Phosphatidic acid and polyphosphoinositide metabolism in rod outer segments. Differential role of soluble and peripheral proteins.
The phosphorylation of endogenous diacylglycerol (DAG) and phosphoinositides by [tau-32P]ATP was studied in bovine rod outer segments (ROS) selectively depleted of soluble or peripheral and soluble proteins by treatment with moderate (100 mM) or low (5 mM) ionic strength medium, respectively. DAG kinase activity was similar in bleached and non-bleached ROS extracted with 100 mM medium, and amounted to 70% of that observed in the corresponding non-extracted ROS. Phosphatidic acid (PtdH) labelling in ROS extracted in the dark with low ionic strength medium was markedly lower than in those extracted in light. Thus, even when a major proportion of DAG kinase was associated to the membrane, a soluble form also occurred. Most of the membrane-bound fraction behaved as a peripherally associated protein, its binding to the membrane being modified by light. Ir ROS extracted at moderate ionic strength the labelling of inositides was similar to that in non-extracted ROS. A marked enhancement in polyphosphoinositide labelling was observed in ROS extracted in the dark with low ionic strength. Alkaline treatment of ROS also produced inhibition of polyphosphoinositide phosphorylation. A peripheral form of a type C phospholipase, or a peripheral protein-mediated activation of a particulate form thereof, is suggested. Labelled polyphosphoinositides were more actively hydrolyzed in the light and in the dark plus GTP tau S than in the dark-incubated membranes. The results of phosphorylation experiments in membranes where differential extraction of the alpha subunit of transducin was carried out suggest that alpha and beta tau subunits may play opposite modulating roles in PtdH and polyphosphoinositide metabolism.
Ilincheta de Boschero
M G
MG
Instituto de Investigaciones Bioquímicas, Universidad Nacional del Sur y Consejo Nacional de Investigaciones Científicas y Técnicas, Bahía Blanca, Argentina.
Giusto
N M
NM
D000818:Animals; D002021:Buffers; D002417:Cattle; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012995:Solubility; D015293:Transducin / Q000378:metabolism
D002021:Buffers; D004075:Diglycerides; D005136:Eye Proteins; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D015293:Transducin