VITAMIN K-DEPENDENT PLASMA PROTEINS

该项目将继续长期调查 γ-羧基谷氨酸(GLA)。这种结构与钙有关 与血浆中依赖维生素K的凝血蛋白结合 以及它们随后与膜表面的相互作用。在 膜结合状态下，凝血级联的酶多 在底物(酵素)转化为活性的过程中更活跃 蛋白酶。这个项目几乎涵盖了这些反应的所有方面。金属 对离子结合进行了调查，并将尝试确定特定的 参与离子螯合或后续蛋白质的氨基酸 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 如果特定的位置受到金属离子的保护，并且如果 这种保护具有金属离子的特异性(例如，钙离子对镁离子)。为 例如，我们已经展示了氨基末端是 膜结合事件，它受钙的保护而不受镁的保护。 另一个位点(残基101或102)受镁或钙的保护。 化学修饰的蛋白质将被研究，以检测它们的金属离子和 膜结合特性。化学修饰也将用于 在特定的位置用光谱探针产生蛋白质。这些探头 将允许对膜结合特性进行研究。一种含有一种 内置的荧光探针是蛋白质Z，这将是许多 研究包括残基1-46的分离(这些残基含有GLA 蛋白质Z)的残留物，并对其金属离子和 膜结合特性。 膜结合是凝血酶原酶最大活性的关键 很复杂。这种酶复合体将通过特殊的尝试进行检测 确定它是否具有碰撞受限的动力学特性 反应。这对于确定所获得的动力学参数是否 对于纯系统来说，体外可以外推到生理 情况。差异可能包括每种类型的复合体的数量 粒子。在体外，用一个或几个酶复合体，反应可能 符合正常的米氏动力学。但是，如果 体内每个颗粒的酶很大，动力学类型可能会改变。 我们将试图通过研究这种行为的动力学 凝血酶原酶，每个小泡有不同数量的酶。这样一种 动力学行为也将与其他几个系统一起研究。 总体而言，这些研究将增加我们对重要血液的了解 凝结血浆蛋白。研究结果可能揭示基本的结构特征 使我们对血液凝结有了新的认识 进程。这些发现还将揭示出 膜结合事件，并为研究一种 不断扩大的外周膜结合蛋白类。

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.