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J Marie

Publications and source records attributed to J Marie.

At least 73 records · Page 4Linked to original sources
PubMed1982

Molecular organization of human L' and L pyruvate kinases.

Extremities, peptide maps and phosphorylatable site localization of human erythrocyte L' and liver L pyruvate kinases (EC 2.7.1.40) were investigated. L' and L subunits seemed to have similar, blocked NH2 termini and differ in their sensitivity to carboxypeptidase A, that is to say in their C-terminal ends. After digestion by Staphylococcus aureus V8 protease, the phosphorylated sites of both L' and L subunits were located on those peptides which were different in L' and L, that is to say on the C-terminal sides. A mild proteolytic attack of the native tetrameric enzymes by trypsin partially degraded the phosphorylatable peptides without removing the phosphoserine residue; in the same conditions, chymotrypsin split off this phosphorylated residue and subtilisin totally degraded the phosphorylated peptides. From these results it appears, therefore, that age-dependent proteolytic degradation of L' subunits in old red cells involves the C-terminal side of the molecules, ultimately resulting in cleavage of the phosphorylated site. Since erythrocyte L' and liver L subunits are encoded by different species of messenger RNAs, our results indicate, in addition, that these messenger RNA species should differ by their 3' coding sequences.

Carboxypeptidases↗
PubMed1981

Research on molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). Use of new protein mapping and immunological techniques.

McArdle's disease is due to the lack of activity of muscle glycogen phosphorylase. We investigated the presence of an inactive protein by two techniques: (a) Bidimensional protein maps, using a modification of the original O'Farrell technique allowing location of phosphorylase. (b) Purification of enzyme from crude muscle extracts, using an immunoaffinity microchromatographic procedure. Protein maps of three patients were obtained. No protein was detected at the normal (97 K) position of phosphorylase but 70 and 60 K spots were visible. Results of enzyme purificaton by immunoaffinity were negative for one patient, whereas a small band of phosphorylase-like material was detected in the other. Our results confirm the molecular heterogeneity of the disease. We think such methods might be useful for investigating other genetic diseases.

Adolescent↗
PubMed1980

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).

Cyclic AMP↗
PubMed1980

Hereditary erythrocyte pyruvate-kinase (PK) deficiency and chronic hemolytic anemia: clinical, genetic and molecular studies in six new Spanish patients.

Clinical, familial and biochemical studies from six unrelated Spanish patients with hereditary hemolytic anemia and erythrocyte pyruvate-kinase (PK) deficiency are described. A remarkable molecular heterogeneity of mutant PK variants involving kinetic properties, molecular stability or electrophoretic mobility is demonstrated. In two patients whose PK variants showed abnormal electrophoretic pattern and strongly aberrant kinetic properties, a chronic hemolytic anemia associated with several other clinical manifestations of chronic hemolysis occurred. In patients whose PK variants showed less abnormal kinetic and electrophoretic characteristics, there was only moderate or mild hemolytic anemia. One patient's PK variant with no obvious kinetic or electrophoretic alterations, showed a markedly decreased heat stability with severe diminution of antigenic concentration of the enzyme. This patient presented a spectacular clinical and hematological improvement after splenectomy. The purpose of the present study is to describe six new PK variants of Spanish origin. In addition, an attempt is made to find relationships between molecular abnormalities of mutant PK variants and the severity of hemolytic anemia, in these patients. The possible role of some kinetic alteration, such as fructose disphosphate (FDP) activation or ATP inhibition of PK variants, in the clinical manifestations of chronic hemolysis is also suggested.

Adult↗
PubMed1980

Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinases. Comparison with human liver L-type enzyme.

Human red cell contain soluble adenosine-3',5'-phosphate-dependent protein kinases, which are able to phosphorylate the L' subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a 'T' allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin-treated L'4 enzyme and accounts for less than 20% of total pyruvate kinase subunits. Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.

Cyclic AMP↗
PubMed1980

Pyruvate kinase hyperactivity genetically determined metabolic consequences and molecular characterization.

Four people from the same family with red cell pyruvate kinase (PK) hyperactivity are described. These people displayed low 2,3-diphosphoglycerate (2,3-DPG) and high adenosine triphosphate (ATP) levels. In vitro metabolism studies of their red cells showed the relationship between the PK activity, low 2,3-DPG, and high ATP levels. The PK electrophoretic pattern of these subjects was abnormal by the presence of several additional bands; one of them migrated like PKM2. PKR from these people was thermounstable and M2-like PK was identical to PKM2 for immunologic reactivity and KO, 5s for phosphoenolpyruvate.

Adenosine Triphosphate↗