deficiency associated with an unstable variant of muscle phosphofructokinase.

A case of chronic non-spherocytic hemolytic anemia due to partial erythrocyte phosphofructokinase deficiency (61% of normal) is reported. Immunological studies in hemolystates, using anti-muscle and anti-leukocyte phosphofructokinase antisera, seemed to indicate that an isozyme of the muscle type was deficient in the patient. This hypothesis was confirmed by the studies of muscle phosphofructokinase; this enzyme was an unstable and fast variant. There was no deficiency in muscle because of the active synthesis of proteins by this tissue, but the deficiency could be detected in erythrocytes, old cells which are no longer able to synthesize proteins.

A new method of inhibiting glycolysis in blood samples.

The maintenance of hydrogen ion concentration in blood samples at pH 5.3-5.9 immediately inhibits glycolysis. This effect is due to the inhibition of all glycolytic enzymes, as shown by measurement of various glycolytic intermediates. At the inhibitory pH at 25 degrees C, the glucose content did not decrease over a period of 8 h, but it did decrease by 1.3 +/- 1.1 mg/dl over 24 h.

Glycolysis measurements in red cells from drug-treated epileptic and anaemia patients.

Glycolytic activity was measured in red cells from epileptic patients treated with anticonvulsant drugs. These results are compared with measurements of red cell glycolytic activity from megaloblastic anaemia patients and normal controls. The differences obtained between epileptic patients and controls, as well as the differences between anaemia, epileptic patients and controls is discussed.

Enzymatic pattern of glucose metabolic pathways in pyruvate kinase-deficient erythrocytes.

It has been shown that in some cases of congenital non-spherocytic haemolytic anaemia (CNSHA) with pyruvate kinase deficiency, the primary defect may be related to diminished magnesium-stimulated ATPase activity, followed by elevation of the erythrocyte ATP level. ATP as the end product of glycolysis inhibits by negative feedback control the activities of key glycolytic enzymes involved in energy production, i.e. pyruvate kinase (PK) and phosphofructokinase (PFK). Erythrocyte-deficient PK, however, is insensitive to the stimulating effect of fructose 1,6-diphosphate (FDP), which is normally a positive effector of PK. Both competing effectors, i.e. ATP and FDP, seem to show specific interaction. PK, inactive in the presence of high concentrations of ATP, seems to lose its sensitivity to FDP. This effect persists until ATP molecules are present in excess. In vitro incubation of deficient PK with ATPase resulted in increased PK activity as well as in recovery of its sensitivity to the stimulating effect of FDP. The same effects were obtained in vivo by administering magnesium levulinate intravenously to CNSHA patients with PK deficiency. This may indicate that magnesium ions stimulate deficient ATPase activity and lead to diminution of ATP as a negative effector for other regulatory enzymes.

Hexokinase, phosphofructokinase and pyruvate kinase isoenzymes in lymphocyte subpopulations.

In order to study the three regulator enzymes of glycolysis, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), in relation to lymphocyte maturation, lymphocytes of different origin were investigated. Lymphocytes from bone marrow, thymus, cord blood, adult peripheral blood and mitogen-stimulated lymphocytes were investigated. The enzyme activities were determined and the isozyme patterns were studied by means of electrophoresis, kinetic measurements and immunoprecipitation. The young lymphocytes from bone marrow and the mitogen-stimulated lymphocytes could be distinguished from the other lymphocytes by a higher residual HK activity in the presence of the inhibitor glucose-1,6-diphosphate. Peripheral blood T lymphocytes differed from non-T lymphocytes in the PK isozymes distribution. All the cells contained PK type K4 and the hybrid K3M. In T cells a smaller amount of the K isozyme was seen than in non-T cells. The PK residual activity in the presence of alanine was significantly higher in peripheral blood T cells than in non-T cells. Thymocytes are characterised by a larger amount of PFK M-subunits than peripheral blood T and non-T lymphocytes. The stimulation of PFK by the positive effector glucose-1,6-diphosphate was higher in thymocytes than in the peripheral blood lymphocytes.

The effect of bilipolinum (Adipiodon), an iodine contrast medium on erythrocyte enzymes.

Bilipolinum (Adipiodon), iodine contrast medium used in cholangiography, showed an inhibitory effect on the activity of human erythrocyte phosphohexoseisomerase, phosphofructokinase, aldolase and glucose-6-phosphate dehydrogenase. The addition of glucose metabolites (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bis-phosphate, pyruvate and lactate) abolished the inhibitory effect of Bilipolinum. In the presence of Bilipolinum purified erythrocyte phosphofructokinase showed a decreased affinity towards substrate, modified allosteric properties and reduced stability at pH below 7.5. Purified erythrocyte glucose-6-phosphate dehydrogenase was also affected by Bilipolinum and its affinity for NADP was decreased. Testing of erythrocyte enzymes in the evaluation of toxicity of iodine contrast media is discussed.

The effect of adrenal steroids on human erythrocyte phosphofructokinase.

Phosphofructokinase isolated from human erythrocytes incubated with cortisol showed alterations in regulatory properties. The enzyme was more sensitive to the inhibition by ATP and citrate. This effect was abolished by alanine. Similar changes in phosphofructokinase properties were found after incubation with aldosterone and corticosterone.

Effect of cortisol on erythrocyte and reticulocyte enzymes: modulation of phosphofructokinase properties.

After incubation of rabbit red blood cells with 10 micrograms/ml of cortisol, a decrease of the activity of glucose-6-phosphate dehydrogenase, phosphofructokinase and acid phosphatase was observed. Glucose-6-phosphate dehydrogenase and phosphofructokinase were isolated from hemolysate and investigated. No changes in the affinity of both enzymes towards substrates and coenzymes were found. Cortisol modified allosteric properties of phosphofructokinase. Differences were seen between the erythrocyte and reticulocyte enzyme. In the erythrocyte enzyme the inhibitory effect of ATP and citrate was enhanced and the activatory effect of AMP was abolished after incubation with cortisol. Cortisol showed no effect on the inhibition of reticulocyte phosphofructokinase by ATP or activation by AMP, and protected the enzyme toward inhibition by citrate.