Identification of guanine nucleotide binding regulatory proteins in bovine tracheal smooth muscle

The identity of G-proteins in airway smooth muscle is not well elucidated. In the present study, by immunoblotting using AS/7 antibody specific for Gi alpha-1/2, EC/2 antibody specific for Gi alpha-3 and RM/1 antibody specific for Gs alpha, we identified, respectively, Mr 39, 41, 46 and 52 KDa, Mr 41 and 43 KDa, and Mr 43 and 46 KDa polypeptides of conventional (heterotrimeric) G-proteins in purified membranes of bovine tracheal smooth muscle. The identity of the Mr 41, 43 and 52 KDa Gi alpha, and the Mr 43 and 46 Gs alpha was also confirmed by ADP-ribosylation with pertussis and cholera toxins, respectively. In addition, the common antibody (AG/1) for both Gi alpha and Gs alpha revealed the presence of all the above polypeptides, except the Mr 52 KDa band. By nitrocellulose blot overlay with [35S]s alpha GTP gamma S, we also detected seven low molecular weight GTP-binding proteins of Mr 18-30 KDa in the bovine tracheal smooth muscle. Photoaffinity crosslinking of [alpha-32P]GTP demonstrated the presence of high molecular GTP-binding proteins of Mr 55, 75 and 110 KDa. It is concluded that plasma membranes of bovine tracheal smooth muscle contain various types of conventional, low molecular weight and high molecular weight G-proteins. This warrantes further attention to elucidate the functional roles of G-proteins in airway smooth muscle.