Abstract
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 1M KCl-washed microsomal fraction by 1% sodium cholate containing 0.15M NaCl, and partially purified by chromatography on a column of Sepharose CL-6 B in the presence of 0.5% sodium cholate and 0.1M 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, Nα-tosyl-L-phenylalanine chloromethyl ketone, Nα-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 protamine, hemoglobin, and casein.
