| The primary function of the rod outer segment (ROS) is to transduce a photon signal into a cellular response. Two sites of cGMP interaction with the ROS phosphodiesterase have direct bearing on the mechanism of phototransduction. In addition to the phosphodiesterase catalytic site, high affinity non-catalytic sites occur that specifically bind cGMP. These non-catalytic binding sites, identified and partially characterized by photoaffinity labeling with {dollar}sp{lcub}32{rcub}{dollar}P-cGMP, are present on both the {dollar}alpha{dollar} and {dollar}beta{dollar}-subunits, and their concentration was determined to exceed the total ROS cGMP content by 4-fold, suggesting a limited availability of cGMP for interaction with the phosphodiesterase catalytic site and the cation channel binding site. Binding of cGMP to these non-catalytic sites was reported by Yamazaki et al. to be inhibited by activation of G-protein, but the possibility was not eliminated that the G-protein-induced increase in phosphodiesterase activity may have limited the availability of cGMP for binding. In the present studies it was shown that this apparent inhibition of cGMP binding by G-protein can be entirely attributed to an increased hydrolysis of cGMP, that activated G-protein does not alter any parameter of these non-catalytic cGMP binding sites. These binding sites are therefore not likely to be involved in any change in cGMP availability upon illumination. In another report (Fesenko and Krapivinsky) it was claimed that illumination, in the presence of ATP, markedly stimulated cGMP binding. This apparent effect was demonstrated to be entirely attributable to a light-stimulated, rapid metabolism of cGMP to GDP and GTP, which were identified as the nucleotides that became bound. If the availability of cGMP is limited, then the standard phosphodiesterase assay using millimolar substrate concentrations does not represent the physiological state, especially since the K{dollar}sb{lcub}rm m{rcub}{dollar} of this enzyme can be shown to increase upon illumination. Quantitative assessment of the light-"stimulated" phosphodieterase activity showed that the large increase in V{dollar}sb{lcub}rm max{rcub}{dollar} was counteracted by a nearly equivalent increase in K{dollar}sb{lcub}rm m app{rcub}{dollar}, resulting in increases of less than 2-fold in this enzyme activity at the micromolar level of cGMP found in the ROS. These results are at odds with the notion that photic stimulation lowers the cGMP concentration and suggests another importance of the phosphodiesterase. |
