Dephosphorylation during bleach and regeneration of visual pigment in carp rod and cone membranes.

ABSTRACT

On absorption of light by vertebrate visual pigment, the chromophore, 11-cis retinal, is isomerized to all-trans retinal to activate the phototransduction cascade, which leads to a hyperpolarizing light response. Activated pigment is inactivated by phosphorylation on the protein moiety, opsin. Isomerized all-trans retinal is ultimately released from opsin, and the pigment is regenerated by binding to 11-cis retinal. In this pigment regeneration cycle, the phosphates incorporated should be removed in order that the pigment regains the capability of activating the phototransduction cascade. However, it is not clear yet how pigment dephosphorylation takes place in the regeneration cycle. First in this study, we tried to estimate the dephosphorylation activity in living carp rods and cones and found that the activity, which is present mainly in the cytoplasm in both rods and cones, is three times higher in cones than in rods. Second, we examined at which stage the dephosphorylation takes place; before or after the release of all-trans retinal, during pigment regeneration, or after pigment regeneration. For this purpose we prepared three types of phosphorylated substrates in purified carp rod and cone membranes phosphorylated bleaching intermediate, phosphorylated opsin, and phosphorylated and regenerated pigment. We also examined the effect of pigment regeneration on the dephosphorylation. The results showed that the dephosphorylation does not show substrate preference in the regeneration cycle and suggested that the dephosphorylation takes place constantly. The results also suggest that, under bright light, some of the regenerated visual pigment remains phosphorylated to reduce the light sensitivity in cones.