Further characterization of the mouse sperm surface zona-binding site with galactosyltransferase activity.
One of the mouse sperm surface binding sites for zona pellucida ligands exhibits galactosyltransferase (GT) enzyme activity. The present study was undertaken to ascertain whether the GT site behaves as a noncatalytic binding site in its physiological capacity, with no glycosylation of zona ligands, or whether glycosylation of zona ligands is an integral part of sperm-zona binding. The effects of Mn2+, the obligatory cation for GT catalysis, on enzyme activity and sperm-zona binding were examined. With uridine-5'-diphosphogalactose (UDPgal) as galactose donor, and N-acetylglucosamine (GlcNAc) as galactose acceptor, increasing concentrations of Mn2+ in the range of 0.1-10 mM increased GT enzyme activity, with half-maximal activation at 0.65 mM Mn2+ (Vmax = 20 pmol/hr/10(6) cells). In the presence of 0-2 mM Mn2+, sperm-zona binding was inhibited in a concentration-dependent manner; 50% inhibition occurred at 1.25 mM Mn2+. At this concentration, GT enzyme activity was at 65% Vmax. To determine the specificity of the GT site for glycoprotein terminal carbohydrate residues, spermatozoa were incubated with, asialo-ovine submaxillary mucin (N-acetylgalactosamine residues), asialo-, -alpha 1-acid glycoprotein (beta 1-4 galactose residues) ovalbumin (Ov; GlcNAc residues), and asialo-agalacto-/alpha 1-acid glycoprotein (AsAgAGP; GlcN-Ac residues). Only Ov and AsAgAGP acted as acceptors for galactose in the enzyme assay and inhibitors in the sperm-zona binding assay. The kinetics of the interaction of AsAgAGP with the GT site were determined: the Km was 3.6 mg/ml, with Vmax of 33 pmol/hr/10(6) cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Benau
D A
DA
Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia 19104-6080.
McGuire
E J
EJ
Storey
B T
BT
D000818:Animals; D001212:Asialoglycoproteins / Q000302:isolation & purification; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D010063:Ovum / Q000502:physiology*; D013084:Sperm-Ovum Interactions / Q000187:drug effects; D013094:Spermatozoa / Q000201:enzymology* / Q000502:physiology; D015044:Zona Pellucida / Q000502:physiology*
D001212:Asialoglycoproteins; D008345:Manganese; D007811:Lanthanum; D005700:Galactosyltransferases