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LIGHT REGULATED RETINAL ENZYMES

光调节视网膜酶

基本信息

批准号：
2710946
负责人：
MARK W BITENSKY
金额：
$ 40.42万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-04-01 至 1999-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2710946
关键词：
G protein animal tissue cyclic GMP light adaptations nucleic acid sequence phosphodiesterases polymerase chain reaction protein sequence retina rod cell site directed mutagenesis tissue /cell culture transducin visual phototransduction

项目摘要

DESCRIPTION (Adapted from applicant's abstract): The long term objective of this application is to understand G-protein function and regulation in retinal rods and to understand light-induced rod membrane conductance changes in terms of the functions of rod gene product ensembles. The specific aims are: to understand the physiological significance and to identify the mechanisms of phosducin regulation of transducin activity; to understand the biochemical mechanisms that permit rod outer segment gene products to continuously function in harmony with the dictates of such second messengers as cAMP, cGMP, and Ca2+; and to understand the physical meaning of, and to identify the molecular ensembles that are responsible for, positive cooperative binding of transducin to rhodopsin. In terms of the overall design and methods, protein components will be isolated, purified, reconstituted into ensembles and functionally evaluated in order to pursue the specific aims and objectives. Biochemical or physical parameters that modify the extent of cooperative transducin interaction will be utilized to study the cooperative mechanism and its kinetic consequences. Site-directed antibodies and peptides and site-directed mutagenesis will be utilized to probe the nature of interactive sites that participate in the phosphorylation-dependent affinity of phosducin for G-protein subunits. Protein chemistry, molecular biology and chromatographic methods will also be utilized to probe protein/protein interactions between transducin and rhodopsin or transducin and phosducin. Amino acid sequencing, PCR and genebank search will be employed to identify the 29 kDa protein that co-purifies with phosducin. Protein biochemistry and gene sequencing techniques will be used to identify and characterize the calcium-activated adenylate cyclase of rod outer segments.

描述（改编自申请人的摘要）：长期目标本申请旨在了解G蛋白的功能和调节，视网膜杆和了解光诱导杆膜电导视杆细胞基因产物集合体功能的变化。的具体目标是：了解其生理意义并确定转导蛋白活性的调节机制; 了解生物化学机制，使杆外节基因产品的持续运作与这些要求的和谐一致，第二信使cAMP，cGMP和Ca 2 +;并了解物理的意义，并确定负责的分子集合对于转导素与视紫红质的正协同结合。在整体设计和方法方面，蛋白质组分将分离、纯化、重组成整体并进行功能评价以实现具体的目标和目的。生物化学或改变协同转导程度的物理参数互动将被用来研究合作机制及其动力学后果定点抗体和肽，以及将利用定点诱变来探测参与磷酸化依赖性亲和力的相互作用位点 G蛋白亚基的表达蛋白质化学、分子生物学层析方法也将用于探测蛋白质/蛋白质转导蛋白和视紫红质或转导蛋白和视紫红质之间的相互作用。氨基酸序列测定、PCR和基因库检索将用于鉴定 29 kDa的蛋白质，它与胰蛋白酶共同纯化。蛋白质生物化学基因测序技术将用于识别和表征视杆外节的钙激活腺苷酸环化酶。