Regulation mechanisms of visual excitation

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In order to understand the molecular mechanism of the adaptation of vertebrate photoreceptors, we have to investigate the functions of various enzymes and their subunits, and also their regulator proteins such as recoverin or S-modulin. In this project, PI has gotten several important findings about this problems as shown below. 1.Cloning of the gene encoding photoreceptor-specific guanylate cyclase(GC)from bovine retina cDNA library, and preparation of anti-GC antibody. In the first year, PI was able to identify several partial amino acid sequence including N-terminal sequence of GC purified from bovine photoreceptor rod outer segments(ROS). On the basis of this finding, PI started to isolate the clone by using polymerase chain reaction method, and got some promising amplified DNAs. However, at that time PI got the information about an another research group succeeded to isolate the photoreceptor GC gene. Thus PI stopped this project. The last fall, the research group in U.S.published … More the whole a.a.sequence of human photoreceptor GC, which was determined by the use of PCR homology search. All of the partial a.a.sequence we have found were included in the sequence. Therefore, the idea that 110kDa membrane protein we have found and succeeded to purify is real photoreceptor GC was substantiated by the molecular biological aspect. PI has tried to prepare the anti-GC antibody, though no able antiserum has generated so far. 2.Function of 26kDa Ca^<2+> binding protein(recoverin : Rec)in GC regulation. Purified GC was incorporated into lipid vesicles and the effect of Ca^<2+>/Rec on GC activity was investigated. No regulatory effect of Rec was observed. On the basis of immunobiochemical experiments, PI suggests the necessity of an approx. 50kDa protein which mediate the putative Rec-GC interaction. 3.Physiological meaning of the phosphorylation of an inhibitory subunit(Pgamma)of cGMP-phosphodiesterase(PDE) PI observed the in vivo phosphorylation of Pgamma in the rod outer segment of intact frog photoreceptors. The phosphorylation of Pgamma was stimulated by a brief light exposure. A novel protein kinase which can phosphorylate Pgamma in phosphatidyl-inositol-dependent manner was released from ROS membranes by Ca^<2+>, and the PK was isolated from another PK, such as PKC, PKA, RhK by an anion-exchange column chromatography. The effect of the phosphorylation on the function of Pgamma has now been under investigation. Less