CYTOSOLIC COMPONENTS OF THE NEUTROPHIL OXIDASE SYSTEM

中性粒细胞(PMN)发挥其关键的杀菌作用 作用在很大程度上是通过氧化代谢的爆发来实现的 通过一种可激活的NADPH氧化酶产生超氧化物和其他 有毒的氧衍生物。无细胞重组技术的最新研究 系统已经证明了两个离散的基本重要性 47 kDa和67 kDa的胞吞蛋白与氧化物酶活性 激活。此外，这些蛋白质的选择性缺失状态 在两种不同形式的常染色体遗传中被检测到 慢性肉芽肿病(CGD)。这些胞浆中的氧化酶成分 将在结构和功能方面表现出特点。结构性 研究将集中在47和67的分离和纯化上 KDA蛋白主要通过层析和电泳法分析 技巧。单特异性多克隆和单抗将是 产生到纯化的蛋白质及其潜能 免疫交叉反应性评估。结构分析将继续进行 蛋白水解酶、肽图、氨基酸分析和 测序。其他伴生胞浆的结构和亲缘关系 蛋白质将被考虑。功能研究将从分析开始 额外的CGD患者以确定缺乏的患病率 47和67 kDa蛋白的状态并搜索患有 其他假定的胞浆氧化酶成分的缺陷。是否 第47和67 kDa蛋白以非共价连接的功能形式存在 复合体将考虑使用天然凝胶、速度沉积和 与亲和力矩阵相关联。最后，我们将进行实验 为了确定氧化酶的激活是否与 胞质蛋白的物理状态(例如，转位到 膜结合组分)或共价键修饰 一种或两种蛋白质的磷酸化(如磷酸化)。这些研究 将提供详细的结构和功能描述 PMN-NADPH氧化酶的两个新的胞液组分 系统。这些蛋白质的关键重要性在以下方面得到了强调 严重反复感染患者细胞中它们的缺失 与常染色体变异的CGD有关。

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.