Georges Schapira

BIOCHEMICAL STUDY OF MUSCLE IN PROGRESSIVE MUSCULAR DYSTROPHY

Current concepts of progressive muscular dystrophy hold that it is a disease in which, through genetic error, there are intrinsic defects in muscle metabolism, although the nature of these hypothetical defects remains unrecognized. It is the purpose of this report to describe one approach to the problem and to show that there are defects in the metabolism of isolated muscle from patients with progressive muscular dystrophy.

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.

Eukaryotic ribosomal proteins. Two-dimensional electrophoretic studies

Although some of the ribosomal proteins are believed to be different from species to species, they are thought to be identical within any one species [l-6]. It has also been reported that ribosomzs, even from the same tissue,-are heterogeneous in their protein content and/or function [7-131. As for the number of proteins in an eukaryotic ribosome, it has been estimated to be similar [ 141 or higher [ 15 ] than in prokaryotic ribosomes. Evidence is reported below which indicates, as expected, that ribosomes from two species differ in some of their protein components. But unexpectedly, some differences are also found in two different organs in the same species. Furthermore eukaryotic ribosomal proteins are shown to be more numerous than prokaryotic ones.

Presence of a new RNA species among the initiation protein factors active in eukaryotes translation

We have isolated a RNA of very small size from 0.5 M KCl wash prepared from rabbit reticulocytes in absence of magnesium. Some physico-chemical properties have been studied: it is single stranded, its base composition in p. 100 is 33 U, 7 G, 46 A and 14 C. The M.W. is approximately 11,000. This RNA is necessary for the biological activity of the dialyzed KCl-wash. It is probably a component either of an initiation factor (IF E3) or of an interference factor. This RNA is required for the hemoglobin chain synthesis in a reticulocyte cell-free system.

An improved technique for preparation of skeletal muscle cell plasma membrane

Abstract 1. 1. An improved technique of plasma membrane isolation from rat skeletal muscle is presented. The procedure is based on the cautious disruption of the muscle fibers, elimination of actomyosin by salt solutions and separation of sarcolemma from organelles by differential centrifugation. This is followed by a centrifugation on a discontinuous sucrose density gradient. The plasma membranes are yielded in the form of vesicles. They are characterized and their purity is assessed by electron microscopy, chemical and biochemical tests. 2. 2. The fraction of pure plasma membrane is found to have a high specific activity in plasma membrane markers: 5′-nucleotidase, (Na+,K+)-stimulated Mg2+-dependent ouabain-sensitive ATPase and a high molar ratio of cholesterol to phospholipids; these markers have lower or negligible specific activities in the other fractions. The enzymatic tests performed to determine the cross-contamination by cell organelles are: succinate dehydrogenase and NADH:cytochrome c- reductase rotenone insensitive, for mitochondria and outer-mitochondrial membrane; NADPH: cytochrome c- reductase for sarcoplasmic reticulum; IDP phosphohydrolase for Golgi apparatus: acid phosphatase for lysosomes; and tentatively lactate-dehydrogenase for cytoplasm. These activities are poorly represented or absent in the plasma membrane fraction.

ABNORMALITIES OF MUSCLE PROTEIN METABOLISM IN MICE WITH MUSCULAR DYSTROPHY

The discovery of a myopathic mutation in a colony of strain 129 mice (1, 2) provides an opportunity for the study of possible abnormalities in the metabolism of muscle proteins with the use of labeled amino acids. Cell proteins miiay show various kinds of behavior. Some of them are constantly being renewed, either secreted, like digestive enzymes or serum albumin from the liver, or subjected to a molecular turnover, as in the case of muscle aldolase and probably all the water-extractable muscle proteins. The other proteins apparently stay unchanged during the life span of the cell, as in the case of hemoglobin in red blood cells (3), of collagen in the rat (4), or of myosin in the myofibril, a subcellular unit (5. 6). We have previously shown in the rat that myosin has a very slow renewal and that its metabolic behavior permits the measurement of the life span of the myofibril, which is close to 30 days in the rat. If, after the injection of a radioactive amino acid, the radioactivity of the protein is plotted against time, the first kind of protein shows an exponential decline, but hemoglobin and m-vosin show a plateau followed by a rapid decline. These two kinds of curves are combined if the protein is slowly renewed and belongs to a cell or part of a cell with a relatively short life span. In this work we have compared the metabolism of myosin and water-extractable muscle proteins in dystrophic mice, in their normal litter mates, and in another normal strain of mice. Wehave not prepared pure aldolase, which would have required too large a number of animals, but we

THE VALUE OF SERUM ENZYME DETERMINATIONS IN THE IDENTIFICATION OF DYSTROPHIC CARRIERS

The increase in serum enzymes is presently the major biochemical symptom in progressive muscular dystrophy (PMD) . After its first being described in two cases (Sibley & Lehninger, 1949) and after the first systematic study (G. Schapira, Dreyfus & F. Schapira, 1953), a controversy arose (Rowlands, 1956) about its value. But negative serum-enzyme determinations were obtained only in older patients in the last evolutive phase of the disease, and overwhelming evidence was presented by numerous workers of the value of an increase in serum enzymes in PMD. This evidence was definitely confirmed at the Conference on Enzymes in Blood held by the New York Academy of Sciences in 1958 (Dreyfus, F. Schapira & G. Schapira, 1958). Soon afterwards a new problem arose: is it possible to detect an apparently healthy carrier using serum enzyme determinations? The enzymes available in 1958, mainly aldolase, were not sensitive enough consistently to detect carriers (Soltan & Blanchaer, 1959). The introduction in 1959 of creatine-kinase (CPK) by Ebashi et al. allowed the use of a much more specific and sensitive enzyme. Ebashi’s technique was still relatively crude. The first technical stride toward detecting the healthy carriers was made by Dreyfus and Schapira, 1961. The first positive reports came from two groups in 1960. Chung et al., using aldolase and transaminase, reported three increases out of 21 mothers of PMD. Our group (Dreyfus et al., 1960; F. Schapira et al., 1960), using both aldolase and creatine-kinase, was able to detect 30 per cent of the known carriers, e.g. 8 out of 53 mothers. On the other hand, Okinaka et al. (1961) found increases in relatives of patients but not in their mothers. Monckton et al. (1963) and Brugsch et aJ. (1960) reported some positive results with aldolase, where Leyburn et al. had been unable to get positive results with this enzyme in 1941. But soon afterwards, the development of better methods of CPK estimation, either spectrophotometric (using a modification of the technique described in 1959 by Tanzer & Gilvarg or colorimetric (Hughes, 1962) led to greater accuracy and reproducibility. Many reports, coming from several countries, have now confirmed these facts, and it is generally admitted that twothirds or three-quarters of the carriers demonstrate a detectable increase in their serum enzyme level. Techniques

The ambiguities in the rabbit hemoglobin: Evidence for a messenger RNA translated specifically into hemoglobin°☆

Abstract Fractional residues of amino acids present in the αTIV peptide from the α chain of rabbit hemoglobin cannot be explained by an ambiguous translation of a unique template; they are the consequence of the translation of at least two different messengers RNA. Fractional residues of amino acids are present in several positions of the α chain of rabbit hemoglobin; the amino acid sequence of the rabbit α chain is not unique, as suggested by non integral values of amino acid residues and demonstrated by sequential degradation (Weisblum et al 1965, Von Ehrenstein 1966). The question arises whether the amino acid multiplicities are due to the ambiguous translation of a unique template, or to a multiplicity of α chain templates. In order to answer this question, we have investigated the role of amino acyl tRNA, activating enzymes, and mRNA, in the ambiguity of the peptide αTIV of rabbit hemoglobin. The experimental evidence demonstrates the role of the template.

Activation de la synthèse acellulaire de l'hémoglobine par l'acide ribonucléique II. Action de fractions f'acide ribonucléique de réticulocytes obtenues par centrifugation en gradient de saccharose

Abstract RNA from rabbit-liver nuclei was fractionated by centrifugation in a sucrose gradient and four fractions were obtained. One of these fractions, which sedimented between 4 S and 16 S, stimulated the biosynthesis of hemoglobin and the incorporation of amino acids into the ribosomal proteins in a cell-free system of rabbit reticulocytes. RNA III obtained from a growing rabbit was much more active than RNA III prepared from either a fetal or a newborn or an adult rabbit. RNA III from ribosomes and from cytoplasm were inactive. Chloramphenicol prevents the stimulation of hemoglobin synthesis by RNA III, but is inactive on the control system. The stimulation therefore definitely concerns hemoglobin biosynthesis as has been shown by comparing the rate of incorporation of three different amino acids in the presence and absence of active RNA III.

Protein kinase and adenylate cyclase of erythrocyte membrane from patients with Duchenne muscular dystrophy.

In freshly prepared erythrocyte membranes from normal individuals and from patients with Duchenne progressive muscular dystrophy the endogenous protein kinase and the cAMP stimulated phosphorylation was identical for the three main 32P proteins including spectrin (protein band II). Another enzyme, adenylate cyclase, was found unchanged. Altered protein kinase and adenylate cyclase has been reported in this disorder. We have no explanation for these discrepancies.