ENZYMES MODULATING SECOND MESSENGERS IN NEUTROPHILS

该项目的重点是催化初始生化反应的酶 刺激中性粒细胞氧化代谢后发生的事件， 吞噬作用或与许多试剂（例如趋化肽、免疫 复合物）。 这种刺激导致产生大量的 超氧化物和过氧化氢-氧依赖性 吞噬细胞的抗菌机制。 虽然整体机制， 哪些外部刺激触发O-2释放仍有待阐明， 现在证明了最初的事件与最近的事件相似 描述了其他细胞类型的某些受体介导的事件。 根据当代的观点，特定受体在细胞上的占据， 中性粒细胞的质膜可以激活细胞内磷脂酶 对含肌醇的磷脂具有特异性的C。 这 磷脂酶被认为优先催化 磷脂酰肌醇-4，5-二磷酸以产生二酰基甘油和肌醇 （1，4，5）-三磷酸盐（Ins（1，4，5）P-3）。 这两种产品可能构成 重要的调节信号或"第二信使"，在产生 生理反应。 甘油二酯是 Ca ++激活的磷脂依赖性蛋白激酶（蛋白激酶 C）。 Ins（1，4，5）P-3促进内质网Ca~（++）释放 嗜中性粒细胞、巨噬细胞和许多其他细胞类型的网状结构。 Ins（1，4，5）P-3磷酸酶催化Ins（1，4，5）P-3降解为 Ins（1，4）P-2和Pi，因此预计将发挥重要作用 在Ca ++介导的细胞事件中。 人们对这些酶知之甚少 任何吞噬细胞的细胞。 该提案的具体目的是纯化和表征 磷脂酶C和肌醇（1，4，5）-磷酸单酯酶活性 中性粒细胞，并确定其亚细胞区域。 特别 重点将放在阐明监管机制， 调节这些酶的活性（即，物理状态 底物、变构修饰剂、共价修饰）。 技术 生物化学（即，酶学）和细胞生物学（即，超微结构 细胞化学）。 这些研究的长期目标是 加深对细胞内发生的最初生物化学事件的了解， 一般的刺激，特别是阐明机制， 其启动吞噬细胞产生O-2。

This project focuses on the enzymes which catalyze the initial biochemical events that occur upon stimulation of neutrophil oxidative metabolism by phagocytosis or with a number of agents (e.g. chemotactic peptides, immune complexes). This stimulation results in the production of large amounts of superoxide and hydrogen peroxide - key components in the oxygen-dependent antimicrobial mechanisms of phagocytes. Although the overall mechanism by which external stimuli trigger O-2 release remains to be elucidated, there is now evidence that the initial events are similar to those recently described for certain receptor-mediated events of other cell-types. According to the contemporary view, the occupation of specific receptors on the plasmalemma of neutrophils may activate an intracellular phospholipase C that is specific for inositol-containing phospholipids. This phopholipase is thought to preferentially catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphate to yield diacylglycerol and inositol (1,4,5)-trisphosphate (Ins (1,4,5)P-3). Both products may constitute important regulatory signals or "second messengers" in generating the physiological response. Diacylglycerol is the physiological activator of the Ca++ activated, phospholipid-dependent protein kinase (protein kinase C). Ins (1,4,5)P-3 promotes the release of Ca++ from the endoplasmic reticulum of neutrophils, macrophages, and a number of other cell types. Ins (1,4,5)P-3 phosphatase catalyzes the degradation of Ins (1,4,5)P-3 to Ins (1,4)P-2 and Pi, and would thus be expected to play a significant role in cellular events mediated by Ca++. Little is known about these enzymes from any type of phagocytic cell. The specific aims of this proposal are to purify and characterize the phospholipase C and inositol (1,4,5)-phosphomonoesterase activities from neutrophils, and to determine their subcellular locales. Particular emphasis will be placed on elucidating the regulatory mechanisms which modulate the activities of these enzymes (i.e., physical state of the substrate, allosteric modifiers, covalent modifications). Techniques of biochemistry (i.e., enzymology) and cell biology (i.e., ultrastructural cytochemistry) will be employed. The long term goal of these studies is to deepen insight into the initial biochemical events that occur upon cellular stimulation in general, and, in particular, to illuminate the mechanism which initiates O-2 production by phagocytes.