Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of Gα12 purified from rat brain

G₁₂ and G₁₃ are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of Gα₁₂ from rat brain membranes. Gα₁₂ was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified Gα₁₂ bound guanosine 5´-[γ-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active Gα₁₂, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G₁₂ subfamily revealed differences in the affinity of Gα₁₂ and Gα₁₃ for Gβγ. Gα₁₂ required a higher Mg²⁺ concentration for AlF₄^--induced dissociation from immobilized Gβγ than did Gα₁₃. In addition, the G₁₂ subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G₁₂ to form supramolecular structures in comparison to G₁₃ and other G-proteins. These G₁₂ structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active Gα₁₂. Despite these distinct biochemical characteristics of G₁₂ and G₁₃, both purified G-proteins coupled to a recombinant thromboxane A₂ (TXA₂) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G₁₂ subfamily, and (2) their specific coupling to TXA₂ receptors.