Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors

Abstract

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer’s solution at 23 ± 1°C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4°C) and the receptor outer segments (ROS) were isolated. Binding of the α- and β-subunits of G-protein to the ROS membranes was analyzed by quantitating Gα and Gβ extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of Gα,β in the HG extract) increased with the extent of bleaching of the visual pigment. Nearmaximal binding was observed after bleaches of ≤30%. With an increasing period of incubation in darkness after ~70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was ~103 s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context ofprevious electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment. © 1986, Rockefeller University Press., All rights reserved.