Regulation mechanisms of visual excitation

为了了解脊椎动物光感受器适应的分子机制，我们必须研究各种酶及其亚基的功能，以及它们的调节蛋白，如恢复素或S-调制素。在这个项目中，PI得到了关于这个问题的几个重要发现，如下所示。1.从牛视网膜cDNA文库中克隆光感受器特异性鸟苷酸环化酶基因及其抗体的制备在第一年，PI能够鉴定几个部分氨基酸序列，包括从牛感光杆外节（ROS）纯化的GC的N-末端序列。在此基础上，PI开始用聚合酶链反应方法分离克隆，并获得了一些有希望的扩增DNA。然而，当时PI得到了另一个研究小组成功分离出感光细胞GC基因的信息。因此，PI停止了这个项目。去年秋天，美国的研究小组发表了 ...更多信息 人光感受器GC的全部a.a.序列，其通过使用PCR同源性搜索确定。我们发现的所有部分a.a.序列都包含在序列中。因此，从分子生物学角度证实了我们所发现并成功纯化的110 kDa膜蛋白是真实的光感受器GC的观点。PI曾尝试制备抗GC抗体，但至今尚未产生有效的抗血清。2. 26 kDa Ca^<2+>结合蛋白（recoverin：Rec）在GC调节中的功能将纯化的GC掺入脂质囊泡中，并研究Ca^<2+>/Rec对GC活性的影响。没有观察到Rec的调节作用。在免疫生物化学实验的基础上，PI提出了一个近似的必要性。50 kDa的蛋白质，其介导推定的Rec-GC相互作用。3. cGMP磷酸二酯酶（PDE）抑制亚基（Pgamma）磷酸化的生理意义PI观察到完整蛙光感受器视杆外节中Pgamma的体内磷酸化。通过短暂的光暴露刺激P γ的磷酸化。利用Ca^2+从ROS膜上释放出一种新的蛋白激酶，该蛋白激酶能以磷脂酰肌醇依赖的方式磷酸化P γ，并通过阴离子交换柱层析从其它蛋白激酶如PKC、PKA、RhK中分离得到。磷酸化对Pgamma功能的影响正在研究中。少

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