Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.

Abstract

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3-kinase are also higher in bleached (light-exposed) ROS (B-ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG-kinase) activity in bovine and rat ROS membranes prepared from dark-adapted (D-ROS) or bleached (B-ROS) retinas. In bovine ROS, DAG-kinase activity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG-kinase activity was due to higher levels of endogenous substrate in B-ROS. DAG-kinase activity was also higher in B-ROS against an exogenous, water-soluable substrate (1, 2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate and saturated at approximately 2 mM. Immunoblot analysis with an anti-DAG-kinase gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG-kinase gamma immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG-kinase gamma was present in B-ROS than in D-ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG-kinase activity than ROS prepared from dark-adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG-kinase from the cytosol to ROS membranes and that the greater DAG-kinase activity in B-ROS is due to the presence of more protein associated with ROS membranes.