Light-stimulated Phosphorylation of Bovine Visual Pigments by Adenosine Triphosphate

Abstract

Abstract Cattle retinal outer segments bind the γ-terminal phosphate residue from [γ-32P]ATP when incubated in the presence of Mg++ ion. The binding involves visual pigment molecules since, following labeling of the visual pigments with 11-cis-[3H]retinaldehyde and reduction on site with sodium borohydride, both the tritium and 32P peaks occur in identical protein bands when the outer segments are dissolved in sodium dodecyl sulfate (SDS), reduced and disaggregated by dialysis against β-mercaptoethanol, and electrophoresed on polyacrylamide gels containing SDS. Phosphorylation of visual pigments is slightly inhibited by Ca++ but is unaffected by Na+, K+, or adenosine 3',5'-monophosphate. It is unrelated to Na,K-ATPase activity present in these preparations. The phosphorylation is increased by a factor of 1.5 to 2.0 by incubation in continuous white light over incubation in darkness; under continuous illumination up to 0.5 to 0.6 mole of Pi per mole of rhodopsin is incorporated. Phosphorylation is maximal after 10 min at 37° and decreases thereafter. Its extent is proportional to the fraction of rhodopsin bleached. The phosphorylation reaction does not appear to have greater specificity for rhodopsin than for other proteins present as minor components in retinal outer segment preparations; however, of a variety of soluble proteins added to the incubation mixture in high concentrations along with outer segment membranes, only calf thymus histone is capable of binding 32P from [γ-32P]ATP. Of possible mechanisms for the light-stimulated phosphorylation, the release of free retinaldehyde, which may activate a kinase or sensitize binding sites, and a light-stimulated Ca++ binding, which could free the Mg++-stimulated phosphorylation from inhibition, have been eliminated experimentally. Visual pigment protein appears capable of binding a single phosphate residue at any step of its bleaching cycle as well as in the dark-adapted state, but the intermediate with the greatest affinity for the γ-phosphate of ATP may be pararhodopsin. The physiological significance of the light-stimulated phosphorylation reaction is uncertain.