SULFATION OF HUMAN PLASMA PROTEINS

人血浆蛋白的硫酸化

• 批准号: 3466246
• 负责人: GLEN L HORTIN
• 金额: $ 8.32万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466246
• 关键词: antithrombin III; blood proteins; high performance liquid chromatography; human tissue; laboratory rabbit; laboratory rat; stoichiometry; sulfation; sulfotransferase; tissue /cell culture

This study investigates the occurrence, physiological function, and enzymology of the sulfation of tyrosine residues in human plasma proteins. Three human plasma proteins-- the fourth component of complement (C4), heparin cofactor II, and alpha2-antiplasmin-- which contain tyrosine sulfate residues will be analyzed in detail. Objectives are: 1) To identify the sites and stoichiometry of sulfation of these proteins, 2) To examine the effects of sulfation on activity and stability of these proteins, 3) To characterize the process of sulfation and the sulfotransferase which mediates it, 4) To investigate the functions of the sulfate-containing domains in these proteins, 5) To determine whether there is physiological or pathological variation in the sulfation of proteins. Peptides containing the sites of sulfation of these proteins have been prepared by chemical synthesis and by cleavage of the purified proteins with cyanogen bromide. These peptides will be used for structural studies, generation of antisera specific for sulfated segments of the proteins, and examination of the function of sulfate-containing domains. The effects of sulfation on the function of C4, heparin cofactor II, and alpha2-antiplasmin will be examined directly by comparing the activity and stability of sulfated and nonsulfated forms of these proteins. Nonsulfated forms of protein will be obtained by incubating HepG2 cells with sulfation inhibitors or by incubating the proteins with arylsulfatase. HepG2 cells will serve as a model system for investigating the sulfation of these proteins and the influence of sulfate concentration, hormones, and drugs on this process. Enzymology of the sulfation of proteins will be analyzed by purification of the sulfotransferase from rat liver. Synthetic peptides corresponding to sites of sulfation will be used as ligands for affinity purification of the enzyme and as substrates for assaying its activity. Finally, this study will examine whether there is physiological or pathological variation in the sulfation of proteins. Plasma specimens will be analyzed by electrophoresis to measure the relative quantities of sulfated and nonsulfated forms of specific plasma proteins.

本研究探讨了其发生、生理功能和 人血浆中酪氨酸残基硫酸化的酶学 proteins. 三种人类血浆蛋白-- 补体（C4）、肝素辅因子II和α 2-抗纤溶酶-- 其含有酪氨酸硫酸盐残基。 目的是：1）确定的网站和化学计量的 这些蛋白质的硫酸化，2）为了检查硫酸化的影响， 对这些蛋白质的活性和稳定性的影响，3）为了表征这些蛋白质， 硫酸化过程和介导该过程的磺基转移酶， 4)研究含硫酸盐结构域的功能 5）为了确定是否存在生理上的 或蛋白质硫酸化的病理变化。 含有这些蛋白质硫酸化位点的肽具有 通过化学合成和裂解纯化的 蛋白质与溴化氰。 这些肽将用于 结构研究，生成硫酸化的特异性抗血清 蛋白质的片段，并检查蛋白质的功能。 含硫酸盐的结构域。 硫酸盐化对 C4、肝素辅因子II和α 2-抗纤溶酶的功能将 通过比较活性和稳定性直接检查 这些蛋白质的硫酸化和非硫酸化形式。 非硫酸化 通过将HepG 2细胞与 硫酸化抑制剂或通过将蛋白质与 芳基硫酸酯酶 HepG 2细胞将作为模型系统， 研究这些蛋白质的硫酸化以及 硫酸盐浓度、激素和药物对这一过程的影响。 蛋白质硫酸化的酶学将通过 从大鼠肝脏中纯化磺基转移酶。 合成 对应于硫酸化位点的肽将用作 用于酶亲和纯化的配体和作为底物 来检测它的活性 最后，本研究将探讨 是否存在生理或病理变化， 蛋白质的硫酸化。 血浆标本将由以下人员进行分析： 电泳以测量硫酸化的和 特定血浆蛋白的非硫酸化形式。