PURIFICATION AND PROPERTIES OF AMINOENDOPEPTIDASE FROM RAT EPIDERMIS

[摘要] An aminoendopeptidase isolated from 2-day-old rat epidermis was purified to apparent homogeneity by the procedures of ammonium sulfate fractionation, DE-52 column chromatography, Sephadex G-200 gel filtration, and CM-52 and DEAE-Sepharose 6B column chromatography. Enyzmatic activity was exhibited only in the presence of sulfhydryl compounds, and was further enhanced by addition of 5 mM EDTA. It was inhibited by p-chloromercuribenzoate, other sulfhydryl blocking reagents, and o-phenanthroline. The monomer form of the enzyme is MW = 52,000 .+-. 2300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was MW 400,000 .+-. 26,000 having an isoelectric point of pH 5.25. Among synthetic substrates, the enzyme hydrolyzed amino acid 2-naphthylamide derivatives and L-leucine amine (L-LeuNH2) most effectively. N-.alpha.-benzoyl-DL-arginine-2-naphthylamide (BANA) was the only endopeptidase substrate for the enzyme, and was a competitive inhibitor for its aminopeptidase activity. Protein substrates have not yet been found. The pH optimum is 7.5, and, in a range of pH 6.5-7.5, it is stable at 37.degree. C for 30 min, but loses .apprx. 50% of its activity at 50.degree. C.