Aeromonas aminopeptidase: Purification and some general properties

Abstract

An aminopeptidase was purified 87-fold from culture filtrates of the marine bacterium Aeromonas proteolytica. The stability of the aminopeptidase at 70 ° permitted the use of a heat treatment to inactivate a proteinase from which the aminopeptidase was otherwise difficult to separate. Purification of the aminopeptidase from heat-treated culture filtrate was accomplished by chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. Moving-boundary electrophoresis, ultracentrifugation, and substrate specificity studies indicated a high degree of homogeneity for the preparations. The enzyme hydrolyzed a number of peptides, amino acid amides, and amino acid β-naphthylamides. It was specific for substrates having a free α-amino group on a residue of the l-configuration; the nature of the amino acid in the N-terminus exerted a considerable effect on hydrolytic rates. The aminopeptidase, as isolated, did not require the addition of activating ions, but was strongly inhibited by ethylenediaminetetraacetate (EDTA). Addition of Zn ++ or Co ++ to the EDTA-inhibited enzyme restored activity to the original level; Mn ++ was partially effective, and Mg ++, Ca ++, and Ni ++ were ineffective. Analysis of several individual preparations revealed the presence of zinc in the aminopeptidase. A molecular weight of 29,500 was calculated from measurements of sedimentation velocity and diffusion.