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Publisher Summary In vertebrate rod outer segments (ROSs), guanosine 3′,5′-cyclic monophosphate (cGMP) appears to be directly involved in the process of visual transduction. The illumination of rhodopsin leads to the activation of cGMP phosphodiesterase (PDE) through transducin. The resulting fall in cytoplasmic cGMP concentration contributes to closure of cGMP-sensitive cation channels, causing the hyperpolarization of plasma membranes. Moreover, recovery of the dark state requires the resynthesis of cGMP by guanylate cyclase. To explain the mechanisms for the recovery of the dark state, isolation and reconstitution of guanylate cyclase in detergent-free systems are essential. The chapter also describes the basic characterization of guanylate cyclase from amphibian and bovine ROS. Guanylate cyclase is analyzed by measuring the amount of [ 32 P]cGMP that is derived from [α- 32 P]GTP in the presence of [ 3 H]cGMP, 3-isobutyl-l-methylxanthine, and a GTP-generating system.

Publisher Summary This chapter discusses the purification and characterization of a light-activated rod outer segment phosphodiesterase. There are abundant data to demonstrate that levels of cyclic guanosine monophosphate (cGMP) in the vertebrate rod outer segment (ROS) are rapidly reduced by light. The explosive hydrolysis of cGMP in vertebrate rods is mediated by a light and guanosine triphosphate (GTP)-dependent cGMP phosphodiesterase (PDE). This PDE is a peripheral membrane protein that has been found in association with amphibian and mammalian rods. Action spectra and reconstitution experiments show that rhodopsin (Rho) is the photopigment that mediates light activation of rod PDE. The purification and characterization of rod PDE has provided significant advantages in studying the mechanism of its activation and its physiological significance. When the PDE is in its native position, activation is supported by light and GTP. The isoelectric point for the purified PDE has been determined. Using an isoelectric focusing technique adapted for nondenaturing polyacryl-amide gels, the purified PDE behaves as a single protein with an isoelectric point of pH.

Publisher Summary This chapter describes a novel and simple single-step chromatographic method for the extensive purification of the subunits of the GTP-binding protein and the phosphodiesterase (PDE). There is a well-established homology between the hormone-sensitive adenylate cyclase and the light-activated cGMP phosphodiesterase in rod outer segments (ROS). The light-activated PDE of ROS has three functional segments. The photopigment rhodopsin, the GTP-binding protein, and the PDE catalytic complex. Signal transduction from rhodopsin to PDE is dependent on the binding of GTP to the α subunit of the GTP-binding protein. Photon capture is necessary for the binding of GTP to the Gα subunit. The GTP/Gα complex is rapidly released from the disk membrane surface. The activation of PDE in ROS prepared from the amphibian retina occurs as a consequence of the presentation of photons and the addition of GTP. Moreover, this activation depends upon the physical release of an inhibitory moiety from the PDE that remains bound to the disk membrane throughout the activation cycle. The major components of the ROS light-sensitive PDE cascade have been isolated and purified. The purification of rhodopsin has been well characterized in a number of laboratories.

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.