Subunit interactions of the Go protein

AbstractThe monoclonal antibody, MONO, recognizes an epitope on the G protein αo-subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTPγS, all βγ-subunits are released from the αo-subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTPγS or GTPγS-induced subunit dissociation. However, we have been unable to induce a similar dissociaton of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent X4 of αo-GDP for binding βγ of 340–390 nM has been reported (1989) J. Biol. Chem. 264, 20688–20696] our results would suggest that binding of MONO to the αo-subunit induces an increased affinity of αo-GDP for βγ. Alternatively, these results could be explained if, under the conditions used, the K4 of αo-GDP for βγ were at least two orders of magnitude lower than estimated previously