Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods.
The abalone sperm adenylate cyclase does not appear to be regulated by guanine nucleotides, but has a Mg2+-supported catalytic activity similar to other hormone- and guanine nucleotide-regulated enzymes (Kopf, G. S., and Vacquier, V. D. (1984) J. Biol. Chem. 259, 7590-7596; Kopf, G. S., and Vacquier, V. D. (1985) Biol. Reprod. 33, 1094-1104). The present studies were undertaken to ascertain whether the abalone enzyme has associated guanine nucleotide-binding regulatory proteins. Membrane fractions were incubated with either islet-activating protein (IAP) or cholera toxin and analyzed by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis for the presence of toxin-catalyzed ADP-ribosylated proteins. The supernatant from a Lubrol PX-extracted 48,000 X g pellet fraction contained a Mr = 41,000 IAP substrate. This substrate could not be ADP-ribosylated prior to detergent extraction. Lubrol PX-solubilized fractions of membrane preparations from mouse, bovine, and human sperm also contained a Mr = 41,000 IAP substrate. These proteins co-migrated on sodium dodecyl sulfate-polyacrylamide gels with the Mr = 41,000 alpha i-subunit of the inhibitory guanine nucleotide-binding regulatory protein (Gi) from transformed chicken embryo fibroblast and mouse S-49 lymphoma membrane extracts. The sperm IAP substrates displayed similar protease digest patterns to alpha i of mouse S-49 lymphoma cells. Sea urchin sperm analyzed in a similar manner contained a Mr = 39,000 IAP substrate. Cholera toxin-catalyzed ADP-ribosylation of specific sperm membrane proteins was not observed in any of the sperm preparations tested. The presence of the beta-subunit common to both the stimulatory and inhibitory guanine nucleotide-binding regulatory heterotrimers was confirmed in sperm using an antiserum directed against the purified beta-subunit of the guanine nucleotide-binding regulatory proteins from bovine brain. It is concluded that all of the sperm tested, with the possible exception of sea urchin sperm, contain a Gi-like protein. Additional properties of these proteins and their role(s) in sperm function are currently being examined.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D037361:Adenylate Cyclase Toxin; D000818:Animals; D002417:Cattle; D002772:Cholera Toxin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D008223:Lymphoma / Q000032:analysis; D008297:Male; D051379:Mice; D008970:Molecular Weight; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D037342:Pertussis Toxin; D012617:Sea Urchins; D013094:Spermatozoa / Q000032:analysis*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D007106:Immune Sera; D009702:Nucleoside Diphosphate Sugars; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins