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 approximately 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.
Kolesnikov
A V
AV
Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, 194223 St. Petersburg, Russia.
Shukolyukov
S A
SA
Cornwall
M C
MC
Govardovskii
V I
VI
D000818:Animals; D010775:Photic Stimulation / Q000379:methods; D011896:Rana temporaria; D012168:Retinal Pigments / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000096:biosynthesis*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D017299:Rod Opsins / Q000378:metabolism; D046509:Tissue Culture Techniques
D012168:Retinal Pigments; D017299:Rod Opsins; D012243:Rhodopsin; D012172:Retinaldehyde