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-insen sitive G-proteins. Here, we describe the isolation of Gee,, from rat b rain membranes. G alpha(12) was purified to apparent homogeneity by th ree steps of conventional chromatography, followed by two cycles of su bunit-exchange chromatography on immobilized G subunits. Purified G al pha(12) bound guanosine 5'-[gamma-thio]triphosphate slowly and substoi chiometrically. For isolation of functionally active G alpha(12), it w as mandatory to use sucrose monolaurate as a detergent. Comparative st udies of both rat-brain-derived members of the G(12) subfamily reveale d differences in the affinity of G alpha(12) and G alpha(13) for G bet a gamma. G alpha(12) required a higher Mg2+ concentration for AlF4--in duced dissociation from immobilized G beta gamma than did G alpha(13). In addition, the G(12) subfamily members differed in their sedimentat ion velocities, as determined by sucrose-density-gradient centrifugati on. 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 mo nolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha(12). Despite these dis tinct biochemical characteristics of G(12) and G(13), both purified G- proteins coupled to a recombinant thromboxane A(2) (TXA(2)) receptor r econstituted into phospholipid vesicles. These data indicate, (1) sign ificant differences in the biochemical properties of native members of the G(12) subfamily, and (2) their specific coupling to TXA(2) recept ors.