Purification and properties of aminoendopeptidase from rat epidermis.

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. Enzymatic activity was exhibited only in the presence of sulfhydryl compounds and 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 Mr = 52,000 +/- 2,300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, but a native form was considered to be Mr = 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 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 degrees C for 30 min but loses about 50% of its activity at 50 degrees C.