STUDIES ON ANTITHROMBIN III-THROMBIN-HEPARIN REACTIONS

拟议项目的长期目标是阐明 抗凝血酶III的化学性质和性质研究 肝素结合部位与蛋白酶结合的结构关系 这一蛋白质中的结合位点。具体来说，目标是：(1) 通过鉴定肝素结合部位来阐明肝素结合部位的性质 与肝素相互作用的特定赖氨酸残基。这一部分是 研究将涉及抗凝血酶III在存在时的赖氨酸修饰 用三硝基苯磺酸和还原剂脱除肝素 甲基化(HCHO/NaBH4)肝素掩盖的特定赖氨酸残基 将通过放射性同位素和氰化物分析进行鉴定 溴化物和蛋白质分解碎片。(2)澄清 酶结合部位的动力学和构象研究 凝血酶和凝血因子Xa与复性抗凝血酶的结合 III--失去60%肝素的抗凝血酶III的中间体 辅因子活性和42%的已知α-螺旋存在于 蛋白质，但仍具有结合凝血酶的能力。这一部分 这项研究将使用构象敏感的工具，如圆二色谱， 荧光光谱和吸收差分光谱。第一个目标 目的是检验肝素结合位点模型(多肽)的有效性 段281-289)。第二 目的是为了证明是否独立和 抗凝血酶中存在非相互作用的蛋白酶和肝素结合部位 三.预计本项目中提出的调查将 将使用一种改变形式的抗凝血酶III将提供有用的 了解抗凝血酶III复杂功能的模型 并最终开发出抗凝血酶的生理调节方法 三.为确定赖氨酸残基所必需的拟议研究 肝素结合有望为更好地理解提供信息 抗凝血酶III影响凝血的机制。 在这项研究的其他可视化结果中，有可能 可被认为是一种构象机制的识别 血浆蛋白水解酶-蛋白酶抑制剂相互作用的一般特征。它是 预计与揭示的分子构象细节一起 通过这些研究中提出的各种生物物理方法，相关性可以 抗凝血酶III的分子顺序与其生物学特性之间的关系 活动。

The long term goal of the proposed project is to elucidate the mechanism of ation of antithrombin III by investigation of the chemical nature and structural relationships of the heparin binding site and protease binding site in this protein. Specifically, the objectives will be: (1) to elucidate the nature of the heparin binding site by identifying the specific lysine residues which interact with heparin. This part of the study will involve lysine modification of antithrombin III in the presence and absence of heparin using trinitrobenzenesulfonic acid and reductive methylation (HCHO/NaBH4). The specific lysine residues masked by heparin will be identified by using radioactive isotopes and analysis of cyanogen bromide and proteolytic fragments. (2) to elucidate the nature of the protease binding site by investigation of the kinetics and conformational aspects of the binding of thrombin and Factor Xa with refolded antithrombin III - an intermediate form of antithrombin III which has lost 60% heparin cofactor activity and 42% of the Alpha-helix known to be present in the protein but still possesses the capacity to bind thrombin. This part of the study will use conformation-sensitive tools such as circular dichroism, fluorescence and absorption difference spectroscopy. The first objective intends to test the validity of the heparin binding site model (peptide segment 281-289) predicted by the principal investigator. The second objective is intended to prove whether or not independent and non-interacting protease and heparin binding sites exist in antithrombin III. It is expected that the investigations proposed in this project which will utilize an altered form of antithrombin III will provide a useful model by which we can understand the complex function of antithrombin III and ultimately develop methods for physiological regulation of antithrombin III. The proposed study to identify the lysine residues essential for heparin binding is expected to provide information for better understanding of the mechanism by which antithrombin III affects blood coagulation. Among other outcomes visualized from this research is the possible identification of a conformational mechanism which might be considered a general feature of plasma protease-proteinase inhibitor interaction. It is expected that together with the details of molecular conformation revealed by various biophysical methods proposed in these studies, correlation can be made between the molecular order of antithrombin III and its biological activities.