Purification and Characterization of an Aminopeptidase, the Enzyme-Hydrolyzing Alanine-p-nitroanilide (APAase), from Euonymus Leaves

An aminopeptidase, the enzyme-hydrolyzing alanine-p-nitroanilide(APAase), from leaves of Euonymus alatus f. ciliato-dentatuswas purified about 1,400-fold by a combination of ion-exchangeand gel filtration column chromatographies. Polyacrylamide gelelectrophoresis showed the purified APAase to be homogenous.The molecular weight of this APAase was estimated to be about100,000, and the optimum pH for its hydrolytic activity againstalanine-p-nitroanilide (APA) was 8.6–9.0. APAase hydrolyzedalanine-ß-naphthylamide (alanine-NA), glycine-NA,lysine-NA and arginine-NA. It was inhibited slightly by p-chloromercuribenzoate(PCMB) and iodoacetic acid and was not activated by thiol reagents.Therefore, a sulfhydryl group could not be present at the activesite of APAase. APAase was inhibited strongly by 1,10-phenanthroline,but was unaffected by EDTA. Of the metal ions tested, Hg²⁺,Zn²⁺ and Mn²⁺ strongly inhibited its activity, and Ca²⁺ stimulatedit to some extent. (Received November 15, 1984; Accepted March 12, 1985)