VASOACTIVE PEPTIDES WHICH STIMULATE SMOOTH MUSCLES

我们的研究集中在两种酶的新功能， 最初命名为激肽酶I和II， 但是现在它们的重要性已经超出了水解肽。 我们的前两个具体目标与羧肽酶N（CPN）有关， 两个活性亚基和两个调节亚基的四聚体。 尼泊尔共产党分裂了 蛋白质和肽的C-末端碱性氨基酸。 我们将 确定每个亚基如何参与肌酸的转化 激酶的亚型。 我们将使用纯化的和重组的 酶，后者的数量足以用于X射线晶体学。 我们的计划是建立这两个亚基的基因组序列， 它们在不同的染色体上合成。 我们亦会探讨 人CPN与纤溶酶-纤溶酶原的复杂相互关系 系统，这可能会影响纤溶酶原激活和凝血。 我们 将决定CPN的亚基被纤溶酶切割的位置， 确定这种切割对稳定性和活性的影响， 并测量四聚体的解离。 此外，我们将看到如何 纤溶酶或纤溶酶原与活性物质形成复合物， 亚单位 我们研究的第三和第四个具体目标是： 血管紧张素转换酶（kininase II，ACE）的作用方式 抑制剂的 这些药物被数百万患者使用，其中一些 它们的有益作用归因于缓激肽的增强作用。 我们将使用转染人ACE和缓激肽cDNA的细胞 B2受体和组成型表达ACE和B2的培养细胞 研究信号释放增强的受体 B2受体配体的转导产物。 抑制剂和缓慢 水解底物能与膜的活性中心联合收割机结合， 结合ACE，从而增强7- 跨膜B2受体，即使激动剂对ACE有抗性。 因此，ACE抑制剂的作用不仅仅是保护缓激肽， 对抗失活，尽管它们不直接作用于其受体。 我们还将采用超微结构分析和显微镜检查， 功能活动的形态学相关性。 我们的最终目标是 为了显示ACE抑制剂如何发挥其对心脏的有益作用， 肾脏等，通过增强受体水平的肽作用。

Our investigation focus on the novel functions of two enzymes, originally named kininase I and II that cleave bonds of vasoactive peptides but now their importance extends beyond hydrolyzing peptides. Our first two specific aims are concerned with carboxypeptidase N (CPN), a tetramer of two active and two regulatory subunits. CPN cleaves the C-terminal basic amino acids of proteins and peptides. We will determine how each of the subunits is involved in converting creatine kinase to its subforms. We will use both purified and recombinant enzyme, the latter in quantities sufficient for X-ray crystallography. Our plan is to establish the genomic sequence of both subunits, although they are synthesized on different chromosomes. We will also explore the complex interrelationship of human CPN with the plasmin-plasminogen system, which may affect plasminogen activation and blood clotting. We will determine where the subunits of CPN are cleaved by plasmin, establish the consequences of this cleavage for stability and activity, and measure dissociation of the tetramer. Furthermore, we will see how a complex of the plasmin or plasminogen is formed with the active subunit. The third and fourth specific aims of our research are concerned with the mode of action of angiotensin converting enzyme (kininase II, ACE) inhibitors. These drugs are used by millions of patients, and some of their beneficial effects are attributed to potentiation of bradykinin. We will employ cells transfected with cDNA of human ACE and bradykinin B2 receptor and cultured cells constitutively expressing ACE and B2 receptors to investigate augmentation of the release of signal transduction products by B2 receptor ligands. Inhibitors and slowly hydrolyzed substrates can combine with the active center of membrane- bound ACE and thereby enhance the activity of ligands of the 7- transmembrane B2 receptor, even if the agonists are resistant to ACE. Thus, the actions of ACE inhibitor go beyond protecting bradykinin against inactivation, although they do not act directly on its receptor. We will also employ ultrastructural analysis and microscopy to establish morphologic correlates of functional activities. Our ultimate goal is to show how ACE inhibitors exert their beneficial effects on heart, kidney, etc., by enhancing peptide action at the receptor level.