Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes.

The equilibria between metarhodopsins I and II (MI and MII) and the binding of MII to retinal G protein (G) were investigated, using the dual wavelength absorbance response of rod disk membrane (RDM) suspensions to a series of small bleaches, together with a nonlinear least-squares fitting procedure that decouples the two reactions. This method has been subjected to a variety of theoretical and experimental tests that establish its validity. The two equilibrium constants, the amount of active G protein (that can bind to and stabilize MII) and the fraction bleached by the flash, have been determined without a priori assumptions about these values, at temperatures between 0 and 15 °C and pHs from 6.2 to 8.2. Binding of G to MII in normal RDM exhibits 1:1 stoichiometry (not cooperative), relatively weak, 2−4 × 104 M-1 affinity on the membrane, with a pH dependence maximal at pH 7.6, and a low thermal coefficient. The reported amount of active G remained constant even when its binding constant was reduced mor...