Major pertussis‐toxin‐sensitive GTP‐binding protein of bovine lung

Two GTP‐binding proteins which can be ADP‐ribosylated by islet‐activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (αβγ), but the α subunits were dissociated from the βγ when they were purified in the presence of AlCl 3 , MgCl 2 and NaF. The molecular mass of the α subunit of the major protein (designated G Lu , with βγ) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of α subunits with a limited proteolysis indicated that G Lu α was entirely different from the α of brain G i or G o , while the 41‐kDa polypeptide was identical with the α of bovine brain G i . The kinetics of guanosine 5′‐[3‐ O ‐thio]triphosphate (GTP[γS]) binding to G Lu was similar to that to lung G i but quite different from that to brain G o . On the other hand, incubation of G Lu α at 30°C caused a rapid decrease of GTP[γS] binding, the inactivation curve being similar to that of G o α but different from that of G i α. The α subunits of lung G i and G Lu did not react with the antibodies against the α subunit of bovine brain G o . The antibodies were raised in rabbits against G Lu α and were purified with a G Lu α‐Sepharose column. The purified antibodies reacted not only with G Lu α but also with the 41‐kDa protein and purified brain G i α. However, the antibodies adsorbed with brain G i α reacted only with G Lu α, indicating antisera raised with G Lu α contained antibodies that recognize both G i α and G Lu α, and those specific to G Lu α. These results further indicate that G Lu is different from G i or G o . Anti‐G Lu α antibodies reacted with the 40‐kDa proteins in the membranes of bovine brain and human leukemic (HL‐60) cells. The βγ subunits were also purified from bovine lung. The β subunit was the doublet of 36‐kDa and 35‐kDa polypeptides. The lung βγ could elicit the ADP‐ribosylation of G Lu α by islet‐activating protein, increase the GTP[γS] binding to G Lu and protect the thermal denaturation of G Lu α. The antibodies raised against brain βγ cross‐reacted with lung β but not with lung γ.