[66] Photoaffinity labeling of high-affinity cGMP-specific noncatalytic binding sites on cGMP phosphodiesterase of Rod Outer segments

Publisher Summary The use of photoaffinity-labeling techniques has gained prominence in many areas of biochemistry. Photoaffinity-labeling reagents have been especially productive for the specific labeling of proteins that contain cyclic nucleotide binding sites. Rod outer segments (ROS) is used for membrane preparations and a crude ROS PDE preparation for the purpose of detecting the noncatalytic cGMP-binding sites with photoaffinity probes. This chapter emphasizes that the binding of cGMP to these noncatalytic high-affinity cGMP-binding sites on PDE is markedly stimulated by the presence of the heat-stable PDE inhibitor. Thus any preparation of PDE from ROS membranes should, for the purpose of demonstrating this class of cGMP-binding sites, contain or be supplemented with a preparation of the heat-stable inhibitory moiety. Moreover, because the PDE preparation often contains the GTP-binding protein subunits, it is essential to exclude either GTP or its nonhydrolyzable analogs from such a preparation inasmuch as the complex formed between GTP and the a subunit of the GTP-binding protein markedly interferes with the binding of cGMP to the noncatalytic sites, presumably as a result of the release of the inhibitory moiety.