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Investigative Ophthalmology & Visual Science, Vol 38, 1873-1882, Copyright © 1997 by Association for Research in Vision and Ophthalmology

ARTICLES AND REPORTS

Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments

X Guo, AJ Ghalayini, H Chen and RE Anderson
Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, USA.

PURPOSE: Phosphatidylinositol 3-kinase (PI 3-kinase) plays important roles in mitogenesis, vesicular trafficking, actin rearrangement, and prevention of apoptotic cell death in nonocular tissues. This investigation looked for whether PI 3-kinase is present in bovine rod photoreceptors and if light has any effect on its activity. METHODS: Bleached (BROS) and dark-adapted (DROS) rod outer segments were prepared from frozen bovine retinas and immunoblotted or immunoprecipitated with antibodies against the regulatory (p85) or catalytic (p110) subunits of PI 3-kinase. Kinase activity was measured in the immunoprecipitates and the reaction products were identified by high-performance liquid chromatography (HPLC). The amount of PI 3- kinase in membrane and cytosol fractions was determined by densitometry of immunoblots. RESULTS: Immunoblot analysis showed the presence of 85 and 110 kDa proteins in ROS. PI 3-kinase immunoprecipitated by anti-p85 antibody converted PI to PI-3-P and PI-4-P to PI-3,4-P2, as determined by HPLC analysis of the deacylated products. The PI 3-kinase activity in these ROS preparations was sensitive to wortmannin, a potent inhibitor of PI 3-kinase, at low concentrations (IC50 3 nM). Immunoprecipitates (IPs) showed activity twice as high in BROS as in DROS. The IPs of ROS membranes but not cytosol maintained the light- dark difference. However, measurement of anti-p85 and anti-p110 immunoreactivities on western blots of ROS, ROS membranes, and ROS cytosol did not show any light-dark differences. CONCLUSIONS: PI 3- kinase is present in bovine rod outer segments and its activity appears to be greater in light-adapted retinas.


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Copyright © 1997 by the Association for Research in Vision and Ophthalmology