title: Kinetic analysis of the activation of transducin by photoexcited rhodopsin. Influence of the lateral diffusion of transducin and competition of guanosine diphosphate and guanosine triphosphate for the nucleotide site.
abstract: The activation of transducin (T) by photoexcited rhodopsin (R*) is kinetically dissected within the framework of Michaelis-Menten enzymology, taking transducin as substrate of the enzyme R*. The light scattering "release" signal (Vuong, T.M., M. Chabre, and L. Stryer, 1984, Nature (Lond.). 311:659-661) was used to monitor the kinetics of transducin activation at 20 degrees C. In addition, the influence of nonuniform distributions of R* on these activation kinetics is also explored. Sinusoidal patterns of R* were created with interference fringes from two crossed laser beams. Two characteristic times were extracted from the Michaelis-Menten analysis: t(form), the diffusion-related time needed to form the enzyme-substrate R*-transducin is 0.25 +/- 0.1 ms, and T(cat), the time taken by R* to perform the chemistry of catalysis on transducin is 1.2 +/- 0.2 ms, in the absence of added guanosine diphosphate (GDP) and at saturating levels of guanosine triphosphate (GTP). With t(form) being but 20% of the total activation time t(form) + t(cat), transducin activation by R* is not limited by lateral diffusion. This is further borne out by the observation that uniform and sinusoidal patterns of R* elicited release signals of indistinguishable kinetics. When (GDP) = (GTP) = 500 microM, t(cat) is lengthened twofold. As the in vivo GDP and GTP levels are comparable, the exchange of nucleotides may well be the rate-limiting process.
authors.affiliation: Departement de Biologie Moleculaire et Structurale, Commissariat a l'Energie Atomique, Grenoble, France.
authors.forename: F
authors.initials: F
authors.lastname: Bruckert
authors.forename: M
authors.initials: M
authors.lastname: Chabre
authors.forename: T M
authors.initials: TM
authors.lastname: Vuong
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D004058:Diffusion; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008433:Mathematics; D008962:Models, Theoretical; D011898:Ranidae; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000378:metabolism*
chemical_list: D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin