BOAR M-ALPHA-ACROSIN - PURIFICATION AND CHARACTERIZATION OF INITIAL ACTIVE ENZYME RESULTING FROM CONVERSION OF BOAR PROACROSIN TO ACROSIN

  • 1 January 1978
    • journal article
    • research article
    • Vol. 253  (23) , 8428-8432
Abstract
The preparation of highly purified m.alpha.-acrosin (EC 3.4.21.10) is described. Purification was achieved by controlled activation of partially purified proacrosin followed by gel chromatography over Sephadex G-100 at pH 3.0. The final m.alpha.-acrosin preparation resulted in a single protein band with a MW of 49,000 as determined by sodium dodecyl sulfate-disc gel electrophoresis. Disc gel electrophoresis at pH 4.3 resulted in a single benzoyl arginine naphthylamide hydrolyzing band with a relative migration of 0.39. m.alpha.-Acrosin catalyzed the hydrolysis of synthetic substrates containing arginine and lysine, but not phenylalanine. Although Ca+2 was not required for enzymatic activity the addition of CaCl2 stimulated the activity through an increased substrate affinity and an increased maximal velocity. Polyamines stimulated the maximal velocity of the reaction but were without effect on the substrate affinity. m.alpha.-Acrosin was inhibited by lima bean, ovomucoid and seminal plasma proteinase inhibitors. Diisopropyl fluorophosphate and 1-chloro-3-tosylamide-7-amino-L-2-heptanone treatment resulted in an irreversible inhibition while L-arginine, benzamidine and p-aminobenzamidine were competitive inhibitors with respect to substrate. These properties of m.alpha.-acrosin are very similar to those previously reported for m.beta.-acrosin and suggest that the portion of the molecule lost during the conversion of m.alpha.-acrosin to m.beta.-acrosin contributes little to the topography of either the active site or regulatory sites of the enzyme.

This publication has 1 reference indexed in Scilit: