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D Cottreau

Publications and source records attributed to D Cottreau.

At least 37 records · Page 2Linked to original sources
PubMed1979

'GPI Roma', a new glucose phosphate isomerase deficient variant: in vivo occurrence of postsynthetic modifications of the mutant enzyme.

In a 5-year-old Italian girl with severe congenital hemolytic anemia, red cell GPI deficiency was proven, and found to be due to a new variant, 'GPI Roma.' The parents are first cousins and have been proven to be heterozygous for this variant. GPI Roma was slightly unstable to heat and exhibited a slightly increased Michaelis constant for fructose-6-phosphate. A single predominant fast-migrating GPI form existed in the patient's white blood cells, while the electrophoretic pattern in the red cells was composed, in addition to this 'fast band,' of a major band migrating as normal GPI and of an additional slow band. It is shown that this phenomenon may be ascribed to postsynthetic events modifying the charge of the mutant enzyme.

Anemia, Hemolytic, Congenital↗
PubMed1979

Phosphofructokinase in human blood cells.

The subunit composition of phosphofructokinase from normal and malignant blood cells has been investigated by means of immunologic, electrophoretic, and chromatographic methods. Immunoprecipitation tests were performed with three specific antisera recognizing each of the basic subunits of human phosphofructokinase: muscle, M-type; liver, L-type; and fibroblast, F-type. Mature polymorphonuclear cells contain mainly L-subunits, while lymphocytes and platelets contain hybrids formed of L and F subunits; these hybrids can be electrophoretically separated. Red cell phosphofructokinase is composed of L and M subunits, as judged by its reactivity with anti-L and anti-M-type antisera. The various M-L hybrids composing red cell phosphofructokinase could be only separated by chromatography on DEAE-Cellulose. Lymphocytes from patients with chronic lymphocytic leukemia and lymphoblasts from patients with acute lymphoblastic leukemia contain phosphofructokinase forms similar to those from normal lymphocytes, while the immature granulocytic cells (leukemic myeloblasts and myeloid cells of chronic myeloid leukemia) are characterized by a reinforcement of enzyme inhibition by anti-F-type antiserum. Lymphoid lines in culture (Epstein-Barr virus (EBV)-induced or malignant lymphoma-derived lines) are characterized by the indistinctive expression of all three basic subunits, similar to that found in some fetal tissues. This article represents the first description of the isozymic nature of phosphofructokinase in platelets and white blood cells and of its changes with malignancy and cell culture. This enzyme might represent a useful marker in the characterization of the leukemic cells.

Antigens↗
PubMed1978

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.

Apoenzymes↗
PubMed1978

G6PD Vientiane: a new glucose-6-phosphate dehydrogenase variant with increased stability.

A new G6PD variant, called G6PD Vientiane, has been discovered in a patient from Laos. The characteristics of this variant are: mild enzyme deficiency (about 50% of the normal activity) in the granulocytes and the red cells, with normal G6PD-related antigen concentration; increased stability; normal Km glucose 6-phosphate and NADP+; increased inhibition constant by NADPH; decreased inhibition by ATP; slightly increased utilization of the substrate analogue; abnormal pH curve, with maximum activity at pH 9.5; slightly reduced starch gel electrophoretic migration. The implications of the molecular stability of a deficient mutant variant are discussed.

Adult↗
PubMed1978

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

Two 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 leads to glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate leads to 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.

Adolescent↗
PubMed1977

'Gd(-) Hôtel Dieu': a new G-6PD variant with chronic hemolysis in a Negro patient from Senegal.

A G-6PD deficiency was detected in a Negro patient from Senegal suffering from congenital nonspherocytic hemolytic anemia. The main characteristics of this variant were: profound defect of G-6PD activity in the red cells, decreased immunologic specific activity, fast electrophoretic mobility, decreased Km-G-6P and normal Km-NADP+, normal inhibition by ATP and NADPH, slightly increased utilization of the substrate analogues, slightly biphasic pH curve, high heat lability, subnormal activation energy. The characteristics of this variant being unique, it was called 'G-6PD Hôtel Dieu.'

Adult↗
PubMed1977

Accuracy of portein synthesis and in vitro aging. Search for altered enzymes in senescent cultured cells from human livers.

The authors have looked for altered proteins in senescent cultured cells from adult liver. Four enzymes (phosphoglycerate kinase, M2 type pyruvate kinase, glucose phosphate isomerase and glucose-6-phosphate dehydrogenase) have been studied by immunological and enzymatic titration and electrofocusing. In addition, heat stability of glucose-6-phosphate dehydrogenase (G6PD) was appraised in cell crude extracts and in partially purified preparations. Enzymatic aactivity as well as immunological reactivity of the four enzymes studied were identical 16 lines in phase II and in 13 lines in phase III. Electrofocusing pattern of the enzymes from 'young cells' was identical to the ones from 'old cells'. Finally, G6PD from old cells seemed to be more unstable than G6PD from young cells when studied in crude extracts. These differences, however, disappeared as G6PD was partially purified from old or young cultured cells. Consequently, no evidence of altered protein, either missynthesized or posttranslationally modified, was found in the senescent cultured cells studied. Moreover, this work indicated that the modification of heat stability of G6PD from old cells was not due to the enzyme molecule itself but rather to the cell medium.

Aging↗
PubMed1977

Immunologic study of the age-related loss of activity of six enzymes in the red cells from newborn infants and adults--evidence for a fetal type of erythrocyte phosphofructokinase.

Blood from 10 normal healthy adults and cord blood from 8 healthy full term infants were infiltrated through a mixture sulfoethylethycellulose-Sephadex G 25 in order to eliminate the platelets and the leukocytes. Then the erythrocytes were fractionated into young and old cells by centrifugation in microhematocrit tubes. The enzyme activity and the immunologic reactivity of glucose phosphate isomerase (EC.5.3.1.9), phosphoglycerate kinase (EC.2.7.2.3), pyruvate kinase (ec.2.7.1.40), glucose 6-phosphate dehydrogenase (EC. 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC.1.1.1.44) were measured in every fraction. As previously reported, the enzyme activities were far higher in cord blood than in adult blood red cells; nevertheless, the age-related loss of enzyme activity was similar in both cord and adult blood. The decrease of the enzyme activity of glucose phosphate isomerase and phosphoglycerate kinase in old cells was singly associated with a lowered concentration of the enzyme-related antigen; by contrast, the age-related decrease of the enzyme activity of pyruvate kinase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase was associated with both a lowered concentration of the enzyme-related antigen and a lowered "molecular specific activity" (i.e., a lowered ratio of enzyme activity to enzyme-related antigen concentration). This phenomenon was especially marked for pyruvate kinase, which had a molecular specific activity in old cells that was 68% of that in young cells. Phosphofructokinase had a lower enzyme activity in cord blood erythrocytes than in adult blood erythrocytes; the difference was especially important in old cells from infants in which phosphofructokinase activity was 53% of that in old cells from adults. Phosphofructokinase from old cells of full term infants and from unfractionated cells from two premature infants (21 and 32 weeks of gestation) was less neutralized by anti-muscle phosphofructokinase serum and more inhibited by ATP than the enzyme from adult blood erythrocytes.

Adenosine Triphosphate↗
PubMed1976

Studies on the nature of different molecular forms of glucose-6-phosphate dehydrogenase purified from human leukocytes.

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon 'native glucose-6-phosphate dehydrogenase' of 'modifying factors' especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the 'modification' of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the 'modifying factors'. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.

Amino Acid Sequence↗
PubMed1976

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.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1976

Human erythrocyte phosphoglycerate kinase deficiency: presence in a deficient patient of a stable variant with lowered catalytic activity.

The phosphoglycerate kinase deficiency found in one boy with hemolytic anemia was associated with an almost normal concentration of phosphoglycerate kinase-immunologically related material in the patient's erythrocytes. Consequently the catalytic activity of the mutant enzyme was drastically lowered. Besides, the abnormal phosphoglycerate kinase was more stable to heat and to urea than normal phosphoglycerate kinase and its isoelectric point was slightly increased.

Adult↗
PubMed1976

Glucose-phosphate isomerase deficiency due to a new variant (GP I Barcelona) and to a silent gene: biochemical, immunological and genetic studies.

A 12-year-old girl of Spanish origin was found to be double heterozygote for a deficient GP I variant (GP I Barcelona) and for a silent GP I gene. The mother was heterozygote for GP I Barcelona and the father was heterozygote for the silent gene. GP I Barcelona was a fast variant (116%) with an increased isoelectric point (9.55), lability to heat and to urea, and shift of the pH curve towards the acidic pH. The other kinetic characteristics were normal. The ratio of enzymatic activity to immunological reactivity was normal in erythrocytes and white blood cells of the father and the mother but decreased to 75% of normal in blood cells of the daughter. The genetic and molecular mechanisms of GP I deficiency of this patient are discussed.

Adult↗
PubMed1976

Human leukocyte glucose-phosphate-isomerase purification by affinity elution and immunological study.

The authors have purified glucose phosphate isomerase (GPI) from human leukocytes ; they used as starting material leukemic leukocytes obtained from a patient with hyper-leukocytic acute myeloid leukemia ; it was possible to obtain several milligrams of pure enzyme from a single patient. The purification procedure includes a two step precipitation by ammonium sulfate and one column chromatography on a cation exchanger with specific elution by 6 phosphogluconate, a ligand of GPI ; of the two cation exchangers tested, phosphocellulose was found to lead to a better yield than CM-Sephadex. The end product of purification had a specific activity of 855 UI/mg and gave only one band in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Anti GPI monospecific rabbit serum was obtained with purified enzyme. GPI from the various blood cells of ten normal controls was studied immunologically and the ratio of the enzymatic activity to the immunological reactivity was measured ; this ratio (i.e. the molecular specific activity) was lower in granulocytes than in lymphocytes and still more depressed in platelets and hemolysate. The significance of such differences is discussed.

Blood Platelets↗
PubMed1976

Modifications of purified glucose-6-phosphate dehydrogenase and other enzymes by a factor of low molecular weight abundant in some leukemic cells.

Highly purified platelet glucose-6-phosphate dehydrogenase (G6PD; D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) can be modified in its isoelectric point and its molecular specific activity by extracts of some leukemic granulocytes. The "G6PD modifying factors" are relatively small molecules (molecular weight slightly under 5000), thermostable, dialyzable, and ultrafilterable. These molecules are destroyed by various endo- and exopeptidases and by serine enzymes present in crude extracts of leukocytes and commercial preparations of ribonuclease. The alterations of platelet G6PD due to the "G6PD modifying factors" are stable and not reversible by dialysis or further chromatography. The leukemic extracts which are able to modify G6PD also can modify the electrophoretic mobility and (or) the enzymatic activity of purified leukocyte pyruvate kinase, 6-phosphogluconate dehydrogenase, and glucosephosphate isomerase. The chemical nature of such modifications and their relationships with post-translational modifications which occur in leukemic or normal cells are discussed.

Blood Platelets↗