CGMP-BINDING PHOSPHODIESTERASE: REGULATORY MECHANISMS

CGMP 结合磷酸二酯酶：调节机制

• 批准号: 3299350
• 负责人: JACKIE David CORBIN
• 金额: $ 27.32万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299350
• 关键词: 3'5' cyclic nucleotide phosphodiesterase; affinity labeling; allosteric site; chemical binding; complementary DNA; cyclic GMP; enzyme induction /repression; enzyme mechanism; enzyme structure; enzyme substrate analog; genetic library; gerbil /jird; molecular cloning; peptide chemical synthesis; phosphorylation; protein kinase; protein purification; protein structure function

cGMP has been implicated in modulation of numerous mammalian systems pertinent to pathophysiology, including smooth muscle tone in blood vessels and other tissues, vision, and platelet aggregation. The objective of this proposal is to examine the regulation of a cGMP-binding phosphodiesterase (B-PDE) which is a probable target of agents that affect the cGMP cascade. This will be done by purifying the bovine lung enzyme and determining its structure and regulatory features. The functions of the B-PDE may be regulated both by an allosteric mechanism and by phosphorylation. The evidence suggests that the enzyme contains at least two functional domains--a highly specific cGMP binding site and a separate hydrolytic site for cGMP. While there is evidence for communication between these putative domains, there is no dir"ct evidence for a function of the binding site. The quaternary structure of B-PDE, number of cGMP binding domains and cyclic nucleotide binding properties of these domains will be ascertained. Effects of binding site- or hydrolytic site-specific cGMP analogs on the enzyme will be tested. Purified protein kinases will be used to study phosphorylation of the enzyme and to determine the mechanism whereby phosphorylation is regulated in a substrate-directed manner by cGMP binding to B-PDE. Preliminary results indicate that the B-PDE is a relatively specific and potent substrate of cGMP-dependent protein kinase, an enzyme for which a physiological substrate has not yet been established. The functional domains of B-PDE will also be studied by generating proteolytic fragments which contain binding activity, hydrolytic activity, or the phosphorylation site. Fragments will be labeled by prior phosphorylation or (32P)cGMP photoaffinity- labeling of B-PDE, and characterized in order to orient the functional domains within the B-PDE. Partial amino acid sequencing of the fragments may reveal features required for function or phosphorylation. Peptides modeled after the sequences of phosphorylation site(s) will be synthesized and tested as substrates for protein kinases in order to identify determinants important for phosphorylation. Oligonucleotide probes based on sequences of peptide fragments, and polyclonal antibodies generated against B-PDE, will be used to screen a bovine lung cDNA library for purposes of cloning. This will permit deduction of the complete primary sequence, assessment of the domain structure, and may indicate evolutionary homologies with other proteins.

CGMP参与了多种哺乳动物的调节 与病理生理学有关的系统，包括平滑肌张力 在血管和其他组织、视力和血小板中 聚合。这项建议的目的是研究 一种cGMP结合的磷酸二酯酶(B-PDE)的调节 影响cGMP级联反应的代理的可能目标。这将是 通过纯化牛肺酶并测定其 结构和监管特征。B-PDE的功能可以 既受变构机制调节，又受 磷酸化。证据表明，这种酶含有 至少两个功能结构域--高度特异的cGMP结合 和一个单独的cGMP水解点。趁有机会 这些假定的域之间通信的证据，在那里 不是结合位点功能的直接证据。 B-PDE的四级结构、cGMP结合域的数目和 这些结构域的环核苷酸结合特性将是 已经确定了。结合部位或水解点特异性的影响 将对该酶上的cGMP类似物进行测试。纯化蛋白 激酶将被用来研究酶的磷酸化，并 确定调节磷酸化的机制。 通过cGMP与B-PDE结合以底物导向的方式。初步 结果表明，B-PDE是一种相对特异和有效的 CGMP依赖的蛋白激酶的底物，一种对其具有 生理底物尚未建立。这个 还将通过生成来研究B-PDE的功能域 含有结合活性的蛋白水解物片段， 水解性，或磷酸化部位。碎片将 被先前的磷酸化或(32P)cGMP光亲和标记- B-PDE的标记，并对其进行表征以定向 B-PDE内的功能域。部分氨基酸序列分析 可以揭示功能所需的特征或 磷酸化。根据以下序列模拟的多肽 磷酸化位点(S)将被合成并测试为 用于确定决定因素的蛋白激酶底物 对磷酸化很重要。基于基因的寡核苷酸探针 多肽片段序列和产生的多克隆抗体 针对B-PDE，将用于筛选牛肺cDNA文库 用于克隆的目的。这将允许扣除 完整的初级序列，结构域结构的评估，以及 可能表明与其他蛋白质在进化上的同源性。