PHOSPHATIDYLINOSITOL 3-KINASE IN BOVINE PHOTORECEPTOR ROD OUTER SEGMENTS

Purpose. Phosphatidylinositol 3-kinase (PI 3-kinase) plays important r oles in mitogenesis, vesicular trafficking, actin rearrangement, and p revention of apoptotic cell death in nonocular tissues. This investiga tion looked for whether PI 3-kinase is present in bovine rod photorece ptors and if light has any effect on its activity. Methods. Bleached ( BROS) and dark-adapted (DROS) rod outer segments were prepared from fr ozen bovine retinas and immunoblotted or immunoprecipitated with antib odies against the regulatory (p85) or catalytic (p110) subunits of PI 3-kinase. Kinase activity was measured in the immunoprecipitates and t he reaction products were identified by high-performance liquid chroma tography (HPLC). The amount of PI 3-kinase in membrane and cytosol fra ctions was determined by densitometry of immunoblots. Results. Immunob lot 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-P-2, as determined by HPLC analysis of the de acylated 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 cyt osol maintained the light-dark difference. However, measurement of ant i-p85 and anti-p110 immunoreactivities on western blots of ROS, ROS me mbranes, and ROS cytosol did not show any light-dark differences. Conc lusions. PI 3-kinase is present in bovine rod outer segments and its a ctivity appears to be greater in light-adapted retinas.