Evidence for Multiple, Ras-like, Guanine Nucleotide-binding Proteins in Swiss 3T3 Plasma Membranes. Stimulation of GTPase Activity by Cytosolic Factors

A novel method for the analysis of putative G-proteins has been developed that reveals the existence of a large family of GTP/GDP-binding proteins with similar characteristics to those of p21ras in 3T3 cell plasma membranes. In the presence of Mg2+, exchange of [alpha-32P]GDP with prebound ligand was very slow, but, as with p21ras, exchange was dramatically accelerated by excess EDTA. In the presence of Mg2+, three classes of binding sites were distinguishable. However, no p21ras was detected in the membranes with the pan-reactive anti-ras antibody, Y13-259. Gel filtration analysis resolved two peaks of binding activity centering at 60 and 21 kilodaltons. High resolution anion exchange chromatography separated at least 11 unique GDP-binding proteins from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-solubilized membranes, none of which cross-reacted with a pan-G alpha antiserum. Analogous to p21ras, the binding activities of 9 of the 11 species were sensitive to the thiol reagent N-ethylmaleimide, and six peaks possessed detectable GTPase activity in the absence of extrinsic factors. Addition of cytosol activated the GTPase activity of four of the peaks. We infer that the 11 peaks represent novel, small molecular weight guanine nucleotide-binding proteins, similar to those recently described in brain membranes.