Distinct biochemical properties of the native members of the G(12) G-protein subfamily - Characterization of G alpha(12) purified from rat brain

G(12) and G(13) are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of Gee,, from rat brain membranes. G alpha(12) was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha(12) bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha(12), it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G(12) subfamily revealed differences in the affinity of G alpha(12) and G alpha(13) for G beta gamma. G alpha(12) required a higher Mg2+ concentration for AlF4--induced dissociation from immobilized G beta gamma than did G alpha(13). In addition, the G(12) subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G(12) to form supramolecular structures in comparison to G(13) and other G-proteins. These G(12) structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha(12). Despite these distinct biochemical characteristics of G(12) and G(13), both purified G-proteins coupled to a recombinant thromboxane A(2) (TXA(2)) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G(12) subfamily, and (2) their specific coupling to TXA(2) receptors.