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 s...

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Veröffentlicht in:Journal of neurochemistry 2000-07, Vol.75 (1), p.355-362
Hauptverfasser: Huang, Zhong, Ghalayini, Abboud, Guo, Xia Xiao, Alvarez, Kathleen M., Anderson, Robert E.
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Sprache:eng
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Zusammenfassung: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 ~2 mM. Immunoblot analysis with an anti‐DAG‐kinase γ polyclonal antibody demonstrated that the γ isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG‐kinase γ immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG‐kinase γ 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.
ISSN:0022-3042
1471-4159
DOI:10.1046/j.1471-4159.2000.0750355.x