SULFATION OF HUMAN PLASMA PROTEINS

人血浆蛋白的硫酸化

• 批准号: 3466248
• 负责人: GLEN L HORTIN
• 金额: $ 10.86万
• 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/3466248
• 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盐化的酶学研究 蛋白质。三种人类血浆蛋白--第四组分 补体(C4)、肝素辅因子II和α2-抗纤溶酶-- 其中含有酪氨酸硫酸盐残基的将被详细分析。 目标是：1)确定位置和化学计量比 这些蛋白质的硫酸盐化，2)检查硫酸盐化的效果 关于这些蛋白质的活性和稳定性，3)表征 硫酸盐化过程和作为其中介的磺基转移酶， 4)研究硫酸盐结构域的功能 在这些蛋白质中，5)确定是否有生理上的 或蛋白质硫酸盐化过程中的病理变化。 含有这些蛋白质的硫化位点的多肽有 通过化学合成和裂解纯化的 含溴氰化物的蛋白质。这些多肽将用于 结构研究，硫酸盐特异性抗血清的制备 蛋白质的片段，并检查其功能 含硫酸盐的结构域。硫酸盐化对黄曲霉毒素的影响 C4、肝素辅助因子II和α2-抗纤溶酶的功能 通过比较活性和稳定性来直接检查 这些蛋白质的硫酸盐化和非硫酸盐化形式。非硫酸盐化 通过与HepG2细胞孵育将获得不同形式的蛋白质 硫酸盐化抑制剂或通过将蛋白质与 芳基硫酸盐酶。HepG2细胞将作为一个模型系统 研究这些蛋白质的硫酸盐化及其影响 硫酸盐浓度、激素和药物对这一过程的影响。 蛋白质硫酸盐化的酶学分析将通过 大鼠肝脏磺基转移酶的纯化。合成的 与硫化部位相对应的多肽将被用作 亲和纯化酶的配体和底物 用来分析它的活动。最后，这项研究将检验 无论是生理上还是病理上的变化 蛋白质的硫酸盐化。血浆样本将通过 用电泳法测定硫酸根和硫酸根的相对含量 特定血浆蛋白的非硫酸盐化形式。