FUNCTIONAL EQUIVALENCE OF METARHODOPSIN-II AND THE GT-ACTIVATING FORM OF PHOTOLYZED BOVINE RHODOPSIN

Absorption of a photon by the visual pigment rhodopsin leads to the formation of an activated conformational state, denoted rho*, which is capable of activating the visual G-protein, G(t). The bleaching of rhodopsin can be resolved into a series of spectrally distinct photointermediates. Previous studies suggest that the photointermediate metarhodopsin II (meta II, lambda-max of 380 nm) corresponds to the physiologically active form rho*. In the studies reported herein, spectral and enzymological data were analyzed and compared so as to evaluate the temporal correspondence between meta II and rho*. This information was obtained by direct observation of the meta Il and rho* decay times in parallel experiments utilizing identical preparations of urea-stripped, bovine retinal rod outer segment disk membranes at pH 8.0, 20-degrees-C. Postflash spectra were deconvolved to resolve the meta II absorbance at 380 nm, and a decay time for the loss of meta 11 of 8.2 min (SD = 0.5 min) was obtained from fitting these data to a single-exponential decay process. The diminishing ability of bleached rhodopsin to activate G(t) was measured by monitoring the level of catalyzed exchange of G(t)-bound GDP for a nonhydrolyzable GTP analogue. Analysis of the decrease in the initial velocity of nucleotide exchange, measured at various postflash incubation times, yielded a rho* decay time of 7.7 min (SD = 0.5 min) when analyzed as a single-exponential process. The similarity of these decay times provides direct evidence that meta 11 and rho* are present over the same time regime, and further supports the equivalence of these two forms of photoactivated rhodopsin. Rho* decay measurements were characterized by a residual level of G(t) activation, which persisted after the dynamic phase of the rho* decay was complete. Spectral measurements, in the presence and absence of added G(t), showed G(t)-induced formation of meta II from the existing pool of meta III; this demonstrates directly the reversibility of the meta II <--> meta III equilibrium. The formation of meta II from meta III under the conditions where the residual level of rho* activity persists provides further evidence for the equivalence of these two forms of photoactivated rhodopsin.