STRUCTURE AND FUNCTION OF HEPARIN COFACTOR II

描述：确定了六个具体目标。其中包括：1) 肝素促进解离机制的研究 凝血酶之间的HCII突变体Leu444 Arg；2)测定 人工合成活性部位多肽对肝素功能的影响 辅因子2作为抑制剂或底物；3)识别 高亲和力结合所需的硫酸皮肤素结构 肝素辅因子II；4)结晶肝素辅因子II及其 X-射线分析用硫酸皮肤素络合物；5)至 内源性肝素辅因子II在人和人中的定位 免疫组织化学染色；6)同源制备 重组，纯合子肝素辅因子II缺陷小鼠。 第一个具体目标是基于调查员的观察 对于HCII Leu444 Arg，与凝血酶的络合物可以逆转 如果有肝素存在，而复合体在 硫酸皮肤素或在没有添加辅因子的情况下。数据 到目前为止获得的结果表明了一个模型，该模型涉及初始 抑制剂和酶的可逆复合体到最初可逆的 共价键，随后形成不可逆的十二烷基硫酸钠共价键。 对该突变体的预期研究将允许澄清和 对这一吸引人的机制的确认。在第二个具体目标中， 模拟所有蛇类中存在的反应位点环的多肽将 根据它们影响HCII抑制的能力进行评估 功能。与放射性标记结合的各种环肽 将使用不同的序列来评估 反应部位环路和测试板将是允许的。vbl.使用 结合和活性测量。该多肽还将允许评估 糖胺多糖对构象连接的影响 在HCII的不同状态之间。凝血酶抑制作用的评价 合成肽与HCII存在下的二元络合物 而糖胺多糖的缺失将通过活性来评估 与125[I]凝血酶形成共价络合物。第三 猪源特效靶向四、五硫化六糖 皮肤硫酸皮肤素将根据它们的 结构及其影响HCII结合和功能的能力。 方法的技术在鉴定中是相当常见的 涉及经典碳水化合物的糖胺多聚糖结构 化学反应。在进一步的具体目标中，HCII及其复合体将 在一次尝试中受到悬滴结晶技术的影响 以生产蛋白质及其复合体的晶体 结晶学评价。第五和第六个具体目标包括 小鼠HCII缺乏症模型的建立。研究涉及 在人类和小鼠系统中进行组织化学以识别 肝细胞癌的血管外部位。小鼠基因缺失的研究进展 胚胎干细胞中同源重组的模型 现在是标准技术。

DESCRIPTION: Six specific aims are identified. These include: 1) investigating the mechanism by which heparin facilitates dissociation between thrombin the HCII mutant Leu444 Arg; 2) to determine the affects of synthetic reactive site peptides on the ability of heparin cofactor 2 to serve as an inhibitor or substrate; 3) to identify structures in dermatan sulfate required for high-affinity binding to heparin cofactor II; 4) to crystallize heparin cofactor II and its complexes with dermatan sulfate for x-ray analysis; 5) to determine the location of endogenous heparin cofactor II in human and murine tissues by immunohistochemistry; 6) to prepare by homologous recombination, homozygous heparin cofactor II-deficient mice. The first Specific Aim is based on an observation by the investigator that for HCII Leu444 Arg, the complex with thrombin can be reversed if heparin is present, while the complex is stable in the presence of dermatan sulfate or in the absence of the added cofactor. The data obtained thus far suggests a model which involves the initial reversible complex of inhibitor and enzyme to an initially reversible covalent bond and subsequently to an SDS irreversible covalent bond. The studies anticipated with this mutant will allow elucidation and confirmation of this attractive mechanism. In the second Specific Aim, peptides which mimic the reactive site loop present in all serpins will be evaluated with respect to their ability to influence HCII inhibitory functions. Various loop peptides incorporating radiolabels with different sequences will be used to evaluate the interaction between the reactive site loop and the beta sheet will be permissible. Using binding and activity measurement. The peptide will also permit evaluation of the influence of glycosaminoglycans on the conformational-linkage between the various states of HCII. An evaluation of thrombin inhibition by binary complexes of synthetic peptide and HCII in the presence and absence of glycosaminoglycans will be evaluated using activity for the formation of covalent complexes with 125[I] thrombin. The third Specific Aim, tetra- and penta-sulfated hexasacchrides from porcine skin dermatan sulfate will be evaluated with respect to their structure and their ability to influence HCII binding and function. The techniques of approach are fairly common in the identification of glycosaminoglycan structure involved classical carbohydrate chemistry. In the further Specific Aim HCII and its complexes will be subjected to hanging drop crystallization techniques in an attempt to produce crystals of both the protein and its complexes suitable for crystallographic evaluation. The fifth and sixth Specific Aims involve the generation of a murine HCII deficiency model. Studies involve histochemistry in both human and murine systems to identify the extravascular sites of HCII. The development of a murine deletion model by homologous recombination in embryonic stem cells using the now standard techniques.