FUNCTIONAL DOMAINS IN PROTHROMBIN AND FACTOR X

这一建议是基于如下假设：凝血酶原和X因子 (因子Xa)包含离散的功能域，涉及 这些生物活性的表达的离散部分 复杂的蛋白质。建议通过组合来识别这些区域 使用特定的化学修饰和有限的蛋白质降解。凝血酶原 将用2-羟基-5-硝基苄基溴或 四硝基甲烷。将对修饰蛋白(S)进行尊重分析 到功能性质的修改和改变的程度。这个 选定的两种试剂可鉴定功能性色氨酸和酪氨酸 残留物，此外，还为 介绍一种光谱和荧光结构探针 属性可以测量修改后的 残留物。存在时天然凝血酶原的有限蛋白分解 以及不存在二价阳离子、磷脂等配体 凝血酶原酶复合体的成分将为 与天然凝血酶原比较。类似的研究也被提议用于 X因子和Xa因子的功能结构域的阐明。初级阶段 我们将致力于Xa因子的研究，因为我们的重点是 用于凝血酶原酶复合体的研究。建议数 实验将提供对凝血酶原酶的分子理解 很复杂。这样的理解对于阐明 正常凝血机制。

This proposal is based on the hypothesis that prothrombin and factor X (factor Xa) contain discrete functional domains which are involved in discrete portions of the expression of the biological activity of these complex proteins. It is proposed to identify these regions by the combined use of specific chemical modification and limited proteolysis. Prothrombin will be modified with 2-hydroxy-5-nitrobenzyl bromide or with tetranitromethane. The modified protein(s) will be analyzed with respect to the extent of modification and changes in functional properties. The two reagents selected can identify functional tryptophanyl and tyrosyl residues respectively and, in addition, provide the basis for the introduction of structural probes whose spectral and fluorescence properties can measure changes in the microenvironment around the modified residues. Limited proteolysis of the native prothrombin in the presence and absence of ligands such as divalent cations, phospholipid and other components of the prothrombinase complex will provide derivatives for comparison with native prothrombin. Similar studies are proposed for the elucidation of functional domains in factor X and factor Xa. The primary effort will be directed toward the study of factor Xa since our emphasis is directed toward the study of the prothrombinase complex. The proposed experiments will provide a molecular understanding of prothrombinase complex. Such understanding is critical to the elucidation of the mechanism of normal blood coagulation.