ENZYMES MODULATING SECOND MESSENGERS IN NEURTROPHILS

这项提议的长期目标是阐明确切的 参与调控的一系列分子事件 吞噬的白细胞产生超氧化物。从以下方面获得的知识 这些研究将与理解宿主防御机制高度相关 机械装置。此外，这里描述的研究可能最终表明 增强抗菌机制的新策略 免疫功能受损的人群。超氧化物是一种关键成分 中性粒细胞的氧气依赖的抗菌剂。这种酶 催化产生超氧化物的系统在 未受刺激的细胞，由膜结合和胞浆组成 组件。在刺激过程中，有一种可溶性的移位 在质膜上组装功能氧化酶的成分。 大量证据表明，蛋白激酶参与了 氧化酶系统的激活。我们最近报道说， 中性粒细胞同时含有可溶性和颗粒蛋白磷酸酶。 这似乎在调节超氧化物的产生方面也起着重要作用。 使用不同的激动剂。这些磷酸酶被有效的 体内和体外的肿瘤促进剂冈田酸和花盏花素A。 具体地说，该项目专注于两个完全未开发的地区 中性粒细胞的信号转导途径。这些是：(1) 猪瘟病毒蛋白丝氨酸/苏氨酸磷酸酶的纯化及性质研究 对肿瘤促进剂敏感的中性粒细胞和(2)阐明 这些酶调节超氧化物产生的机制。 生物化学和细胞免疫定位研究技术)将是 受雇的。经典和现代程序(亲和色谱柱、高效液相色谱法 系统)将用于提纯这些酶。特性研究 将包括确定四元结构、底物 特殊性和辅因要求。我们将关注以下问题 揭示调节这些磷酸酶的调节机制。 翻译后修饰(例如，磷酸化)和可能的 含氧脂肪酸作为生理抑制剂的作用将是 被追寻。最终目标是在两国之间建立牢固的联系 分离酶的调控特性及对其的控制 在完整的细胞中产生超氧化物。

The long term objective of this proposal is to elucidate the exact sequence of molecular events that are involved in the regulation of superoxide production by phagocytic leukocytes. Knowledge gained from these studies will be highly relevant to an understanding of host-defense mechanisms. Moreover, studies described here may ultimately suggest novel strategies for enhancing antimicrobial mechanisms in immunocompromised populations. Superoxide is a key component of the oxygen-dependent antimicrobial armamentarium of neutrophils. The enzyme system that catalyzes the generation of superoxide is dissociated in unstimulated cells and consists of both membrane-bound and cytosolic components. During stimulation, there is a translocation of the soluble components to the plasmalemma where the functional oxidase is assembled. A good deal of evidence indicates that protein kinases are involved in the activation of the oxidase system. We have recently reported that neutrophils contain both soluble and particulate protein phosphatases that also appear to have major roles in modulating superoxide production with different agonists. These phosphatases are inhibited by the potent tumor promoters okadaic acid and calyculin A both in vivo and in vitro. Specifically, this project focuses on two completely unexplored areas in the signal transduction pathways of neutrophils. These are: (1) purifying and characterizing the protein serine/threonine phosphatases of neutrophils that are sensitive to tumor promotors and (2) elucidating the mechanisms by which these enzymes modulate superoxide production. Techniques of biochemistry and cell immunolocalization studies) will be employed. Classical and modern procedures (affinity columns, HPLC systems) will be used to purify the enzymes. Characterization studies will include determination of quaternary structure, substrate specificities and cofactor requirements. Attention will be paid to uncovering the regulatory mechanisms that modulate these phosphatases. Post-translational modifications (e.g., phosphorylation) and possible roles of oxygenated fatty acids as the physiological inhibitors will be sought. The ultimate goal is to forge a solid link between the regulatory properties of the isolated enzymes and the control of superoxide production in intact cells.