The isolation of acrosome-reaction-inducing glycoproteins from sea urchin egg jelly

Abstract

The solubilized jelly coat of Strongylocentrotus purpuratus eggs was boiled in SDS and mercaptoethanol, precipitated in ethanol, washed in 70% ethanol, lyophilized, and redissolved in seawater. The sample retained its activity to induce the sperm acrosome reaction (AR), showing that the AR inducer was resistant to harsh denaturing conditions. Egg jelly (EJ) was separated into its component macromolecules by denaturing polyacrylamide gel electrophoresis and the gel silver-stained. Several macromolecules ranging from approximately 30 to 380 kDa were consistently observed. These macromolecules were not seen by Coomassie blue staining. With the exception of the fucose sulfate polymer (FSP; 380 kDa) and the 300-kDa component, the other molecules were of minor abundance. Sephacryl-500 gel-filtration chromatography in detergents and disulfide reducing agents separated EJ into three fractions. The FSP and 300-kDa components were purified to homogeneity. A third fraction consisted of components of 30, 82, 116, and 138 kDa. The purified FSP and 300-kDa components had insignificant AR-inducing activity. Substantial AR activity was found only in the 30 to 138-kDa fraction. Ion-exchange chromatography and Centricon filtration separated the 82- and 138-kDa glycoproteins from the other components, but these two components could 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 enter the stacking gel. Of those macromolecules that were visible on gels after silver staining, only the fraction containing the 80- and 138-kDa proteins had significant AR-inducing activity.