Immunological identification of G protein α- and β-subunits in tail membranes of bovine spermatozoa

Abstract

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 G13 and detecting all known pertussis toxin-sensitive α- subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against α-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the β2-subunit of G proteins and an antiserum reacting with both β1- and β2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa β1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein α-subunits nor G protein β-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 α-subunits, whereas the putative β-subunit could be identified as a β2-subunit.