High affinity (K(D) ~ 1 x 10-9 M) monoclonal antibodies (ROS-1 and ROS-2) were prepared to bovine photoreceptor outer segment cGMP phosphodiesterase. ROS-1 immunoadsorbed >95% of the cGMP phosphodiesterase activity from a detergent-solubilized bovine retine extract while ROS-2 immunoadsorbed only a subfraction of the same activity. Sodium dodecyl sulfate gel analysis of these immunoadsorbates demonstrated that ROS-1 and ROS-2 specifically adsorbed only peptides that comigrated with purified rod outer segment phosphodiesterase. Limited trypsin digestion of purified rod outer segment phosphodiesterase greatly reduced its affinity for ROS-1 but not ROS-2. When a crude heat-stable inhibitor fraction was added back to the activated enzyme, the affinity for ROS-1 was restored, suggesting that the inhibitor was necessary for ROS-1 binding. ROS-1 but not ROS-2 was found to inhibit cGMP phosphodiesterase which had been activated either by dilution or guanyl nucleotide. The inhibitory property of ROS-1 may provide a useful probe for directly studying the effects of this phosphodiesterase on the phototransduction response in the retina. Sodium dodecyl sulfate gel analysis demonstrated that the ROS-1 immunoadsorbates from mammals, fish, and amphibia contained peptides of similar mobility. Immunocytochemistry performed with ROS-1 and fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG localized the antigenic determinant to both rod and cone outer segments suggesting the presence of an antigenically similar phosphodiesterase in both types of photoreceptors.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology