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J M Robin

Publications and source records attributed to J M Robin.

11 recordsLinked to original sources
PubMed1980

[Neutral calcium dependent proteinase from rabbit skeletal muscle: activity on myofibrillar proteins].

Experiments have been carried out to explore the proteolytic cleavage of rabbit skeletal myofibrils by a calcium dependent neutral proteinase (CaANP). Polyacrylamide gel elctrophoresis on great slabs showed the ability of CaANP to degrade myofibrils more readily than supposed. Besides the hydrolysis of troponin T and the apparition of degradation product of 30,000 molecular weight, the activity of this enzyme is obvious too on some components of the M-line and on heavy subunits of tropomyosin as well as on three unidentified proteic fractions. The variety of the degradation products which appear suggest that the specificity of CaANP is not as selective as presumed. The participation of this proteinase in the postmorten evolution of muscle and its intervention in the turnover of myofibrillar proteins is discussed.

Animals↗
PubMed1979

Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle.

Ca(2+)-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial-Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or alpha(1)-casein were hydrolysed with a maximum rate at 30 degrees C, pH7.5, and with 5mm-CaCl(2), but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, alpha-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry15, 2150-2158]. The Ca(2+)-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca(2+)-activated neutral proteinase.

Animals↗
PubMed1976

[Cathepsin D from horse spleen. I. Purification and study of certain physicochemical properties].

Horse spleen cathepsin D (3.4.23.5.) was purified from crude extract by sodium chloride and ethanol precipitation, column chromatography fractionation on DEAE cellulose and CM Sephadex, re-chromatography on DEAE cellulose and gel filtration. The enzyme has been purified about 3.000 folds with a yield of 30 per cent. The purified enzyme seems to be homogeneous on Sephadex G100, one protein band is apparent on disc electrophoresis. Determined by dansylation the N-terminal amino acid is glycine. A molecular weight of 42,500 +/- 3,000 was obtained with Sephadex G100 gel filtration and light scattering measurements. Amino acid analysis and chemical determinations were performed: cathepsin D is a glycoprotein (2 or 3 osamine residues) including 344 amino acids and 4 disulfide bonds. Spectrophotometric data show that E1cm/1 mg/ml = 1.01 at lambda = 280 nm. ORD measurements indicate about 20 per cent of helicoidal content in the molecule.

Amino Acids↗
PubMed1976

[Cathepsin D from horse spleen. II. Study of certain enzymatic properties].

This work reports some enzymatic properties of highly purified horse spleen cathepsin D. Hydrolysis rate of several proteins are compared. The Kinetic constants (Km = 4.95 10(-5) M and Vm = 1,76 delta DO/mn/mug) have been determined in the presence of a denatured haemoglobin substrate. Stability of the enzymatic preparation is discussed according to the pH, concentration and time of storage. Some investigations concerning the active site are described. Enzymatic and chemical results show that dicarboxylic and tryptophanyl residues seem to be involved in the hydrolytic process. Catalysis does not depend on sulfhydryl or seryl residues. Different salts, particularly nitrate, nitrite and polyphosphate are potent inhibitors of enzymatic activity.

Animals↗