PURIFICATION AND MECHANISM OF INTERFERON RECEPTORS

干扰素受体的纯化及其机制

• 批准号: 3445548
• 负责人: ANDREW A BRANCA
• 金额: $ 5.42万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1988-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3445548
• 关键词: affinity chromatography; affinity labeling; analytical chemistry; antibody formation; bactericidal immunity; carbohydrate structure; cell type; covalent bond; density gradient ultracentrifugation; electrofocusing; gel filtration chromatography; high performance liquid chromatography; human tissue; interferon inducers; interferons; ion exchange chromatography; molecular biology; monoclonal antibody; radiotracer; surface antigens; tissue /cell culture

The research in this application will accomplish three specific objectives regarding the biochemistry and molecular biology of the Type I interferon (IFN) receptor. The first objective is to develop procedures for the preparation of highly purified interferon receptor from human and bovine tissues. There are, to our knowledge, no receptor studies deomonstrating the binding of radiolabeled IFN to receptors derived from the homogenates of human and animal tissues. The procedures in this proposal include: the preparation of membranes by differential centrifugation of tissue homogenates; extraction of the interferon receptor from the membranes with non-denaturing detergents and the subsequent purification of the receptor by ion-exchange and gel-permeation chromatography, preparative electrophoresis, isoelectric focusing, high performance liquid chromatography (HPLC), lectin-specific and affinity chromatography. The second objective is to provide a rigorous biochemical characterization of the Type I interferon receptor. The characterization will include: the use of density gradient centrifugation, gel-filtration, isoelectric focusing, covalent and photoaffinity labeling and determination of putative carbohydrate moieties of the receptor. Information about the molecular weight, possible subunit composition and carbodydrate composition of the receptor will be obtained. The third objective is to examine the role of the Type I IFN receptor in the interferon response in cultured cells. Through the use of photoaffinity labeling the determination of Type I interferon receptor structures in a series of cell lines which exhibit differential responses to interferon will be compared to the effect(s) of antireceptor monoclonal antibodies on the biological activities of interferon. It is intended that possible differences in the structure of the receptors will be related to the biological activities of interferon and that this correlation will serve to establish that there are separate and distinct effector systems which can interact with the Type I interferon receptor and elicit the well-established variety of biological responses of interferons. The existence of different effector systems for the Type I interferon receptor would provide for an explanation of the observed deficiencies of certain cell types with respect to the antiviral and, in particular, the antiproliferative activities of interferon.

该应用程序中的研究将实现三个特定目标 关于I型干扰素的生物化学和分子生物学 （IFN）受体。 第一个目标是为 从人和牛制备高度纯化的干扰素受体 组织。 据我们所知，没有受体研究去启示 放射性标记IFN与源自匀浆的受体的结合 人类和动物组织。 该提案中的程序包括： 通过组织的差异离心制备膜 匀浆；从膜上提取干扰素受体与 非污染洗涤剂和随后的受体纯化 由离子 - 交换和凝胶 - 渗透色谱法，制备者 电泳，等电聚焦，高性能液体 色谱（HPLC），凝集素特异性和亲和力色谱法。 这 第二个目标是提供严格的生化特征 I型干扰素受体。 特征将包括： 使用密度梯度离心，凝胶过滤，等电的使用 聚焦，共价和光性标签和假定的确定 受体的碳水化合物部分。 有关分子的信息 重量，可能的亚基组成和碳水化合物的组成 将获得受体。 第三个目标是检查 培养细胞中干扰素反应中的I型IFN受体。 通过使用光亲和标记I型的确定 一系列细胞系中的干扰素受体结构 将对干扰素的差异反应与效果进行比较 对生物学活性的抗受体单克隆抗体 干扰素。 打算在结构上可能存在差异 受体将与干扰素的生物学活性有关 并且这种相关性将有助于确定有不同的 以及可以与I类干扰素相互作用的独特效应系统 受体并引起良好的生物学反应的种类 干扰素。 I型I类型的效应系统的存在 干扰素受体将提供观察到的 某些细胞类型的缺陷相对于抗病毒，在 特别是干扰素的抗增殖活性。