MULTIPLE GTP-BINDING PROTEINS IN SEA-URCHIN SPERM - EVIDENCE FOR GS AND SMALL G-PROTEINS

Sea urchin sperm plasma membranes isolated from heads and flagella wer e used to examine the presence of Gs (stimulatory guanine nucleotide-b inding regulatory protein) and small G-proteins. Flagellar plasma memb ranes incubated with [P-32]NAD and cholera toxin (CTX) displayed radio labeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [P-32]ADP -ribosylation in conjunction with immunoprecipitation with anti-Gs, fo llowed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertus sis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX w as higher in flagellar than in head membranes. Small G-proteins were i nvestigated by [P-32]GTP-blotting. Both head and flagellar plasma memb ranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [alpha-P-3 2]GTP-binding in a concentration-dependent manner. A monoclonal antibo dy against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protei n that shares characteristics with mammalian Gs and four small G-prote ins, including Res. Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distri bution along the sea urchin sperm plasma membrane. The role of these G -proteins in sea urchin sperm is presently under investigation.