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.

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