REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

人血中性粒细胞产生氧自由基是至关重要的 对于成功的主机防御，也可能对主机不利 在某些炎症过程中，即在成人呼吸窘迫中 综合征、关节炎等。了解氧自由基的调节 生产是该项目的长期目标，对 开发控制各种疾病状态的方法。人体内的酶 产生氧自由基的中性粒细胞是一种膜结合的、多细胞的 组分电子传递链，称为NADPH氧化酶，最近 显示由至少4-5个多肽组成。它的作用机制 这个复合体的激活及其组件的确切功能是 尚不清楚，但激活可能涉及两种磷酸化反应 以及组件的组装。NADPH的一种磷酸蛋白组分 氧化酶是一种47 kDa的蛋白(Pp47)，已被鉴定，但其功能 它的磷酸化作用尚不清楚。具体目标1 应用建议研究磷酸化反应在 Pp47和其他氧化酶组分的功能和调节。vbl.使用 一种基于新开发的免疫小球的氧化物酶组装新方法 技术，两者都参与了磷酸化反应 丝氨酸/苏氨酸和酪氨酸蛋白激酶的组装和 将分析NADPH氧化酶的激活情况。此外，无手机 系统将被用来研究特定的 蛋白激酶和重组和纯化的天然氧化物组分。一个 大量数据表明，蛋白激酶C(PKC)是一种重要的 NADPH氧化酶的调节因子，但涉及的机制尚不清楚。它 最近认识到PKC由一组同工酶组成， 它们在不同的细胞中不同地分布和调节 类型，我们已经在人类中鉴定出至少两种PKC同工酶 中性粒细胞。本申请的具体目标2提出了一个全面的 为了了解这些细胞中的PKC家族的特征 个体同工酶在NADPH激活中的生物学作用 氧化物酶。利用特异性抗体和生化分析，PKC 人类中性粒细胞中的同工酶将被鉴定、分离和它们的 表征了调节和自动磷酸化特性。这个 分离的同工酶将用于无细胞的磷酸化系统 含有NADPH氧化酶成分。总体而言，这些研究应该会提供新的 对呼吸爆发的生化调节的洞察。

The production of oxygen radicals by human blood neutrophils is critical for the success of host defense, but also may be detrimental to the host in certain inflammatory processes, i.e., in the adult respiratory distress syndrome, arthritis, etc. Understanding the regulation of oxygen radical production is the long-term goal of this project and is important for developing methods of controlling various disease states. The enzyme in neutrophils which generates oxygen radicals is a membrane-bound, multi- component electron transport chain, termed NADPH oxidase, and recently shown to consist of at least four-five polypeptides. The mechanisms of activation of this complex and the exact functions of its components are still unclear, but activation may involve both phosphorylation reactions and assembly of components. One phosphoprotein component of NADPH oxidase, a 47 kDa protein (pp47), has been identified, but the functional role of its phosphorylation is unknown. Specific Aim 1 of this application propose to examine the role of phosphorylation reactions in the functions and regulation of pp47 and other oxidase components. Using a novel assay for oxidase assembly, based on a newly-developed immunobead technique, the participation of phosphorylation reactions by both serine/threonine and tyrosine protein kinases in the assembly and activation of NADPH oxidase will be analyzed. In addition, cell-free systems will be utilized to study the interactions between specific protein kinase and recombinant and purified native oxidase components. A large body of data implicates protein kinase C (PKC) as an important regulator of NADPH oxidase, but the mechanisms involved are unknown. It has recently been recognized that PKC consists of a family of isozymes, which are differentially distributed and regulated in different cell types, and we have identified at least two PKC isozymes in human neutrophils. Specific Aim 2 of this application proposes a comprehensive characterization of the PKC family in these cells in order to understand the biological roles of individual isozymes in the activation of NADPH oxidase. Using specific antibodies and biochemical analysis, the PKC isozymes in human neutrophils will be identified, separated, and their regulatory and autophosphorylation properties characterized. The separated isozymes will be utilized in cell-free phosphorylation systems with NADPH oxidase components. Overall, these studies should provide new insights into the biochemical regulation of the respiratory burst.