INTERFACIAL ACTIVATION OF WATER-SOLUBLE PHOSPHOLIPASES

拟议的工作试图理解复杂的界面行为 三种水溶性脂解酶：细菌非特异性 磷脂酶C(PLC)和磷脂酰肌醇特异性磷脂酶C (PI-PLC)和细菌和甘蓝磷脂酶D(PLD)。核磁共振，QLS， SANS和荧光技术与动力学技术结合使用 研究磷脂底物、类似物和抑制剂的分析 与每种酶的相互作用。一般问题包括：(I)如何 磷脂界面在不同的聚集结构中变化，并且可以 这些与磷脂酶的易感性有关吗？(Ii)如何 第二信使亲脂产物对磷脂酶作用的影响 底物聚集体？它们能潜在地调节磷脂酶吗？ (Iii)磷脂与这些物质结合的关键参数是什么？ 酶--无论是从磷脂结构还是从磷脂结构的角度 酶活性部位？(四)我们能否根据以下条件设计特定的抑制剂 这些结果呢？(5)能否制定一个统一的模式，说明 磷脂酶作为单体、胶束、洗涤剂对底物的活性 混合胶束和双层囊泡？ 具体目标包括：(1)测定合成物的构象 与PLC和PI-PLC结合的底物类似物；建议的PLC的检测 三方双锥反应中间体的时间分辨固体- 国家31P核磁共振技术；(2)底物头基的鉴定 使用表征良好的短链单体与PLC相互作用， 用于动力学的胶束和双层底物系统；用于克隆PLC Glu-4可能与胆碱相互作用的定点突变 N(CH3)3基团；(3)PLC对钒的抑制作用 建议的环状磷酸二酯中间体的模型；(4)测定 衬底横向和纵向扩散对PLC、PI-1的重要性。 可聚合磷脂的PLC和PLD；(5)合成和 肌醇C2‘和C6’修饰短链PI‘s TO的表征 定义环如何与PI-PLC相互作用；以及(6)荧光的使用- 标记DAGs用于监测不同磷脂之间的混合和转移 PA的聚集体及其表征对PLD的抑制作用及PA的作用 在不同的脂质聚集体上。 这些研究的结果应该会让我们更好地理解 这些磷脂酶的反应机理和产物， 特别是脂质第二信使和底物物理 特性影响磷脂酶活性。因为这些脂质第二次 信使激活细胞生长，它们与广泛的 疾病以及正常的细胞功能。

The proposed work seeks to understand the complex interfacial behavior of three water-soluble lipolytic enzymes: bacterial nonspecific phospholipase C (PLC) and phosphatidylinositol-specific phospholipase C (PI-PLC), and bacterial and cabbage phospholipase D (PLD). NMR, QLS, SANS, and fluorescence techniques are used in conjunction with kinetic analyses to investigate phospholipid substrate, analog, and inhibitor interactions with each enzyme. General questions include: (i) How do phospholipid interfaces vary in different aggregate structures, and can these be correlated with susceptibility to phospholipases? (ii) How do the second messenger lipophilic products of phospholipase action affect the substrate aggregate? Can they potentially regulate phospholipases? (iii) What are the critical parameters for phospholipid binding to these enzymes - both from the point of view of phospholipid structure and enzyme active sites? (iv) Can we design specific inhibitors based on these results? (v) Can a unified model be developed that accounts for phospholipase activity toward substrate as monomer, micelles, detergent mixed micelles, and bilayer vesicles? Specific aims include: (1) Determination of the conformation of synthetic substrate analogs bound to PLC and PI-PLC; detection of the proposed PLC trigonal bipyramidal reaction intermediate using time-resolved solid- state 31P NMR techniques; (2) Elucidation of substrate headgroup interactions with PLC using well-characterized short-chain monomer, micellar, and bilayer substrate systems for kinetics; cloning PLC for site-specific mutagenesis of Glu-4 suggested to interact with the choline N(CH3)3 moiety; (3) Characterization of vanadate inhibition of PLC as a model for a proposed cyclic phosphodiester intermediate; (4) Determining the importance of substrate lateral and vertical diffusion for PLC, PI- PLC, and PLD with polymerizable phospholipids; (5) Synthesis and characterization of inositol C2' and C6' modified short-chain PI's to define how the ring interacts with PI-PLC; and (6) Use of fluorescent- labeled DAGs to monitor mixing and transfer among different phospholipid aggregates and characterization of PA inhibition of PLD and effect of PA on different lipid aggregates. The results of these studies should provide a better understanding of the reaction mechanisms for these phospholipases and how products, specifically lipid second messengers, and substrate physical characteristics affect phospholipase activity. Since these lipid second messengers activate cell growth, they have relevance to a wide range of diseases as well as to normal cell functions.