THE ISOLATION OF ACROSOME-REACTION-INDUCING GLYCOPROTEINS FROM SEA-URCHIN EGG JELLY

The solubilized jelly coat of Strongylocentrotus purpuratus eggs was b oiled in SDS and mercaptoethanol, precipitated in ethanol, washed in 7 0% ethanol, lyophilized, and redissolved in seawater. The sample retai ned its activity to induce the sperm acrosome reaction (AR), showing t hat the AR inducer was resistant to harsh denaturing conditions. Egg j elly (EJ) was separated into its component macromolecules by denaturin g polyacrylamide gel electrophoresis and the gel silver-stained. Sever al macromolecules ranging from approximately 30 to 380 kDa were consis tently observed. These macromolecules were not seen by Coomassie blue staining. With the exception of the fucose sulfate polymer (FSP; 380 k Da) and the 300-kDa component, the other molecules were of minor abund ance. Sephacryl-500 gel-filtration chromatography in detergents and di sulfide reducing agents separated EJ into three fractions. The FSP and 300-kDa components were purified to homogeneity. A third fraction con sisted of components of 30, 82, 116, and 138 kDa. The purified FSP and 300-kDa components had insignificant AR-inducing activity. Substantia l AR activity was found only in the 30 to 138-kDa fraction. Ion-exchan ge chromatography and Centricon filtration separated the 82- and 138-k Da glycoproteins from the other components, but these two components c ould not be separated from each other. There may be other AR-inducing molecules in sea urchin EJ that were not visible after silver staining of gels. Also, some EJ components are so large that they did not ente r the stacking gel. Of those macromolecules that were visible on gels after silver staining, only the fraction containing the 80- and 138-kD a proteins had significant AR-inducing activity. (C) 1994 Academic Pre ss, Inc.