REGULATION OF OXYGEN METABOLISM IN BLOOD NEUTROPHILS

人体血液中性粒细胞产生氧自由基是至关重要的 对于宿主防御的成功，也可能对宿主不利 在某些炎症过程中，即，成人呼吸窘迫 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，对于 开发控制各种疾病状态的方法。 中的酶 产生氧自由基的中性粒细胞是一种膜结合的，多 组成电子传递链，称为NADPH氧化酶，最近 显示由至少4 - 5种多肽组成。 的机制 该复合物的激活及其组分的确切功能是 目前还不清楚，但激活可能涉及两种磷酸化反应 和组件的组装。 NADPH的一种磷蛋白组分 氧化酶，一种47 kDa的蛋白质（pp 47），已被确定，但功能性 其磷酸化的作用是未知的。 具体目标1 该申请提出研究磷酸化反应在 pp 47等氧化酶组分的功能及调节。 使用 基于新开发的免疫珠的氧化酶组装的新测定法 技术，磷酸化反应的参与， 丝氨酸/苏氨酸和酪氨酸蛋白激酶， 将分析NADPH氧化酶的活化。 此外，无细胞 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酶组分。 一 大量数据表明蛋白激酶C（PKC）是一种重要的 NADPH氧化酶的调节剂，但涉及的机制尚不清楚。 它 最近认识到PKC由同工酶家族组成， 它们在不同的细胞中有不同的分布和调节 型，我们已经确定了至少两个PKC同工酶在人类 中性粒细胞 本申请的具体目标2提出了一种全面的 这些细胞中PKC家族的特征，以了解 单个同工酶在NADPH活化中的生物学作用 氧化酶。 利用特异性抗体和生化分析， 人类中性粒细胞中的同工酶将被鉴定、分离， 调节和自磷酸化特性的特点。 的 分离的同工酶将用于无细胞磷酸化系统 与NADPH氧化酶成分。 总的来说，这些研究应该提供新的 深入了解呼吸爆发的生化调节。

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.