Abstract
Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.
Highlights
Lightstimulatesphospholipase Az activity inrod ety of systems [5,6,7], I examined the role of transducin in the outer segments(ROS) of bovineretina as measured by light activation of phospholipases A2 and C in bovine ROS
Neither the amount of rhodopsin, an intrinsic ROS protein, nor the total amount of phospholipase A, activity associated with ROS membranes is significantly altered by the purification procedures. This was determined by spectrophotometric assay of rhodopsin and by analysis of the total phospholipase A2 activity present in dark-adapted transducin-richandtransducin-poor membranes following treatment w500ith the phospholipase A, activator, melittin
While the extent of activation by GTPyS varies with the ROS preparation, for the reasons previously addressed, the stimulatory effect of 100 ~ L MGTPyS on crude darkadapted ROS is consistently 3-4 times basal activity, 80% that achieved by light activation of those membranes
Summary
Vol 262, No., Issue of January 5, ~ npn1t:e6d3-m168U,1.S98.A7. Light Activation of Phospholipase A2 in Rod OuterSegments of Bovine Retina and Its Modulation by GTP-bindingProteins*. Transdu- Preparation of Rod Outer Segments and Isolation of Transdwincin, the major G protein of the ROS, mediatesthe activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. The light-sensitive phospholipase ROS prepared from light-activated retina, are transducin depleted. The retinal G protein, to the radiolabeled phosphatidylcholine (lo7dpm/ml) and thesolvent transducin, is highlylocalized in the rod outer segments evaporated under Nz. After addition of 1 ml of 0.12 M Tris-HC1, pH (ROS)’ [8],where it couples light activation of rhodopsin to stimulation of cGMP phosphodiesterase activity [9]. G. and Gi, the regulatory GTP-binding components of adenylate texed, centrifuged (10min, 1000 X g), and theradioactivity of a 1-ml cyclase that mediate stimulationorinhibition, respectively
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