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: 3240109
• 负责人: JACKIE David CORBIN
• 金额: $ 20.45万
• 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/3240109
• 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，已经指出cGMP依赖性蛋白 激酶作为cGMP诱导的血管平滑肌舒张的介质 响应于激动剂如心房利钠因子， 硝基血管扩张剂（例如，硝酸甘油），甲基黄嘌呤（例如，咖啡因） 和代谢物（例如，EDRF）。这一建议主要基于两个 本实验室发现的cGMP依赖性蛋白激酶的特征 （1）血管平滑肌组织可溶性组分 含有一种新的同工酶形式，命名为I型β，除了 在其他组织中发现的特征良好的I型α;（2） 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。组织和种类分布 将检查I型α和I型β。新发现的I型β 将被纯化至同质并确定它是否是不同的基因 I型α的产物。其他物理和动力学特性 比较两种酶的底物，包括蛋白质和肽 的特异性如果确定I型β和I型α一样， 两个不同的cGMP结合位点上的每两个亚基，然后一个彻底的 cGMP结合动力学和cGMP类似物对每个位点特异性的研究 将进行。在可能的情况下，两种同工酶将用于研究 每个cGMP结合位点的功能，通过分离cGMP缺陷和 cGMP结合形式的酶。对于其中一些实验，酶 含有在特定位点结合的cGMP的药物将表征如下 最近，通过cGMP诱导的DEAE“电荷移位”程序分离 确立了习“电荷转移”也将被用作一种新的方法， 确定cGMP或cAMP是否与I型α或I型β的每个位点结合 在血管平滑肌的基础状态，这可能是一个 “启动”装置用于激活;并确定各种试剂是否 调节cGMP水平的药物，以及 调节cAMP和钙，将改变环核苷酸的量 绑定到每个站点。链内与链间相互作用在 cGMP激活机制也将通过研究 单体激酶（天然酶是二聚体），最近发现是 由天然酶的蛋白水解产生。还补充了 测序和cGMP结合位点的未来研究将是 尝试克隆I型α和I型β的cDNA和基因组DNA。