Phosphatidylinositol 3-kinase in bovine photoreceptor rod outer segments.
OBJECTIVE
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.
Guo
X
X
Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Ghalayini
A J
AJ
Chen
H
H
Anderson
R E
RE
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D003623:Dark Adaptation; D004591:Electrophoresis, Polyacrylamide Gel; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D010775:Photic Stimulation; D010786:Photoreceptor Cells / Q000201:enzymology / Q000528:radiation effects; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000528:radiation effects
D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)