Partial purification and characterization of sperm receptor hydrolase, a cortical granule proteoesterase, from eggs of the sea urchin Strongylocentrotus purpuratus

Abstract

AbstractSperm receptor hydrolase, one of two classes of cortical granule proteoesterases (E.C.3.4.4.4) was purified approximately 30‐fold with 80% yield from Strongylocentrotus purpuratus cortical granule exudate. Sperm receptor hydrolase preparations were free of vitelline delaminase activity (the other class of cortical granule proteoesterase) and had less than 1% of the starting levels of cortical granule peroxidase and β‐1,3‐glucanohydro‐lase activities. Native polyacrylamide gel electrophoresis coupled with a protease activity stain showed that three proteases were present in the most highly purified preparations of sperm receptor hydrolase. Each of the three proteases has the same molecular weight of 60,000, but different isoelectric points of 2.4, 3.8, and 5.5. The Km value of the mixture of proteases for α‐N‐benzoyl‐L‐arginine ethyl ester as substrate was 263 μM at pH 8.4 and 30°C; the pH dependence of Vm showed a single prototrophic group with a pK of 6.7 and an enthalpy of ionization of 8.6 kcal‐mol−1. The values of these kinetic parameters are consistent with an enzyme‐active site containing histidine. Phenylmethyl sulfonyl fluoride, tosyl lysine chloromethyl ketone, several proteinaceous trypsin inhibitors, and p‐aminobenzamidine inhibited the esterase activity of the proteases. These data suggest that sperm receptor hydrolases are serine proteases.