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

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by lig ht 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 s tudy, we investigated the effect of bleaching on diacylglycerol (DAG) kinas e (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 acti vity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Qua ntification of DAG in both sets of membranes showed that the levels were th e same, eliminating the possibility that the greater DAG-kinase activity wa s due to higher levels of endogenous substrate in B-ROS. DAG-kinase activit y was also higher in B-ROS against an exogenous, water-soluble substrate (1 ,2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate a nd saturated at similar to 2 mM. Immunoblot analysis with an anti-DAG-kinas e gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal secti ons 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 tha n 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 da rk-adapted animals. Taken together, these data suggest that light exposure reads 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.