G-proteins in rat blood vessels-I. identification

1. 1. The purpose of this investigation was to identify various types of conventional, low and high molecular weight G-proteins in purified membranes of rat aorta and mesenteric artery. 2. 2. In both blood vessels, immunoblotting of G-proteins using AS/7 antibody specific for G_{i- 1 2}, EC/2 antibody specific for Gi-3 and RM/1 antibody specific for Gs-type conventional G-proteins demonstrated the presence of M_r 28-43 kDa, M_r 41 and 48 kDa, and M_r 36-46 kDa polypeptides, respectively. Immunoblotting using a common antibody (GA/1) for the Gs and Gi α-subunits also revealed the existence of multiple polypeptides (M_r 24-52 kDa) in both aorta and mesenteric artery, some of which were identified by the specific antibodies. The intensity and number of bands detected by most of the antibodies were greater in mesenteric artery than in aorta. 3. 3. Cholera toxin (CT) catalyzed ADP-ribosylation of two G_sα (M_r 43, 46 kDa) in both types of blood vessels with similar intensities of bands. Pertussis toxin (PT), on the other hand, catalyzed ADP-ribosylation of one G_iα (M_r 41 kDa) polypeptide, and the intensity of this band was greater in aorta than in mesenteric artery. The polypeptides ADP-ribosylated by the toxins corresponded with their identification by antibodies. 4. 4. Nitrocellulose blot overlay with [³⁵S]GTPγS identified at least seven low molecular weight G-proteins (M_r 21-30 kDa) in both aorta and mesenteric artery, with greater intensity of bands in mesenteric artery. 5. 5. Photoaffinity labeling with [α-³²P]GTP detected at least nine types of GTP-binding proteins (M_r 18-110 kDa), including low, conventional and high molecular weight proteins in both blood vessels, and some of these were recognized by the other methods. 6. 6. Our data suggest that purified membranes of rat aorta and mesenteric artery contain various types of heterotrimeric, low and high molecular weight G-proteins some of which appear to vary with the type of blood vessel.