Abstract
Light-induced conformational changes of the dim-light photoreceptor rhodopsin promote efficient binding and activation of the intracellular guanine nucleotide-binding protein, transducin. This activation event initiates GDP/GTP exchange and subsequent dissociation of transducin, from the activated photoreceptor. In this work we have developed a method to assemble and purify an activated rhodopsin/transducin complex in both detergent micelles and lipid bilayers. Activated rhodopsin was immobilized on an affinity resin, allowed to bind to transducin, and extensively washed to remove nonbinding material prior to elution. Evaluation of the eluate by SDS-PAGE and UV-visible absorption spectoscopy confirm the presence of rhodopsin and transducin. The incubation of inactive rhodopsin with transducin in a control experiment resulted in the elution of rhodopsin but no transducin. Activity of the rhodopsin/transducin complex was measured by rhodopsin-dependent GDP release, the uptake of GTPγS, and the nucleotide-dependent release of transducin.