DIFFERENT ISOZYMIC FORM OF CGMP-DEPENDENT PROTEIN KINASE

CGMP 依赖性蛋白激酶的不同同工形式

• 批准号: 3240107
• 负责人: JACKIE David CORBIN
• 金额: $ 19.96万
• 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: 1989-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240107
• 关键词: atrial natriuretic peptide; calcium; chemical binding; complementary DNA; cyclic AMP; cyclic GMP; cyclic nucleoside monophosphate; enzyme mechanism; enzyme structure; enzyme substrate; guinea pigs; high performance liquid chromatography; ion exchange chromatography; isozymes; laboratory rabbit; laboratory rat; molecular cloning; nitroglycerin; nucleic acid probes; protein kinase; protein purification; protein sequence; vascular smooth muscle

Recent studies, including our own, have pointed to cGMP-dependent protein kinase as the mediator of cGMP-induced relaxation of vascular smooth muscle in response to agonists such as atrial natriuretic factor, nitrovasodilators (e.g., nitroglycerin), methylxanthines (e.g., caffeine) and metabolites (e.g., EDRF). This proposal is based primarily on two features of cGMP-dependent protein kinase discovered in this laboratory over the past year: (1) Soluble fractions of vascular smooth muscle tissues contain a novel isozymic form named type I beta, in addition to the well-characterized type I alpha found in other tissues; (2) The cGMP-dependent protein kinase exists as chromatographically separable cGMP-deficient and cGMP-bound forms. The tissue and species distribution of types I alpha and I beta will be examined. The newly discovered type I beta will be purified to homogeneity and determined if it is a different gene product from type I alpha. Other physical and kinetic characteristics of the two enzymes will be compared, including protein and peptide substrate specificity. If it is established that type I beta, like type I alpha, has two different cGMP binding sites on each of two subunits, then a thorough study of cGMP binding kinetics and cGMP analog specificity for each site will be undertaken. Where possible, both isozymes will be utilized to study the function of each cGMP binding site by isolating cGMP-deficient and cGMP-bound forms of the enzymes. For some of these experiments, enzyme containing cGMP bound at particular sites will be characterized following isolation by the cGMP-induced DEAE "charge shift" procedure recently established. The "charge shift" will also be used as a novel approach to determine if cGMP or cAMP is bound to each site of type I alpha or I beta in the basal state of vascular smooth muscle, which might act as a "priming" device for activation; and to determine if the various agents mentioned above which modulate cGMP levels,.as well as agents which modulate cAMP and calcium, will change the amount of cyclic nucleotide bound to each site. The role of intra-versus interchain interactions in the cGMP activation mechanism will also be investigated by studies of the monomeric kinase (the native enzyme is dimeric), found recently to be produced by proteolysis of the native enzyme. Also complementary for sequencing and for future studies of the cGMP binding sites will be the attempted cloning of the cDNA and genomic DNA for type I alpha and I beta.

最近的研究，包括我们自己的研究，都指出了 cGMP 依赖性蛋白质 激酶作为 cGMP 诱导血管平滑肌松弛的介质 响应心房钠尿因子等激动剂， 硝基血管扩张剂（例如硝酸甘油）、甲基黄嘌呤类药物（例如咖啡因） 和代谢物（例如 EDRF）。该提议主要基于两个 本实验室发现的cGMP依赖性蛋白激酶的特征 近一年：（1）血管平滑肌组织可溶性部分 含有一种名为 I 型 beta 的新型同工酶形式 在其他组织中发现的特征明确的 I 型 α； (2) 的 cGMP 依赖性蛋白激酶以色谱分离形式存在 cGMP 缺陷型和 cGMP 结合型。组织和物种分布 将检查 I alpha 和 I beta 型。新发现的I型β 将被纯化至同质性并确定它是否是不同的基因 I 型 alpha 的产品。其他物理和动力学特性 将比较两种酶，包括蛋白质和肽底物 特异性。如果确定 I 型 beta 与 I 型 alpha 一样，具有 两个亚基各有两个不同的 cGMP 结合位点，然后进行彻底的 研究每个位点的 cGMP 结合动力学和 cGMP 类似物特异性 将进行。在可能的情况下，两种同工酶都将用于研究 通过隔离 cGMP 缺陷和每个 cGMP 结合位点的功能 酶的 cGMP 结合形式。对于其中一些实验，酶 含有结合在特定位点的 cGMP 的特征如下 最近通过 cGMP 诱导的 DEAE“电荷转移”程序进行隔离 已确立的。 “电荷转移”也将被用作一种新颖的方法 确定 cGMP 或 cAMP 是否与 I 型 α 或 I β 的每个位点结合 在血管平滑肌的基础状态下，这可能充当 用于激活的“启动”装置；并确定各个代理是否 上面提到的调节 cGMP 水平的药物，以及 调节cAMP和钙，会改变环核苷酸的量 绑定到每个站点。链内与链间相互作用的作用 cGMP 激活机制也将通过以下研究进行调查： 单体激酶（天然酶是二聚体），最近发现 由天然酶的蛋白水解产生。也补充了 测序和 cGMP 结合位点的未来研究将是 尝试克隆 I 型 α 和 I β 的 cDNA 和基因组 DNA。