A neutral proteinase of monkey liver microsomes. Solubilization, partial purification, and properties.
Conditions for the solubilization of membrane-bound neutral proteinase associated with monkey liver microsomes were investigated. Among the reagents tested, deoxycholate, cholate, and some nonionic detergents, including Triton X-100, with hydrophilic-lipophilic balance values of around 13, were effective. The solubilization profile indicated that the enzyme is bound to the microsomal membranes by strong hydrophobic interaction. The enzyme was partially purified from monkey liver microsomal fraction, previously washed with 1 M KCl and 0.05% sodium dodecyl sulfate, by Triton X-100 extraction, followed by chromatography on columns of hydroxylapatite and Sepharose CL-6B. The apparent molecular weight of the enzyme was estimated to be about 88,000 from the elution position on Sepharose CL-6B column chromatography in the presence of 0.5% sodium cholate. It was optimally active at pH 8.0 with heat-denatured casein as a substrate. It was strongly inhibited by diisopropyl phosphorofluoridate and phenylmethanesulfonyl fluoride, indicating that the enzyme is a serine proteinase. EDTA, EGTA, and chymostatin also inhibited the enzyme strongly. Among urea-denatured protein substrates tested, calf thymus histone was hydrolyzed most rapidly, followed by casein, hemoglobin, and bovine serum albumin, whereas practically no hydrolysis occurred with denatured ovalbumin, fibrinogen, and gamma-globulin as substrates.
D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D003902:Detergents / Q000494:pharmacology; D000882:Haplorhini; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008251:Macaca; D008862:Microsomes, Liver / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
D003902:Detergents; D010447:Peptide Hydrolases; D000626:Aminopeptidases