Rhodopsin phosphorylation in the frog retina: Analysis by autoradiography

Light microscopic autoradiography was used to localize phosphorylated rhodopsin and to examine rate and extent of light activated rhodopsin phosphorylation in frog rod outer segments and in the isolated frog retina. The even distribution of label in totally bleached rod outer segments and partial labeling of rod outer segments after local illumination indicates that the light induced transfer of (32)P from exogenous (?-(32)P) ATP to rhodopsin is not restricted to particular parts of the outer segment. Light activated phosphorylation of rhodopsin in retinas incubated in (32)P(i) starts with the formation of a gradient of labeled rhodopsin suggesting that either light affects the formation of (32)P labeled ATP or that outer segments contain a gradient of a diffusable metabolite involved in the phosphorylation of rhodopsin. Evaluation of autoradiograms by microdensitometry shows that rate and extent of phosphate incorporation in individual rod outer segments significantly differ from the mean value of phosphate incorporation into opsin. Phosphorylation of rhodopsin in single rod outer segments may occur at a rate of 2 moles of phosphate incorporated per mol rhodopsin per minute with a half-time of less than 30 seconds.