Dominique Cottreau

Post-translational modifications of human glucose-6-phosphate dehydrogenase.

Summary Immunological, kinetic and electrophoretic (electrofocusing) properties of glucose-6-phosphate dehydrogenase have been studied in various hemopoietic tissues. The influence of molecular aging on these properties has been followed. o 1. The molecular specific activity defined as the ratio : enzymatic activity/immunological reactivity decreased during aging of cells which are no more capable of protein synthesis (platelets, red blood cells). 2. Each type of cells (polymorphonuclear, lymphocytes, platelets, red cells) shows a constant and particular pattern upon electrofocusing. Aging of erythrocytes is accompanied by the appearance of active bands with a lower isoelectric point. 3. In vitro incubation leads to the appearance of forms with a lower isoelectric point. This phenomene requires the presence of cellular factors in the tissue. It is not prevented by the addition of inhibitors of proteolysis. 4. The electrofocusing pattern is independent from the saturation in NADP+, the presence of reducing or oxidizing agents, the protein concentration. 5. The platelet enzyme shows several peculiarities, e.g. increased Km glucose-6-phosphate, abnormal pH curve, and a smaller effect of in vitro incubation on the electrophoretic pattern. 6. A connection between kinetic, immunological and electrophoretic properties is demonstrated for most cells. The forms with the lowest isoelectric point have also the lowest molecular specific activity and the highest Km for glucose-6-phosphate. Finally, the nature and meaning of postranslational alteration of glucose-6-phosphate dehydrogenase are discussed.

Purification and partial characterization of different forms of phosphofructokinase in man.

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from human muscle, brain, heart and granulocytes has been purified using a two or three step purification procedure. The main step is Blue Dextran-Sepharose 4B chromatography with selective elution of phosphofructokinase by formation of the ternary complex ADP or ATP-fructose-6-P-enzyme. Muscle and heart contain only enzyme subunits with a molecular weight of 85,000. This type of subunit is predominnant in brain, where it co-exists with subunits of about 80,000 daltons. A single type of subunits is found in the granulocytes, with a molecular weight of 80,000. Anti-muscle phosphofructokinase antiserum reacts only with M-type enzyme. Anti-granulocyte enzyme antiserum, absorbed by pure brain phosphofructokinase, exhibits a narrow specificity against the so-called L-type enzyme. Anti-brain antiserum, absorbed by pure muscle phosphofructokinase and partly purified red cell enzyme, exhibits a narrow specificity against a phosphofructokinase form predominant in fibroblasts and present in brain (F-type).

Gd(--) Abrami: a deficient G-6PD variant with hemizygous expression in blood cells of a woman with primary myelofibrosis.

SummaryA new deficient G-6PD variant, Gd(-) Abrami, was found in granulocytes, platelets and red blood cells of a 65-year-old woman with myelofibrosis. Enzyme and immunological titrations showed that only the deficient variant was present in blood cells whereas both the normal and abnormal enzymes were found in the fat cells of this patient.These results seem to indicate that the granulocytes, platelets and erythrocytes of this woman with myelofibrosis have arisen from a single abnormal precursor the functional X chromsome of which is the one carrying the abnormal G-6PD gene.

Assignment of the ǵene for F-type phosphofructokinase to human chromosome 10 by somatic cell hybridization and specific immunoprecipitation

A method of specific immunoprecipitation of minor isozymes was developed and applied to the detection of human F‐type phosphofructokinase in man‐rodent somatic cell hybrids.

Heat lability of glucose-6-phosphate dehydrogenase in some senescent human cultured cells. Evidence for its postsynthetic nature.

Summary The nature of heat lability of glucose-6-phosphate dehydrogenase in senescent cells has been studied in human liver-derived cultured cells and in human fetal lung fibroblasts. The main data obtained were the followings : 1. When purified, glucose-6-phosphate dehydrogenase from either young or senescent cells exhibited similar heat stability. 2. In some cases, previous dialysis of the crude extracts from senescent cells improved heat stability of glucose-6-phosphate dehydrogenase. 3. The crude extracts from senescent cells deprived of endogenous glucose-6-phosphate dehydrogenase by specific immunoneutralization were able to alter heat stability of exogenous pure enzyme secondarily added to the extracts. 4. Some proteic fractions purified from senescent cell extracts were also able to alter heat stability of exogenaus enzyme.

Molecular and functional anomalies in two new mutant glucose-phosphate-isomerase variants with enzyme deficiency and chronic hemolysis

SummaryTwo new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal.GPI ‘Paris’ was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI “Enfants malades’ exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate»glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate»fructose-6-phosphate).No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other.Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.

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.

Gd (−) Matam, an african glucose-6-phosphate dehydrogenase variant with enzyme deficiency. Biochemical and immunological properties in various hemopoietic tissues☆

Two unrelated Senegalese patients, both native of the Matam province, were found to have the same deficient G6PD variant. One has no hematological history, the other had several induced acute hemolytic episodes. The deficiency was almost complete in red blood cells and 20-30 percent of the normal level in leukocytes and platelets; in leukocytes the deficiency was due to a decrease in the molecular specific activity of the enzyme to which a molecular instability was added, explaining the greater deficiency in red blood cells. The electrophoretic mobility was slightly fast in leukocytes and platelets but normal in red blood cells. This pattern was confirmed by electrofocusing in ampholine-acrylamide gel. From a kinetic point of view, these enzymes were characterized by a lowered Km (G6P) (13 to 20 muM) a normal Km (NADP+), a Ki (NADPH) increased about twice, a thermal instability, a biphasic pH curve and an increased activation energy (15 kcal/mole). The polymorphonuclear cells were functionally strictly normal: engulfment, nitroblue tetrazolium (NBT) reduction test, induced iodination, and oxygen consumption were normal. The authors discuss the importance of post-synthetic modifications of the muted enzymes and their repercussions on the enzyme characteristics.

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.

Hyperanodic forms of human glucose-6-phosphate dehydrogenase

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into hyperanodic forms when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some NADP+ modifying proteins purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.