A GTPASE-ACTIVATING PROTEIN FOR THE G-PROTEIN G-ALPHA(Z) - IDENTIFICATION, PURIFICATION, AND MECHANISM OF ACTION

Citation
J. Wang et al., A GTPASE-ACTIVATING PROTEIN FOR THE G-PROTEIN G-ALPHA(Z) - IDENTIFICATION, PURIFICATION, AND MECHANISM OF ACTION, The Journal of biological chemistry, 272(9), 1997, pp. 5732-5740
Citations number
48
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
272
Issue
9
Year of publication
1997
Pages
5732 - 5740
Database
ISI
SICI code
0021-9258(1997)272:9<5732:AGPFTG>2.0.ZU;2-W
Abstract
A GTPase-activating protein (GAP) specific for G alpha(2) was identifi ed in brain, spleen, retina, platelet, C6 glioma cells, and several ot her tissues and cells. G(z) GAP from bovine brain is a membrane protei n that is refractory to solubilization with most deter-gents but was s olubilized with warm Triton X-100 and purified up to 50,000-fold. Acti vity 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 e qual to 20 min(-1) at 30 degrees C. It does not alter rates of nucleot ide association or dissociation. When co-reconstituted into phospholip id vesicles with trimeric G(z) and m2 muscarinic receptor, G(z) GAP ac celerates 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 inhib ited by G alpha(z)-guanosine 5'-O-thiotriphosphate (G alpha(z)-GTP gam ma S) or by G alpha(z)-GDP/AlF4 with K-i similar to 1.5 nM for both sp ecies; 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) bet a 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 ad enylyl cyclase isoforms display G(alpha) GAP activity.