Recombination reaction of rhodopsin in situ studied by photoconversion of “indicator yellow”

We measured the kinetics of recombination of 11- cis-retinal with opsin in intact frog rod outer segment (ROS). The rhodopsin in ROS was bleached and allowed to decay to “indicator yellow,” a photoproduct where all- trans-retinal is partly free, and partly bound to non-specific amino groups of disk membranes. By briefly illuminating the “indicator yellow” by an intense 465 or 380-nm flash, we then photoconverted all- trans-retinal to (mostly) the 11- cis- form thus introducing into ROS a certain amount of cis-chromophore. The recombination of cis-retinal with opsin and the formation of rhodopsin were followed by fast single-cell microspectrophotometry. Regeneration proceeded with a time constant of ∼3.5 min; up to 27% of bleached visual pigment was restored. The regenerated pigment consisted of 91% rhodopsin (11- cis-chromophore) and 9% of presumably isorhodopsin (9- cis-chromophore). The recombination of 11- cis-retinal with opsin inside the ROS proceeds substantially faster than rhodopsin regeneration in the intact eye and, hence, is not the rate-limiting step in the visual cycle.