Purification and properties of rabbit spermatozoal acrosomal neuraminidase

Abstract

Treatment of rabbit spermatozoa with 50 mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromatography. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 μmol of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2→6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72x10-6M with Cowper's-gland mucin, 1.17x10-5M with fetuin and 8.8x10-4M with sialyl-lactose as substrates. The Vmax. was 0.112μmol/min per mg with the Cowper's-gland mucin, 0.071 μmol/min per mg with fetuin and 0.038 μmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH5.0 on the Cowper's-gland mucin and at pH 4.3 on sialyl-lactose. The enzyme activity was unaffected by 20 mM-Na+ and -K+, but was inhibited by 20 mM-Ca2+, -Mg2+, -Mn2+, -Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at -20°C.