Interactions between photoexcited rhodopsin and GTP-binding protein: kinetic and stoichiometric analyses from light-scattering changes.
In rod outer segments, photoexcited rhodopsin (R*) activates a cyclic GMP phosphodiesterase through a sequence of reactions involving a GTP-binding protein. By measuring light-scattering changes above 700 nm, we have studied the kinetics and stoichiometry of the association of R* with this protein and of the dissociation of the complex upon GDP/GTP exchange. Two light-scattering signals were obtained upon photoexcitation of rhodopsin in bovine rod outer segment membranes as well as in a reconstituted system consisting of purified GTP-binding protein and washed disc membranes; both signals depended specifically on the presence of GTP-binding protein. A "binding signal" that was observed in the absence of gTP as an increase in turbidity became saturated when a number of rhodopsin molecules equal to the number of GTP-binding protein molecules present (congruent to 10% in rod outer segments) has been bleached, suggesting that the protein binds to R* in a 1:1 complex. A "dissociation signal" of opposite sign, observed in presence of GTP at greater than or equal to 1 microM, is half maximal at 0.04% bleaching and saturated at 0.5% bleaching; it is interpreted as reflecting the dissociation of GTP-binding protein-R* complexes after GDP/GTP exchange on the GTP-binding protein, one R* being able to interact sequentially with about 100 GTP-binding protein molecules. The early time course of the binding signal is faster than that of the dissociation signal, and both signals take place in the 100-msec range at 20 degrees C.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008027:Light; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D012542:Scattering, Radiation
D011956:Receptors, Cell Surface; D012168:Retinal Pigments; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D019204:GTP-Binding Proteins