Attachment of the ascidian sperm surface egg receptor N-acetylglucosaminidase to the cell membrane.
Ascidian sperm bind to vitelline coat N-acetylglucosamine groups of the egg via sperm surface N-acetylglucosaminidase. This sperm surface egg receptor remains anchored throughout penetration. Localization to the 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 judged by resistance to release by Kl and high pH. Linkage of the enzyme to the sperm surface was probed through differential solubilization followed by measuring released enzymatic activity with a fluorogenic substrate. Nonionic detergents released 90% of the activity. Proteases released about 40%. No activity was released by a phosphatidylinositol specific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including Triton X-100 and octylglucoside, 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 released from the membrane. Thus, although clearly an integral membrane protein, the enzyme has limited hydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation.
Downey
J C
JC
Department of Biological Science, California State University, Fullerton 92634.
Lambert
C C
CC
D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003902:Detergents / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D005260:Female; D006031:Glycosylation; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000187:drug effects / Q000201:enzymology*; D010738:Type C Phospholipases / Q000494:pharmacology; D014561:Urochordata / Q000378:metabolism*; D014817:Vitelline Membrane / Q000378:metabolism*
D003902:Detergents; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C035027:egg surface sperm receptor; D010738:Type C Phospholipases; D000118:Acetylglucosaminidase; D010450:Endopeptidases