Guanine nucleotide binding characteristics of transducin: essential role of rhodopsin for rapid exchange of guanine nucleotides
Fawzi, A.B.; Northup, J.K.
Biochemistry 29(15): 3804-3812
ISSN/ISBN: 0006-2960 PMID: 2187531 DOI: 10.1021/bi00467a030
Transducin (Gt) is a member of a family of receptor-coupled signal-transducing guanine mucleotide (GN) binding proteins (G-proteins). Light-activated rhodopsin is known to catalyze GN exchange on Gt, resulting in the formation of the active state of the Gt.alpha.-GTP complex. However, purified preparations of Gt have been shown to exchange GN in the absence of activated receptors [Wessling-Resnick, M., and Johnson, G. L. (1987) Biochemistry 26, 4316-4323]. To evaluate the role of rhodopsin in the activation of Gt, we studied GN-binding characteristics of different preparations of Gt. Gt preparations obtained from the supernate of GTP-treated bovine rod outer segment (ROS) disks, followed by removal of free GTP on a Sephadex G-25 column, bound GTP.gamma.S at 30.degree. C in the absence of added exogenous rhodopsin with an activity of 1 mol of GTP.gamma.S bound/mol of Gt (Gt-I preparations). Binding of GTP.gamma.S to Gt-I preparations closely correlated with the activation of ROS disk cGMP phosphodiesterase. GN-binding activity of Gt-I preparations was dependent on reaction temperature, and no binding was observed at 4.degree. C. In the presence of 10 .mu.M bleached rhodopsin, Gt-I preparations bound GTP.gamma.S at 4.degree. C. However, hexylagarose chromatography of Gt-I preparations led to a preparation of Gt that showed < 0.1 mol/mol binding activity following 60-min incubation at 30.degree. C in the absence of rhodopsin (Gt-II preparations). In the presence of as low as 0.03 .mu.M bleached rhodopsin, Gt-II preparations rapidly bound GTP.gamma.S at 30.degree. C. Equimolar mixtures of Gt-I and Gt-II preparations showed a 1.0 mol/mol binding activity in the absence of added rhodopsin. While treatment of Gt-I preparations with hydroxylamine, which is known to convert rhodopsin to opsin, greatly diminished the rate of GTP.gamma.S binding to the preparation, addition of 0.03 .mu.M bleached rhodopsin following removal of hydroxylamine restored the binding activity. These results indicate that (1) rhodopsin is essential for rapid GN exchange on Gt and (2) rapid GN exchange in the absence of exogenous rhodopsin observed in some Gt preparations is stimulated by undetected rhodopsin contamination.