PURIFICATION OF THE G-PROTEIN G(13) FROM RAT-BRAIN MEMBRANES

Significant amounts of G(13), a member of the recently described G(12)-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G(13) (G alpha(13)) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G(13) from most of the other G-proteins, including G(q/11). Moreover, G alpha(13)-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha(13) retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha(13) to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha(13) provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5'-[gamma-[S-35]thio]triphosphate (GTP[S-35]). Accordingly, dissociation of G alpha(13) from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G(13) exhibits properties highly distinct from those of other G-proteins.