Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product.

In bovine aortic smooth muscle, about 50% of total GTP-binding activity was present in the cytosol fraction. A major GTP-binding protein (G protein) with a Mr value of about 21,000 (21K G) in this fraction was purified to near homogeneity and characterized. 21K G bound maximally about 0.8 mol of [35S]guanosine 5'-(3-O-thio)triphosphate/mol of protein with a Kd value of about 20 nM. 21K G showed GTPase activity with a turnover number of about 0.007 min-1. 21K G was ADP-ribosylated by botulinum ADP-ribosyltransferase and about 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of 21K G. 21K G and the bovine brain rhoA gene product (rhoA p21) were eluted at the same retention time on C4 reversed-phase high performance liquid chromatography and migrated at the same positions on two-dimensional gel electrophoresis. These results indicate that the major G protein in bovine aortic smooth muscle cytosol is rhoA p21.