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J. Biochem, 1982, Vol. 91, No. 1 87-95
© 1982 Japanese Biochemical Society

research-article

Occurrence and Some Properties of Membrane-Bound Neutral Proteinase in the Microsomal Fraction of Rat Skeletal Muscle1

Yoshikazu ICHIHARA, Kazuhiro SOGAWA and Kenji TAKAHASHI

Department of Biochemistry, Primate Research Institute, Kyoto University Inuyama, Aichi 484

Membrane-bound neutral proteinase was found in the microsomal fraction of rat skeletal muscle as assayed with heat-denatured casein as a substrate. The enzyme was solubilized from 1 M KC1-washed microsomal fraction by 1% sodium cholate containing 0.1 M NaCl, and partially purified by chromatography on a column of Sepharose CL-6B in the presence of 0.5% sodium cholate and 0.1 M NaCl. The enzyme was eluted from the Sepharose column as a single but rather broad peak at a position corresponding to a molecular weight of about 190,000. The pH optimum for hydrolysis of heat-denatured casein was about 8.0. It was inhibited to significant extents by various reagents including diisopropyl phosphorofluoridate, phenylmethanesulfonyl fluoride, N{alpha}-tosyl-L-phenylalanine chloromethyl ketone, N{alpha}-tosyl-L-lysine chloromethyl ketone, p-chloromercuriphenyl sulfonate, chymostatin, EDTA, EGTA, and O-phenanthroline. This inhibition profile suggests that the present muscle proteinase is a mixture of proteinases, such as a serine proteinase and a metallo-proteinase similar to those occurring in the microsomal membranes of liver and kidney (or small intestine), respectively. Among urea-denatured proteins tested as substrates, calf thymus histone was hydrolyzed most rapidly, followed by prota mine, hemoglobin, and casein.

1 This study was supported in part by grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare, Japan, and the Toray Science Foundation. A preliminary account of this work has appeared elsewhere (1).


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