Summary
Formation of the complex between photoreceptor G-protein (G) and photoactivated rhodopsin (R M ) leads to a change in the light scattering of the disk membranes (binding signal or signalP). The signal measured on isolated disks (so-calledP D signal) is exactly stoichiometric in its final level to bound G-protein but its kinetics are much slower than theR M G binding reaction. In this study on isolated disks, recombined with G-protein, we analyzed theP D -signal level and kinetics as a function of flash intensity and compared it to theR M G-complex formation monitored spectroscopically (by extra metarhodopsin II). The basic observation is that the initial slopes of theP D 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 theR M G 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 (R M ). The experimental evidence for this two-state model is the following: (1) The intensity dependence of the initial rate of theP D 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 aK D of 10−5 M. (2) 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 withR M . (3) 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. (4) The temperature dependence of theP D -signal rate leads to an activation energy of the membrane-association process ofE a =44 kJ/mol.
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Schleicher, A., Hofmann, KP. Kinetic study on the equilibrium between membrane-bound and free photoreceptor G-protein. J. Membrain Biol. 95, 271–281 (1987). https://doi.org/10.1007/BF01869489
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DOI: https://doi.org/10.1007/BF01869489