Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.
Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.
Hinsch
K D
KD
Zentrum für Dermatologie und Andrologie, Justus-Liebig-Universität Giessen, Germany.
Hinsch
E
E
Aumüller
G
G
Tychowiecka
I
I
Schultz
G
G
Schill
W B
WB
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000032:analysis*; D015151:Immunoblotting; D046911:Macromolecular Substances; D008297:Male; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D013082:Sperm Tail / Q000737:chemistry* / Q000648:ultrastructure; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins