Purification and immunochemical characterization of the major pertussis‐toxin‐sensitive guanine‐nucleotide‐binding protein of bovine‐neutrophil membranes

Abstract

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as α_n, was purified to near homogeneity from this fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified α_n was shown to interact with β_γ subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified α_n on SDS/polyacrylamide gels was intermediate between those of the α subunits of G_i and G_o, purified from bovine brain, and slightly lower than the mobility of the α subunit of transducin (G_t). Several polyclonal antisera against the α subunits of bovine G_t and G_o did not react with α_n on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine α_i, reacted only minimally with α_n. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated G_n, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.