Boar sperm aminopeptidase: Purification and partial characterization

Abstract

An aminopeptidase has been isolated by detergent treatment of boar spermatozoa at pH 7.25 after the spermatozoa were washed through 1 M sucrose containing 50 m M benzamidine. The enzyme has been purified and partially characterized. The purification scheme consisted of ammonium sulphate fractionation, Sephadex G-200 gel filtration and affinity chromatography on thiopropyl-Sepharose 6B. The final preparation is highly purified as judged by sodium dodecyl sulphate (SDS) disc gel electrophoresis. The enzyme shows a molecular weight of 126 000 by gel filtration on Sephadex G-200, having two subunits as revealed by SDS disc gel electrophoresis. The enzymatic activity was enhanced by dithiothreitol and was not inhibited by N-α-p- tosyl- l- lysine chloromethyl ketone (TLCK) or l-1-tosylamide-2-phenyl ethyl chloromethyl ketone (TPCK), indicating that the enzyme is a thiopeptidase having no serine involved in enzymatic catalysis. Boar sperm aminopeptidase is a non-specific exopeptidase as it hydrolyses acidic, basic and neutral amino acid-β-naphthylamides, showing the highest rate of hydrolyses with valine-β-naphthylamide. It is a glycoprotein as it binds tightly though reversibly to concanavalin-A-Sepharose. The enzyme also binds to liposomes made up of aqueous dispersions of synthetic phospholipids in the form of anionic vesicles. The binding is 19%, 32% and 74% with liposomes containing 100% phosphatidyl choline, 70% phosphatidyl choline +30% phosphatidyl serine and 50% phosphatidyl choline + 50% phosphatidyl glycerol, respectively. Since the binding of enzyme to liposomes is dissociated (68%) with 0.5 M sodium chloride, it is, therefore, possibly the result of ionic interaction with the membranes.