Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments

• Published in: Investigative ophthalmology & visual science Vol. 38; no. 9; pp. 1873 - 1882
• Main Authors: Guo, X, Ghalayini, AJ, Chen, H, Anderson, RE
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01-08-1997; Association for Research in Vision and Ophtalmology
• Subjects: Animals; Biological and medical sciences; Blotting, Western; Cattle; Cell Membrane - enzymology; Chromatography, High Pressure Liquid; Cytosol - enzymology; Dark Adaptation; Electrophoresis, Polyacrylamide Gel; Eye and associated structures. Visual pathways and centers. Vision; Fundamental and applied biological sciences. Psychology; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Photic Stimulation; Photoreceptor Cells - enzymology; Photoreceptor Cells - radiation effects; Rod Cell Outer Segment - enzymology; Rod Cell Outer Segment - radiation effects; Vertebrates: nervous system and sense organs; Enzyme; Transferases; Rod; Photoreceptor; Ox; Retina; Activation; 1-Phosphatidylinositol 3-kinase; Eye; Visual system; Vertebrata; Mammalia; Outer segment; Enzymatic activity; Light; Immunoblotting assay; Artiodactyla; Ungulata
• ISSN: 0146-0404; 1552-5783

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.
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.
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.
PI 3-kinase is present in bovine rod outer segments and its activity appears to be greater in light-adapted retinas.