Occurrence and some properties of membrane-bound neutral proteinase in the microsomal fraction of rat skeletal muscle.
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 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.
D000818:Animals; D002793:Cholic Acids; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D011480:Protease Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013379:Substrate Specificity
D002793:Cholic Acids; D011480:Protease Inhibitors; D010450:Endopeptidases