Purification and immunochemical characterization of the major pertussis-toxin-sensitive guanine-nucleotide-binding protein of bovine-neutrophil membranes.
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 alpha n, was purified to near homogeneity from this fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified alpha n was shown to interact with beta gamma subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified alpha n on SDS/polyacrylamide gels was intermediate between those of the alpha subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the alpha subunit of transducin (Gt). Several polyclonal antisera against the alpha subunits of bovine Gt and Go did not react with alpha n on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine alpha i, reacted only minimally with alpha n. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated Gn, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.
D000246:Adenosine Diphosphate Ribose / Q000097:blood; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000097:blood* / Q000302:isolation & purification; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000097:blood; D007124:Immunoenzyme Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D009504:Neutrophils / Q000378:metabolism*; D037342:Pertussis Toxin; D013873:Thionucleotides / Q000097:blood; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D046911:Macromolecular Substances; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins