Abstract
Abstract: :
Purpose: To measure the rapid time course of carp cone visual pigment phosphorylation and to explore the mechanism of this rapid phosphorylation in cones. Methods: Carp cones (mixture of red, green, blue and UV cones) and rods were isolated (Tachibanaki et al., PNAS, 98:14044–49, 2001), and their visual pigment phosphorylation was measured under in vitro conditions. In one type of the studies, rapid phosphorylation time course in cone membranes was measured with a rapid quench apparatus. In another type of the studies, both visual pigment membranes devoid of kinase activities and kinase membranes devoid of pigment activities were prepared from isolated rod and cone membranes. They were fused to measure the efficiency of phosphorylation of rod/cone pigments by rod/cone kinases (GRK1/GRK7). Results: (1) The carp cone visual pigment phosphorylation is rapid: phosphorylation was detected within 150 msec after a light flash bleaching a few % of cone pigments and was almost completed within 1 sec with a half maximum phosphorylation at 0.5 sec. Under similar conditions, rod pigment phosphorylation continued for more than 30 sec. (2) This rapid phosphorylation in cones is due to high GRK7 activities in cones, because the phosphorylation was rapid independently on the type of the pigment when GRK7 was present. (3) The potent GRK7 activity is very high: under extreme conditions where the kinase/visual pigment ratio was very low, the GRK7 activity per unit amount of visual pigment was almost 500 times higher than the GRK1 activity. (4) Even at high bleaching levels, visual pigments can be phosphorylated readily in cones, because the amount of phosphorylation at 10 sec after a light flash increased as the bleaching level increased up to 90% in cones. In rods, however, the phosphorylation became saturated and was constant at >5% bleach. (5) According to our immunochemical quantification, the content of the kinase molecule per unit amount of visual pigment seemed to be more than several times higher in cones than in rods. Together with the finding of (3), this observation indicates that the specific activity of GRK7 is more than 10 times higher than that of GRK1. Conclusions: The visual pigment phosphorylation in cones is very powerful: the time course is rapid and massive amount of bleached pigment can be phosphorylated readily. These are due to high specific activity and high content of the kinase in cones. These characteristics of GRK7 probably explain some of the features of cone photoresponses.
Keywords: photoreceptors • phosphorylation • opsins