Mild proteolytic digestion restores exocytotic activity to N-ethylmaleimide-inactivated cell surface complex from sea...

The Ca2+-stimulated release of cortical vesicle (cortical granule) contents from the cell surface complex (CSC) of the sea urchin egg is an in vitro model for exocytosis. To gain insight into the molecular mechanism of exocytosis we investigated the sensitivity of this model to sulfhydryl modification and proteolytic digestion. Our findings include the following: (a) Proteolytic treatment with trypsin or pronase of CSC prepared from the eggs of Strongylocentrotus purpuratus increased the free Ca2+ concentration required to elicit exocytosis. Although a small increase in the Ca2+ threshold was detected after mild proteolysis, high concentrations of trypsin (0.5 mg/ml) and prolonged incubation (3 h) were required to render the CSC unresponsive to high concentrations of Ca2+ (0.5 mM). Despite the severity of the proteolytic digestions required to inactivate the CSC, the individual cortical vesicles remained intact, as gauged by the latency of ovoperoxidase, a cortical vesicle enzyme. (b) As previously shown (Haggerty, J. C., and R. C. Jackson, 1983, J. Biol. Chem. 258:1819-1825), cortical exocytosis can be blocked by sulfhydryl-modifying reagents such as N-ethylmaleimide (NEM). In this report we demonstrate that NEM inhibits by increasing the Ca2+ threshold required for exocytosis. When CSC that had been completely inactivated by NEM modification was briefly digested, on ice, with a low concentration of trypsin (or several other proteases), exocytotic activity was restored. Although the Ca2+ threshold of the reactivated CSC was slightly higher than that of untreated CSC, it was nearly identical to that of control CSC, which was trypsinized but not treated with NEM. We discuss the significance of these results with regard to the molecular mechanism of exocytosis.