Selective release and purification of two periplasmic Alteromonas B-207 aminopeptidases

Abstract

Two periplasmic aminopeptidases were selectively released from Alteromonas B-207 when its outer membrane and peptidoglycan layer were systematically removed. Neither enzyme was detected in cytoplasmic materials. The first enzyme (aminopeptidase II) was isolated and purified 160-fold from the supernatant of osmotically shocked cells. The second enzyme (aminopeptidase I) was obtained from the peptidoglycan fraction of lysozymetreated mureinoplasts and purified 15-fold. The two enzymes are distinguished by molecular weights, subunit structures, temperature profiles, pH optima, effects of EDTA and substrate specificities. Aminopeptidase I has a molecular weight of approx. 450 000 and appears to be a tetramer; while aminopeptidase II is a stable monomer with a molecular weight of 81 000–88 000. Aminopeptidase II has higher pH and temperature optima than does aminopeptidase I. Aminopeptidase II has broader peptide specificity than aminopeptidase I. This is particularly evident when amino acid β-naphthylamides are used as substrates. Aminopeptidase I shows its greatest activity against l-α-Asp-β-naphthylamide and l-Ala-β-naphthylamide, whereas aminopeptidase II shows a decided preference for l-Leu-β-naphthylamide. Aminopeptidase I appears to be more sensitive to EDTA than aminopeptidase II, but both enzymes apparently require Zn²⁺.