The object of this proposal is to further elucidate the stimulatory and control mechanisms that govern the light stimulated changes of c-GMP in photoreceptors, especially the roles played by GDP and Ca ion in these mechanisms. Some of the proposed experiments are based on our observation that for certain ranges of light intensity and concentrations of GTP, GDP modulates the sensitivity of PDE to stimulation by light intensity. The proposed experiments are designed to measure the effects of Ca ions on the PDE response to light with especial attention on its effects in the presence of GDP. Experiments will be done on bovine photoreceptors isolated to preserve as much as possible components which may be involved in regulating responses to light stimulation. The kinetics of light induced changes in the activity of the GTP-binding protein (GBP) and phosphodiesterase (PDE) will be monitored by biochemical and radioisotope methods for different levels of light intensity and concentrations of GTP, GDP and Ca ions. PDE activity will be monitored continuously after light stimulation by continuous recording of the small changes in pH which occur when c-GMP is hydrolyzed by PDE. Our experiments indicate that important transient changes in PDE activity occur immediately on light stimulation and that these transients are missed by the usual one time point activity determinations done several minutes after light stimulation. Transient changes in GBP activity stimulated by light will be measured by periodic sampling of the amount of tracer radionucleotide incorporated into GBP following light stimulation. The effects of GDP and Ca ions on the light activated GTPase activity will also be determined through measurements of their effects on maximum reaction velocity and Km. Finally, the results of the proposed experiments will be used to develop a mechanism for the mode of action of GDP and Ca ions in the light response of PDE. These experiments are intended to open the way for approaches which will lead to understanding the roles played by regulatory agents in phototransduction processes.
