CELL DEVELOPMENT IN A MODEL SYSTEM

刺激人中性粒细胞引起“呼吸爆发” 导致产生02-和H202。这一反应对 正常的细菌杀灭，但不适当的反应 中性粒细胞介导的组织损伤在许多疾病中。刺激 呼吸爆发涉及到电子的激活 运输复合体，NADPH氧化酶。酶的作用机制 激活在很大程度上是不确定的。即使是组件的 氧化物酶是有争议的，尽管数据强烈支持它 至少涉及与NADPH结合的黄素蛋白和细胞色素。 其他地方对细胞色素的研究也很深入，但 黄素蛋白尚未得到大量纯化及其机制 激活和电子转移的过程尚不清楚。 该项目将完成一种新型NADPH的开发- NADPH结合酶的亲和纯化方法 黄素蛋白。它还将采用新的评估方法 提纯：1)形成可还原的硼氢化物的能力 蛋白质与2，3-二醛-NADPH之间的加合物。2) 重建依赖NADPH的02-生产的能力 脂质体。在这个模型中，黄素蛋白纯化组分 将与增溶的膜结合(膜 功能NADPH结合黄素蛋白的特异性耗尽，但 提供氧化酶络合物的所有其他组件)，用于CO- 去除法重建脂质体中的所有氧化物组分 洗涤剂。 氧化还原对(FAD、FMN、硫醇、铁硫、过渡金属) 在黄素蛋白中，我们是确定的。光谱，荧光 和电子顺磁共振方法将用于 NADPH与二亚硫酸盐滴定黄素蛋白的比较 测定NADPH对黄素电子转移的化学计量比 中间氧化还原态的形成。平行研究 来自静息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说 亲和力(厌氧NADPH结合)或催化(电子 转移)。 从蛋白质中提取的胰蛋白酶多肽将被测序。这个 序列将允许合成多肽以提高抗- 黄素蛋白抗体和合成cDNAs探针，每个 可用于黄素蛋白基因的分子克隆。

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.