SEX-DEPENDENT CARBOXYLESTERASE EXPRESSION IN THE REPRODUCTIVE-SYSTEM OF BIVALVE MOLLUSKS - AN APPROACH TO SUBSTRATE-SPECIFIC DETECTION OF MALE ASSOCIATED POLYPEPTIDE (MAP) AFTER SDS-ELECTROPHORETIC SEPARATION OF CRUDE GONAD EXTRACTS

We showed that male-associated polypeptide (MAP) (Mikhailov et al.,199 5) identified in the gonad (mantle) tissue of Mytilus galloprovinciali s is characterized by immunochemical similarity with the protein of ej aculatory bulb (esterase S) (Korochkin et al.,1995) of Drosophila viri lis, belonging to the carboxylesterase family. Accordingly, we address ed the question concerning the carboxylesterase activity of MAP and de vised an approach to substrate-specific in situ detection of mussel go nad carboxylesterase after SDS-PAGE. In this regard, we demonstrated t hat: (1) urea and beta-mercaptoethanol should be omitted from the buff er using for SDS-PAGE gonad sample preparation; (2) denaturation of go nad polypeptides may be achieved by SDS pre-treatment of the samples a t 20-37 degrees C for 5-30 min; (3) Triton X 100 and/or Tween 20, but not urea, can be used to remove SDS from gels after SDS-PAGE of gonad extracts; and (4) buffer conditions of substrate-specific staining sho uld be evaluated carefully. Using the optimized protocol, we could rec over the carboxylesterase activity after SDS-PAGE of mussel gonad extr acts. In male gonads substrate-stained bands were located in gels at t he position typical for MAP. Moreover, immunoblot analysis of the same samples showed that these bands contain MAP. In female gonads no or v ery weak MAP immunoreactivity was found. In addition, we could not det ect the carboxylesterase activity in MAP containing fractions of femal e gonads separated by SDS-PAGE. Taken together, the results indicate t hat MAP of M. galloprovincialis is a new member of male-predominant ca rboxylesterase family, and strongly suggest that mussel gonad MAP is s tructurally and functionally related to esterase S of the Drosophila e jaculatory bulb.