ATTACHMENT OF THE ASCIDIAN SPERM SURFACE EGG RECEPTOR N-ACETYLGLUCOSAMINIDASE TO THE CELL-MEMBRANE

Ascidian sperm bind to vitelline coat N-acetylglucosamine groups of th e egg via sperm surface N-acetylglucosaminidase. This sperm surface eg g receptor remains anchored throughout penetration. Localization to th e sperm surface was verified by biotinylation of intact sperm followed by solubilization in Triton X-100 and binding to streptavidin agarose . The enzyme was determined to be an integral membrane protein as judg ed by resistance to release by KI and high pH. Linkage of the enzyme t o the sperm surface was probed through differential solubilization fol lowed by measuring released enzymatic activity with a fluorogenic subs trate. Nonionic detergents released 90% of the activity. Proteases rel eased about 40%. No activity was released by a phosphatidyl-inositol s pecific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including Triton X-100 and octyl glucoside, argues against a phosphatidyl-inositol linkage. The release form enters the hydrophilic phase of a Triton X-114 phase separation experiment. This observation, coupled with the findings of release by nonionic detergents, suggests that the protein is hydrophilic once rel eased from the membrane. Thus, although clearly an integral membrane p rotein, the enzyme has limited hydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation. (C) 19 94 Wiley-Liss, Inc.