Jean-Claude Dreyfus

Serum enzymes in the physiopathology of muscle.

In 1949 Sibley and Lehninger, in a general study on serum aldolase, reported an increased activity in two patients with progressive muscular dystrophy (PMD). We thought then that an elevation in serum enzymes might represent an interesting basis for the diagnosis and, perhaps, for the search for a biochemical lesion of this disease, and we undertook a systematic survey. We studied in turn : aldolase (1953), phosphohexoisomerase (1954), transaminases (19.55), and lacticodehydrase (1957). We could show that such enzyme determinations undoubtedly justify a clinical interest and allow a description of certain factors involved in prognosis. They also give rise to hypotheses on the physiopathology of PMD.

Posttranslational modifications of enzymes.

Publisher Summary This chapter describes enzyme alterations in some selected systems. The isocitrate lyase from old Turbatrix aceti ( T. aceti ) consists of a mixture of active and inactive molecules. The nematode T. aceti , the eye lens, and red blood cells are the systems that allow recognition of the most frequent and unequivocal postsynthetic changes in enzymes. G6PD shows two types of modifications: (1) a nonproteolytic lowering of the isoelectric pH and (2) a limited proteolysis of the COOH-terminal end, which takes place in the red blood cells. Aldolase undergoes a specific deamidation. Changes in enzyme level and properties can be because of changes in rates of proteolysis. Salivary amylase in humans is present as multiple isozymes, which is derived from a single gene product through posttranscriptional modifications. The lens of the eye is a very peculiar organ in many respects. It is a nonvascularized tissue, receiving nutrients from aqueous and vitreous humors. It is an organ of choice in the study of some aspects of postsynthetic changes in proteins.

Phosphofructokinase (PFK) isozymes in man

SummaryIsozymic heterogeneity of human phosphofructokinase was investigated by means of ATP inhibition, immunoneutralization by antihuman muscle-type and antiliver-type phosphofructokinase antisera, solubility in (NH4)2SO4 solutions, and starch gel and polyacrylamide slab gel electrophoresis. The enzymes studied by these methods were purified from various normal and malignant human adult tissues by chromatography on blue Dextran Sepharose 4 B columns. From the results of these studies we suggest that three basic phosphofructokinase isozymes could exist: muscle-type, fibroblast-type, and liver-type isozymes.Muscle-type isozyme is the single form found in adult muscle, and is involved in the enzymes from heart, brain, red cell, and testis.Fibroblast-type isozyme is found mainly in the placenta, fibroblasts, kidney, and some malignant tissues.Liver-type phosphofructokinase seems to be very definitely the predominant form in mature polymorphonuclear cells, platelets, and liver.Testis and red cell phosphofructokinase enzymes definitely include muscle-type and liver-type subunits, associated in various hybrid forms.

Biochemie der progressiven Muskeldystrophie

SchlußfolgerungIn der biochemischen Aufklärung von Myopathien wurden große Erfolge erzielt und es sind empfindliche und genaue Teste gefunden worden. Man kann die Hoffnung haben, mit Hilfe dieser Teste auch die Krankheitsüberträger zu erkennen. Das Hauptproblem jedoch bleibt das des Krankheitsmechanismus sowie das der Therapie. Die wichtigsten Vorgänge, die man beobachten kann, sind das Verschwinden der Fermente im Sarkoplasma und der Zusammenbruch des Myofibrillensystems. Jedoch bleibt der eigentliche Mechanismus, der diese Reaktionen auslöst, noch unbekannt.

Endogenous, cyclic 3′-2-5′ AMP-dependent phosphorylation of human red cell pyruvate kinase

Abstract ATP-depleted human red cells have been incubated in a glucosecontaining medium with ortho [ 32 P] phosphate in the presence and in the absence of cyclic AMP and dibutyril cyclic AMP. Then pyruvate kinase has been purified to homogeneity. It was found that pyruvate kinase was intensively phosphorylated in the presence of cyclic 3′–5′ AMP and 5 times less in its absence. The phosphorylated site was readily cleaved by subtilisin.

Evidence for structural differences between human glucose-6-phosphate dehydrogenase purified from leukocytes and erythrocytes.

Summary The C-terminal end of pure glucose-6-phosphate dehydrogenase from human leukocytes and red cells has been determined by subjecting these enzymes to partial proteolysis by carboxypeptidase A and B. The C-termini of the leukocyte enzyme were Lysine-Leucine, while Glycine was found as the C-terminal residue of erythrocyte glucose-6-phosphate dehydrogenase. From the results reported in this paper and from data previously reported we may conclude that aging of glucose-6-phosphate dehydrogenase in the red cells is associated with partial proteolysis of the C-terminal end of the enzyme molecules.

Characterization of messenger RNA for aldolase B in adult and fetal human liver

Abstract High molecular weight cellular RNA was isolated from adult and fetal human liver tissue by a procedure of ethanol precipitation in concentrated guanidine-HCl solutions. About 5 mg of RNA were obtained from one gram of liver. RNA was fractionated by sucrose gradient ultracentrifugation. Aldolase B neosynthesized in a reticulocyte lysate cell-free system under the direction of total or fractionated RNA was purified by immunoaffinity microchromatography. Messenger RNA specifying synthesis of aldolase B exhibited a sedimentation coefficient of 16 S both in adult and fetal liver. This enzyme represented 1.3 % of the total neosynthesized proteins in adult liver, 0.1 % in the liver of a 6-month-old fetus and less than 0.01 % in the liver of a 4.5 month-old fetus.

Kinetic properties of human F4 phosphofructokinase: A poor regulatory enzyme

Phosphofructokinase (ATP:D-fructose 6-P-l phosphotransferase, EC 2.7.1 .l l), one of the key enzymes of the glycolytic pathway, is an allosteric enzyme subjected to a complex regulation by various metabolites, i.e., fructose 1,6-P, fl], ATP [2] AMP and ADP [3], citrate f4], NHf [5] and SOi[6]. In addition to the already known muscle type (M-type) and liver type (L-type) forms [7], we have recently proven the existence of a third basic form of phosphofructo~nase, called ~broblast (of F) type enzyme 181. This newly discovered isozyme is predominantly expressed in fibroblasts, platelets, brain, lymphocytes and kidney. The homotetramer F4 has been purified to homogeneity from human platelets f93. The goal of this work was to characterize, on a kinetic point of view, human F4 phosphofructokinase as compared to the other forms, L4 and Mq.

Activation des acides aminés par les microsomes et l'enzyme pH5 de réticulocytes de lapin

Abstract Microsomes from reticulocytes are able to activate aminoacids, as it is established by the measurement of the increase of the amino acid dependent ATP- 32 PP exchanges. The activating properties of the microsomes are compared to those of the pH-5-enzyme from rabbit reticulocytes. They are similar, but not identical, and not additive.

Evidence for a distinct fraction of hemoglobin, specifically associated with the young erythrocytes.

Labelling hemoglobin in vivo with 59Fe or 14C and fractionating on alumina (Schapira et al., 1950, 1955) have given evidence for a metabolic heterogeneity of rabbit hemoglobin. By electrophoresis on starch block, heterogeneity was also observed and has been explained by the “aging” of a fraction of the hemoglobin. The minor anodic fraction was shown to consist of hemoglobin molecules whose electrical charge was modified during the life span of the erythrocytes (Kunkel and Bearn, 1957; Rosa et al., 1960). In the present work, we study the metabolic significance of heterogeneity by the use of ion exchanger Amberlite IRC 50, according to the technic of Allen, Schroeder and Balog (1958). We have been able to characterize, next to the “old” fraction, a “young” hemoglobin.