DIFFERENT ISOZYMIC FORM OF CGMP-DEPENDENT PROTEIN KINASE

最近的研究，包括我们自己的研究，已经指出cGMP依赖的蛋白质 肌动蛋白作为环鸟苷诱导的血管平滑肌松弛的介导物 作为对心钠素等激动剂的反应， 硝基血管扩张剂(如硝化甘油)、甲基黄嘌呤(如咖啡因) 和代谢物(例如EDRF)。这项建议主要是基于两个 本实验室发现的cGMP依赖蛋白激酶的特性 过去一年：(1)血管平滑肌组织的可溶性部分 包含一种新的同工酶形式，命名为类型Iβ，除了 在其他组织中发现的具有良好特征的I型α；(2) CGMP依赖的蛋白激酶以层析分离的形式存在 CGMP缺乏和cGMP结合形式。组织和物种的分布 将检查I型阿尔法和I型贝塔。新发现的I型贝塔病毒 将被提纯到同质性，并确定它是否是一个不同的基因 来自第一类阿尔法的产品。其他物理和动力学特征 将对两种酶进行比较，包括蛋白质和多肽底物。 专一性。如果确定I型贝塔，就像I型阿尔法一样， 在两个亚基上各有两个不同的cGMP结合位点，然后彻底 CGMP结合动力学及各部位cGMP类似物特异性的研究 将会被承担。在可能的情况下，两种同工酶都将被用于研究 每个cGMP结合位点通过分离cGMP缺陷和 CGMP结合形式的酶。在其中一些实验中，酶 含有结合在特定位点的cGMP的特征如下 新近通过cGMP诱导的DEAE“电荷转移”程序分离 已经成立了。“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结合 在血管平滑肌的基础状态下，这可能作为一种 “预置”装置用于激活；并确定各种试剂是否 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。链内相互作用与链间相互作用在 CGMP的激活机制也将通过对 单体激酶(天然的酶是二聚体)，最近发现是 由天然酶的蛋白质分解产生的。也是对 对cGMP结合位点的测序和未来的研究将是 尝试克隆I型α和I型β的cDNAs和基因组DNA。

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.