Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.
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.
Huang
Z
Z
Departments of Ophthalmology, Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center Dean A. McGee Eye Institute, Oklahoma City, Oklahoma, USA.
Ghalayini
A
A
Guo
X X
XX
Alvarez
K M
KM
Anderson
R E
RE
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D019852:Diacylglycerol Kinase / Q000032:analysis / Q000378:metabolism*; D004075:Diglycerides / Q000032:analysis / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D005615:Freezing; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis; D008027:Light; D051381:Rats; D012160:Retina / Q000201:enzymology; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000201:enzymology*
D004075:Diglycerides; D007527:Isoenzymes; D019852:Diacylglycerol Kinase