CELL DIVISION AND SURFACE GROWTH IN A MODEL SYSTEM

模型系统中的细胞分裂和表面生长

• 批准号: 3124875
• 负责人: MICHAEL L HIGGINS
• 金额: $ 17.76万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-05-01 至 1994-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3124875
• 关键词: Enterococcus; NAD(P)H dehydrogenase; bacterial genetics; binding proteins; biological models; carbon; cell cycle; cell growth regulation; cell wall; density gradient ultracentrifugation; developmental genetics; electron microscopy; electron transport; flavoproteins; immunochemistry; leukocyte oxidative burst; macromolecule; membrane activity; neutrophil; nucleic acid probes; radionuclides; radiotracer; stoichiometry; superoxides; tritium

Stimulation of human neutrophils induces a "respiratory burst" causing production of 02- and H202. This response is crucial to normal bacterial killing but an inappropriate response contributes to neutrophil-mediated tissue damage in many diseases. Stimulation of the respiratory burst involves activation of an electron transport complex, the NADPH oxidase. Mechanisms of oxidase activation are largely undefined. Even the components of the oxidase are controversial, although data strongly support that it at least involves an NADPH-binding flavoprotein and a cytochrome. The cytochrome is being intensively studied elsewhere, but the flavoprotein has not been purified in quantity and its mechanisms of activation and electron transfer are unknown. This project will complete the development of a novel NADPH- affinity method for purification of the NADPH-binding oxidase flavoprotein. It will also employ new methods for assessing purification: 1) The ability to form a borohydride-reducible adduct between the protein and 2,3-dialdehyde-NADPH. 2) The ability to reconstitute NADPH-dependent 02- production in liposomes. For this model, flavoprotein purification fractions will be combined with solubilized membrane (the membrane being specifically depleted of functional NADPH-binding flavoprotein but providing all other components of the oxidase complex), for co- reconstitution of all oxidase components in liposomes by removal of detergent. Redox couples (FAD, FMN, thiols, iron-sulfur, transition metal) within the flavoprotein we be determined. Spectral, fluorescent and electron paramagnetic resonance methods will be used for titration of the flavoprotein with NADPH versus dithionite to determine stoichiometry of NADPH to flavin electron transfer and formation of intermediate redox states. Parallel studies of flavoprotein from resting and stimulated cells will test the hypothesis that oxidase activation involves increased substrate affinity (anaerobic NADPH binding) or catalysis (electron transfer). Tryptic peptides derived from the protein will be sequenced. The sequences will allow synthesis of peptides for raising anti- flavoprotein antibodies and for synthesizing cDNA probes, each of which can be used for molecular cloning of the flavoprotein cDNA.

刺激人类中性粒细胞诱发“呼吸爆发” 导致产生02-和H2O2。 这一回应至关重要 正常的细菌杀灭作用，但不适当的反应会导致 许多疾病中中性粒细胞介导的组织损伤。 刺激 呼吸爆发涉及电子的激活 运输复合物，NADPH 氧化酶。 氧化酶的机制 激活很大程度上是未定义的。 甚至连组件 氧化酶存在争议，尽管数据强烈支持它 至少涉及 NADPH 结合黄素蛋白和细胞色素。 其他地方正在对细胞色素进行深入研究，但 黄素蛋白尚未大量纯化及其机制 活化和电子转移的过程尚不清楚。 该项目将完成新型 NADPH-的开发 亲和法纯化 NADPH 结合氧化酶 黄素蛋白。 它还将采用新的方法来评估 纯化：1) 形成硼氢化物还原性的能力 蛋白质与 2,3-二醛-NADPH 之间的加合物。 2) 的 重建 NADPH 依赖性 02- 生产的能力 脂质体。 对于该模型，黄素蛋白纯化级分 将与溶解膜结合（该膜是 专门耗尽了功能性 NADPH 结合黄素蛋白，但 提供氧化酶复合物的所有其他成分），用于共 通过去除重建脂质体中的所有氧化酶成分 的洗涤剂。 氧化还原对（FAD、FMN、硫醇、铁硫、过渡金属） 在黄素蛋白中我们被确定。 光谱、荧光 和电子顺磁共振方法将用于 用 NADPH 与连二亚硫酸盐滴定黄素蛋白 确定 NADPH 与黄素电子转移的化学计量 中间氧化还原态的形成。 平行研究 来自静息细胞和刺激细胞的黄素蛋白将测试 氧化酶激活涉及底物增加的假设 亲和力（厌氧 NADPH 结合）或催化作用（电子 转移）。 来自蛋白质的胰蛋白酶肽将被测序。 这 序列将允许合成肽以提高抗 黄素蛋白抗体和用于合成 cDNA 探针，每个 可用于黄素蛋白cDNA的分子克隆。