Immunotitration analysis of the rod outer segment phosphodiesterase.
Two high-affinity monoclonal antibodies (ROS-1, ROS-2) have been produced to the rod outer segment phosphodiesterase (ROS PDE). These antibodies bind at different antigenic determinants. ROS-2 absorbs a subset of the total PDE activity from a detergent-solubilized retina extract, whereas ROS-1 adsorbs nearly all of the PDE activity. DEAE-cellulose chromatography separates two peaks of activity from a hypotonic extract of rod outer segments. Peak I activity is adsorbed only by ROS-1, whereas peak II activity is adsorbed by both ROS-1 and ROS-2. Both peaks of activity are activated by histone H2B and limited trypsin digestion, and both peaks of activity contain a heat-stable, trypsin-sensitive inhibitor. When analyzed by SDS gel electrophoresis, ROS-1 adsorbed a single peptide from peak I, which comigrated with phosphorylase B, whereas ROS-1 adsorbed two slightly faster migrating peptides from peak II. Histone H2B activated at least 80% of the PDE activity bound to ROS-2 but was less effective in activating the PDE bound to ROS-1. ROS-1 but not ROS-2 was an effective inhibitor of PDE activity, suggesting that ROS-1 may be a specific probe to study the effects of ROS PDE on the light response.
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D000939:Epitopes / Q000276:immunology; D007163:Immunosorbent Techniques; D051379:Mice; D010786:Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015106:3',5'-Cyclic-GMP Phosphodiesterases