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
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.