Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state.

The interaction of the rod GTP binding protein, Transducin (G(t)), with bleached Rhodopsin (R()) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound G(t) by reconstituting G(t) containing bound GDP (G(t-)GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate (G(t-)p[NH]ppG) with R under physiological conditions. Release of GDP and p[NH]ppG from G(t) occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of G(t) without bound nucleotide (G(t-)) were generated. When ROS containing bleached rhodopsin (R()) were centrifuged in low ionic strength buffer, G(t-) remained associated with the membrane fraction, whereas G(t-)GDP remained in the soluble fraction. These results suggest that G(t-)GDP and G(t-)p[NH]ppG have similar affinities for R(). The results also suggest that G(t-), rather than G(t-)GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.