VITAMIN K-DEPENDENT PLASMA PROTEINS

维生素 K 依赖性血浆蛋白

• 批准号: 3335045
• 负责人: Gary L Nelsestuen
• 金额: $ 23.02万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3335045
• 关键词: blood coagulation disorders; calcium binding protein; coagulation factor V; coagulation factor VIII; complement; conformation; cow; fluorescence; gamma carboxyglutamate; light scattering; membrane activity; membrane proteins; nuclear magnetic resonance spectroscopy; phospholipids; protein C; protein S; prothrombin; vitamin K

This project will continue a long-standing investigation into the role of gamma-carboxyglutamic acid (Gla). This structure is involved in calcium binding to the vitamin K-dependent blood clotting proteins of the plasma and their subsequent interaction with membrane surfaces. In the membrane-bound state, the enzymes of the blood coagulation cascade are much more active in conversion of their substrates (zymogens) to active proteases. This project covers nearly all aspects of these reactions. Metal ion binding is investigated and attempts will be made to identify specific amino acids involved in ion chelation or in subsequent protein conformational changes. These studies will involve modification with chemical reagents followed by analysis of protein structure to determine if specific sites are modified, if they are protected by metal ions and if there is metal ion specificity for the protection (e. g. Ca versus Mg). For example, we have already shown that the amino terminal is required for the membrane-binding event, it is protected by calcium but not by magnesium. Another site (residues 101 or 102) is protected by either Mg or Ca. Chemically modified proteins will be studied to examine their metal ion and membrane binding properties. Chemical modification will also be used to generate proteins with spectroscopic probes at specific sites. These probes will allow investigation of membrane-binding properties. A protein with a built-in fluorescent probe is protein Z and this will be the focus of many studies including the isolation of residues 1-46 (these contain aH the Gla residues of protein Z) and thorough examination of its metal ion and membrane-binding properties. Membrane binding is essential for maximum activity of the prothrombinase complex. This enzymatic complex will be examined with special attempts to determine if it has the kinetic properties of a collisionally limited reaction. This is important to determine if the kinetic parameters obtained for pure systems in vitro can be extrapolated to physiological circumstances. The difference might consist of the number of complexes per particle. In vitro, with one or a few enzyme complexes, the reaction may conform to normal Michaelis-Menton kinetics. However, if the number of enzymes per particle is large in vivo, the type of kinetics may be altered. We will try to demonstrate this behavior by studying the kinetics of prothrombinase with various numbers of enzymes per vesicle. This kind of kinetic behavior will also be investigated with several other systems. Overall, these studies will add to our knowledge of the important blood clotting plasma proteins. Findings may reveal essential structural features of these proteins and lead to new understanding of the blood clotting process. The findings will also reveal general characteristics of membrane-binding events and provide a basis for investigation of an expanding class of peripheral membrane-binding proteins.

该项目将继续对以下方面的作用进行长期调查： γ-羧基谷氨酸（Gla）。这种结构与钙有关 与血浆中维生素K依赖性凝血蛋白结合 以及它们随后与膜表面的相互作用。在 在膜结合状态下，血液凝固级联的酶多 在将其底物（酶原）转化为活性物质方面更有活性 蛋白酶该项目几乎涵盖了这些反应的所有方面。金属 离子结合进行了调查，并试图确定具体的 参与离子螯合或随后蛋白质中的氨基酸 构象变化这些研究将涉及修改， 化学试剂，然后分析蛋白质结构，以确定 如果特定位点被金属离子保护， 存在金属离子对保护的特异性（例如，G. Ca对Mg）。为 例如，我们已经表明，氨基末端是必需的， 在膜结合事件中，它受到钙的保护，但不受镁的保护。 另一个位点（残基101或102）被Mg或Ca保护。 化学修饰的蛋白质将被研究，以检查它们的金属离子， 膜结合性能。化学修饰也将用于 在特定位点用光谱探针产生蛋白质。这些探针 将允许研究膜结合特性。一种蛋白质 内置的荧光探针是蛋白质Z，这将是许多人关注的焦点。 包括残基1-46（这些含有α H Gla）的分离的研究 蛋白质Z的残留物），并对其金属离子进行彻底检查， 膜结合特性。 膜结合对于凝血酶原酶的最大活性是必需的 复杂.这种酶复合物将被特别尝试检查， 确定它是否具有碰撞限制的动力学特性 反应这对于确定所获得的动力学参数是否 对于体外纯系统，可以外推到生理 情节不同之处可能在于每个复合体的数量 粒子在体外，用一种或几种酶复合物，反应可以 符合正常的Michaelis-Menton动力学。但是，如果 如果每个颗粒的酶在体内是大的，则动力学的类型可以改变。 我们将尝试通过研究以下的动力学来证明这种行为： 每个囊泡具有不同数量的酶的凝血酶原酶。这种 还将用几种其它系统研究动力学行为。 总的来说，这些研究将增加我们对重要血液的认识， 凝血血浆蛋白研究结果可能揭示了基本的结构特征 并导致对血液凝固的新认识 过程研究结果还将揭示 膜结合事件，并提供了一个基础的调查， 扩展类外周膜结合蛋白。