1 Reproduction Research Laboratories, Department ofBiochemistry, University ofGeorgia, Athens, GA 30602, U.S.A.
2Department of Biochemistry
3Division of Basic Health Science, Emory University, Atlanta, GA 30322, U.S.A.
Tóm tắt
Treatment of rabbit spermatozoa with 50mM-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 chromato. 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 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6′-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/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 pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.
