CELL DIVISION AND SURFACE GROWTH IN A MODEL SYSTEM

刺激人中性粒细胞诱导“呼吸爆发” 导致O2-和H2 O2的产生。 这一反应至关重要 正常的细菌杀死，但不适当的反应有助于 在许多疾病中由嗜酸性粒细胞介导的组织损伤。 刺激 呼吸爆发的一部分涉及到电子的激活 运输复合物，NADPH氧化酶。 氧化酶的机制 激活在很大程度上是不确定的。 即使是 氧化酶是有争议的，虽然数据强烈支持，它 至少涉及NADPH结合黄素蛋白和细胞色素。 细胞色素正在其他地方进行深入研究，但 黄素蛋白尚未大量纯化，其作用机制 活化和电子转移是未知的。 该项目将完成一种新型NADPH的开发- 亲和纯化NADPH结合氧化酶的方法 黄素蛋白 它还将采用新的方法来评估 纯化：1）形成硼氢化物可还原的 蛋白质与2，3-二醛-NADPH之间的加合物。 2)的 在细胞中重建NADPH依赖性O2产生的能力 脂质体。 对于该模型，黄素蛋白纯化级分 将与溶解的膜（该膜被 特异性耗尽功能性NADPH结合黄素蛋白， 提供氧化酶复合物的所有其它组分），用于共- 通过去除脂质体中的所有氧化酶组分来重建 洗涤剂。 氧化还原对（FAD、FMN、硫醇、铁硫、过渡金属） 我们可以确定其中的黄素蛋白。 光谱、荧光 和电子顺磁共振方法将用于 用NADPH对连二亚硫酸盐滴定黄素蛋白， 确定NADPH到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 静息和刺激细胞中的黄素蛋白将测试 氧化酶活化涉及底物增加的假说 亲和力（厌氧NADPH结合）或催化（电子 transfer）。 将对蛋白质衍生的胰蛋白酶肽进行测序。 的 序列将允许合成肽来提高抗- 黄素蛋白抗体和用于合成cDNA探针， 可用于黄素蛋白cDNA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.