CGMP-BINDING PHOSPHODIESTERASE: REGULATORY MECHANISMS

cGMP参与调节许多哺乳动物的 与病理生理学相关的系统，包括平滑肌张力 在血管和其他组织、视力和血小板中 聚合来 这项建议的目的是审查 cGMP结合磷酸二酯酶（B-PDE）的调节， 影响cGMP级联的药物的可能靶点。 这将 通过纯化牛肺酶并测定其 结构和监管特点。 B-PDE的功能可以 受变构机制和 磷酸化 证据表明这种酶含有 至少有两个功能结构域， 位点和单独cGMP水解位点。 虽然 这些假定域之间的通信的证据，有 没有直接证据表明结合位点的功能。 的 B-PDE的四级结构、cGMP结合结构域的数量和 这些结构域的环核苷酸结合性质将是 确定。 结合位点特异性或水解位点特异性 将测试酶上的cGMP类似物。 纯化蛋白 激酶将用于研究酶的磷酸化， 确定磷酸化调节的机制， 通过cGMP与B-PDE结合的底物导向方式。 初步 结果表明，B-PDE是一种相对特异、有效的 cGMP依赖性蛋白激酶的底物， 生理底物尚未建立。 的 B-PDE的功能结构域也将通过生成 含有结合活性的蛋白水解片段， 水解活性或磷酸化位点。 片段将 通过预先磷酸化或（32 P）cGMP光亲和标记- B-PDE的标记，并进行表征，以定向 B-PDE内的功能域。 部分氨基酸测序 可能揭示功能所需的特征， 磷酸化 肽模仿的序列 将合成磷酸化位点并进行测试， 蛋白激酶的底物，以确定决定簇 对磷酸化很重要。 寡核苷酸探针基于 肽片段的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 为了克隆。 这将允许扣除 完整的一级序列，结构域结构的评估，以及 可能表明与其他蛋白质的进化同源性。

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.