Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein.
Formation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (RM) leads to a change in the light scattering of the disk membranes (binding signal or signal P). The signal measured on isolated disks (so-called PD signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than the RMG binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed the PD-signal level and kinetics as a function of flash intensity and compared it to the RMG-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of the PD signals decrease with flash intensity when the signals are normalized to the same final level. This finding prevents an explanation of the scattering signal by a slow postponed reaction of the RMG complex. We propose to interpret the scattering change as a redistribution of G-protein between a membrane-bound and a solved state. The process is driven by the complexation of membrane-bound G to flash-activated rhodopsin (RM). The experimental evidence for this two-state model is the following: The intensity dependence of the initial rate of the PD signal is explained by the model. Under the assumption of a bimolecular reaction of free G with sites at the membrane, equal to rhodopsin in their concentration, the measured rates yield a KD of 10(-5) M. Evaluation of the extra MII kinetics yields a biphasic rise at saturating flashes. The measured rates fit to the supply of free and membrane-bound G-protein for the reaction with RM. Quantitative estimation of the expected scattering intensity changes gives a comprehensive description of binding signal and dissociation signal by the gain and loss of G-protein scattering mass. The temperature dependence of the PD-signal rate leads to an activation energy of the membrane-association process of E alpha = 44 kJ/mol.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D007700:Kinetics; D008027:Light; D008433:Mathematics; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D012542:Scattering, Radiation; D015293:Transducin
D005136:Eye Proteins; C034809:GNAT1 protein, human; D008565:Membrane Proteins; D012168:Retinal Pigments; C045414:photorhodopsin; D012243:Rhodopsin; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin