VASOACTIVE PEPTIDES WHICH STIMULATE SMOOTH MUSCLES

我们的研究重点是两种酶的新功能， 最初命名为激氨酸酶I和激氨酸酶II，它断裂血管活性的键 多肽，但现在它们的重要性已经超越了水解肽。 我们的前两个具体目标与羧基肽酶N(CPN)有关， 由两个活性亚基和两个调节亚基组成的四聚体。CPN裂解了 蛋白质和多肽的C末端碱性氨基酸。我们会 确定每个亚基如何参与肌酸的转化 将其转化为其亚型。我们将同时使用纯化和重组 酶，后者的数量足以进行X射线结晶学研究。 我们的计划是建立这两个亚基的基因组序列，尽管 它们是在不同的染色体上合成的。我们还将探索 人CPN与纤溶酶-纤溶酶原的复杂关系 系统，这可能会影响纤溶酶原激活和血液凝固。我们 将确定CPN的亚基被纤溶酶切割的位置， 确定这种分裂对稳定和活动的影响， 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体 亚单位。 我们研究的第三和第四个具体目标涉及 血管紧张素转换酶(Kininase II，ACE)的作用方式 抑制剂。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对缓激肽的增强。 我们将使用转染人血管紧张素转换酶和缓激肽的细胞 B2受体及其组成性表达ACE和B2的培养细胞 受体研究信号释放的增强 B2受体配体的转导产物。缓释剂和缓释剂 水解后的底物可以与膜的活性中心结合- 结合血管紧张素转换酶，从而增强7- 跨膜B2受体，即使激动剂对血管紧张素转换酶具有耐药性。 因此，血管紧张素转换酶抑制剂的作用超出了保护缓激肽的范围 抗失活，尽管它们不直接作用于其受体。 我们还将使用超微结构分析和显微镜来确定 功能活动的形态关联。我们的最终目标是 为了展示血管紧张素转换酶抑制剂如何对心脏发挥有益作用， 通过在受体水平上增强多肽的作用而发挥对肾脏等的作用。

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.