CYTOSOLIC COMPONENTS OF THE NEUTROPHIL OXIDASE SYSTEM

多形核白细胞（PMN）发挥其关键的杀微生物作用， 在很大程度上是通过氧化代谢介导的 通过可激活的NADPH氧化酶产生超氧化物和其他 有毒的氧衍生物 重组无细胞 系统已经证明了两个离散的重要性 47和67 kDa的细胞体积蛋白，导致氧化酶 activation. 此外，这些蛋白质的选择性缺陷状态 在两种不同形式的常染色体遗传的 慢性肉芽肿病（CGD）。 这些胞浆氧化酶成分 将在结构和功能方面进行描述。结构 研究将集中在47和67的分离和纯化， 主要通过色谱和电泳分析的kDa蛋白 技术. 单特异性多克隆和单克隆抗体将被 产生的纯化蛋白质及其潜力 评估免疫交叉反应性。 结构分析将继续进行， 蛋白水解消化、肽图谱、氨基酸分析和 测序其他相关细胞质的结构和相关性 将考虑蛋白质。 功能研究将从分析开始开始 额外的CGD患者，以确定缺乏症的患病率 47和67 kDa蛋白质的状态，并寻找患有 缺乏其他假定的胞质氧化酶组分。 是否 第47和67 kDa蛋白质以非共价连接功能性 将考虑使用天然凝胶，速度沉降和 与亲和矩阵的关联。 最后进行实验 为了确定氧化酶活化是否与 胞质蛋白的物理状态（例如，易位到 膜结合部分）或具有共价修饰 在一些实施方案中，所述方法包括使一种或两种蛋白质的磷酸化（例如磷酸化）。 这些研究 将提供详细的结构和功能特征， 两种新描述的PMN NADPH氧化酶的胞质组分 系统 这些蛋白质的关键重要性被强调， 严重复发性感染患者的细胞中没有它们 与CGD的常染色体变种相关。

The polymorphonuclear leukocyte (PMN) exerts its critical microbicidal effects in large part through a burst of oxidative metabolism mediated by an activatable NADPH oxidase that generates superoxide and other toxic oxygen derivatives. Recent studies in a reconstituted cell-free system have demonstrated the essential importance of two discrete cytoscolic proteins of 47 and 67 kDa in events leading to oxidase activation. Moreover, selective deficiency states of these proteins have been detected in tow different forms of autosomally inherited chronic granulomatous disease (CGD). These cytosolic oxidase components will be characterized in terms of structure and function. Structural studies will focus on the separation and purification of the 47 and 67 kDa proteins primarily by chromatographic and electrophoretic techniques. Monospecific polyclonal and monoclonal antibodies will be generated to the purified proteins and their potential immunocrossreactivity assessed. Structural analyses will proceed with proteolytic digestion, peptide mapping, amino acid analysis and sequencing. The structure and relatedness of other associated cytosolic proteins will e considered. Functional studies will begin with analysis of additional CGD patients to determine the prevalence of deficiency states for the 47 and 67 kDa proteins and to search for patients with deficiencies of other putative cytosolic oxidase components. Whether the 47 nd 67 kDa proteins exist as a non-covalently linked functional complex will be considered using native gels, velocity sedimentation and association with affinity matrices. Finally, experiments will be done to determine whether oxidase activation is associated with changes in the physical state of cytosolic proteins (e.g. translocation to a membrane-associated fraction) or with covalent modification (e.g.phosphorylation) of one or both of the proteins. These studies will provide a detailed structural and functional characterization of two newly described cytosolic components of the PMN NADPH oxidase system. The critical importance of these proteins is underscored by their absence from cells of patients with severe recurrent infections associated with autosomal varieties of CGD.