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Multiple GTP‐binding proteins in sea urchin sperm: Evidence for Gs and small G‐proteins

1995; Wiley; Volume: 37; Issue: 2 Linguagem: Inglês

10.1046/j.1440-169x.1995.t01-1-00006.x

1440-169X

Patricia Cuéllar‐Mata, Guadalupe Martı́nez-Cadena, Laura E. Castellano, Gustavo Aldana‐Veloz, Irma Vargas, Alberto Darszon, Jesús Garcı́a-Soto,

Neuroscience and Neuropharmacology Research

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide‐binding regulatory protein) and small G‐proteins. Flagellar plasma membranes incubated with [ 32 P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX‐catalyzed [ 32 P]ADP‐ribosylation in conjunction with immunoprecipitation with anti‐Gs, followed 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 pertussis toxin (PTX) ADP‐ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP‐ribosylation by PTX was higher in flagellar than in head membranes. Small G‐proteins were investigated by [ 32 P]GTP‐blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α‐ 32 P]GTP‐binding in a concentration‐dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G‐proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G‐proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G‐proteins in sea urchin sperm is presently under investigation.