A GTPase-activating protein for the G protein G alpha(z) - Identification, purification, and mechanism of action

A GTPase-activating protein (GAP) specific for G alpha(2) was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. G(z) GAP from bovine brain is a membrane protein that is refractory to solubilization with most deter-gents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of M(r) similar to 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble G alpha(2), the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min(-1) at 15 degrees C, or to greater than or equal to 20 min(-1) at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric G(z) and m2 muscarinic receptor, G(z) GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the K-m of the GAP for G alpha(z)-GTP is 2 nM. Its activity is inhibited by G alpha(z)-guanosine 5'-O-thiotriphosphate (G alpha(z)-GTP gamma S) or by G alpha(z)-GDP/AlF4 with K-i similar to 1.5 nM for both species; Ga alpha(z)-GDP does not inhibit. G protein beta gamma subunits inhibit G(z) GAP activity, apparently by forming a GTP-G alpha(z) beta gamma complex that is a poor CAP substrate. G(z) CAP displays little GAP activity toward G alpha(i1) or G alpha(o), but its activity with G alpha(z) is competitively inhibited by both G alpha(i1) and G alpha(o) at nanomolar concentrations when they are bound to GTP gamma S but not to GDP. Neither phospholipase C-beta 1 (a G(q) GAP) nor several adenylyl cyclase isoforms display G(alpha) GAP activity.