HIGH-AFFINITY INTERACTIONS OF GTP-GAMMA-S WITH THE HETEROTRIMERIC G-PROTEIN, TRANSDUCIN - EVIDENCE AT HIGH AND LOW-PROTEIN CONCENTRATIONS

A well known difference in nucleotide binding characteristics between heterotrimeric G proteins and small GTP binding proteins of the Ras su perfamily is that the former bind GTP or guanosine 5'-O-(3-thiotriphos phate) (GTP gamma S) with a much lower affinity (K-d similar to 10(-8) -10(-7) M) than the latter (K-d similar to 10(-11)-10(-10) M). We repo rt here that the alpha subunit of the heterotrimeric G protein transdu cin (G(t)) binds GTP gamma S with an affinity comparable to that of Ra s. High affinity binding was suggested by GTP gamma S titrations of ro d outer segment samples with G(t) concentrations in the range of 7 nM to 300 nM; the results were more consistent with a dissociation consta nt for GTP gamma S in the subnanomolar range, than with one in the 10( -8)-10(-7) M range typically reported for heterotrimeric G proteins. E quilibrium binding experiments with G protein concentrations in the su bnanomolar to nanomolar range confirmed this conclusion and revealed a dissociation constant of 50 pM, Thus, transducin's affinity for GTP g amma S, and by inference, for GTP, appears to be approximately three o rders of magnitude higher than previously reported. These results rais e the possibility that some results obtained with high concentrations of nucleotide analogues may be due to minute traces of contaminants su ch as GTP, GTP gamma S, or GTP alpha S, that have high affinities for G(t alpha).