IMMUNOCHEMISTRY OF STREPTOCOCCAL MEMBRANE PROTEINS

链球菌膜蛋白的免疫化学

• 批准号: 3129160
• 负责人: IVO J VAN DE RIJN
• 金额: $ 12.03万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-06-01 至 1991-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3129160
• 关键词: Streptococcus; bacterial proteins; enzyme linked immunosorbent assay; gel electrophoresis; genetic strain; glomerulonephritis; hybridomas; immunochemistry; laboratory mouse; laboratory rabbit; membrane activity; membrane proteins; membrane structure; microorganism culture; microorganism growth; microorganism immunology; monoclonal antibody; rheumatic fever

Group A and C streptococci have the ability to surround themselves with a capsule formed by hyaluronic acid (HA) which serves as a virulence factor. The structure of streptococcal HA is identical to mammalian HA where it is a ubiquitous component of connective tissue. HA is also synthesized and secreted by many differentiated cells in the body and plays a role in the morphogenesis of embryonic tissue, the invasiveness of carcinoma cells, and virus transformation of fibroblasts and chondrocytes. Finally, deregulation of the synthesis of HA appears to play an important role in Marfans syndrome and osteogenesis imperfecta. Therefore an examination of the mode of synthesis of this biological molecules is required in order to understand its role in the above processes. The cell free synthesis of HA was demonstrated in 1955 but attempts at solubilization of the synthetase have all failed until recently. Our laboratory is the first group to solubile the HA synthetase with its enzymatic activity intact. Furthermore, a requirement for phospholipid was demonstrated for both reactivation and enhancement of the enzsymatic activity. The overall objective of this proposal is to purify and characterize the HA synthetase from streptococci. Toward this objective we plan to isolate the streptococcal enzyme using affinity chromatography and specific probes. In addition the synthetase will be cloned into E. coli to engineer an overproducer of the synthetase. As the synthetase is isolated it will be characterized as to its physical properties, phospholipid requirements, substrate kinetics, size of product, role of primer, and location of addition of monosaccharides (reducing or nonreducing end). The completion of these specific aim should provide us with the information to understand the regulation of the production of this molecule in procaryote cells. Finally, these studies should add information useful in understanding the role of this molecule as a virulence factor in the poststreptococcal sequelae (acute rheumatic fever, glomerulonephritis) and its deregulation in such disease states as Marfans syndrome, osteogenesis imperfecta, and cancer.

A组和C链球菌有能力与 由透明质酸（HA）形成的胶囊，作为毒力因子。 链球菌HA的结构与哺乳动物HA相同 结缔组织的无处不在成分。 HA也已合成， 由体内许多分化细胞分泌，并在 胚胎组织的形态发生，癌细胞的侵入性和 成纤维细胞和软骨细胞的病毒转化。 最后， 放松HA合成的管制似乎在 Marfans综合征和成骨症Imperfecta。 因此检查 为了使该生物分子的合成模式是为了 了解其在上述过程中的作用。 HA的无细胞合成 在1955年被证明，但尝试溶解合成酶 直到最近都失败了。 我们的实验室是第一个 溶于HA合成酶，其酶活性完整。 此外，证明了对磷脂的要求 重新激活和增强结构合理活性。 总体 该建议的目的是净化和表征HA合成酶 来自链球菌。 朝向这个目标，我们计划隔离 使用亲和色谱和特定探针的链球菌酶。 在 补充合成酶将被克隆到大肠杆菌中以设计 合成酶的过度生产剂。 由于合成酶是孤立的，它将是 特征在其物理特性，磷脂需求中， 底物动力学，产品的大小，底漆的作用和位置 添加单糖（还原或非还原端）。 完成 这些特定目的应该为我们提供以了解的信息 调节该分子在procaryote细胞中的产生。 最后，这些研究应添加有关理解的信息 该分子作为毒力因子的作用 后遗症（急性风湿热，肾小球肾炎）及其放松管制 在这样的疾病状态下 癌症。