ENZYMES MODULATING SECOND MESSENGERS IN NEUTROPHILS

这个项目的重点是催化初始生化的酶。 在刺激中性粒细胞氧化代谢时发生的事件 吞噬作用或与多种制剂(如趋化性多肽、免疫 复合体)。这种刺激导致产生大量的 超氧化物和过氧化氢--氧依赖中的关键成分 吞噬细胞的抗菌机制。尽管总体机制是通过 哪些外部刺激触发O-2释放仍有待阐明 现在有证据表明，最初的事件与最近的事件相似 对其他细胞类型的某些受体介导的事件进行了描述。 根据当代的观点，对特定受体的占领 中性粒细胞的质膜可能激活细胞内的磷脂酶 C是含肌醇磷脂特有的。这 磷脂酶被认为优先催化水解酶 磷脂酰肌醇-4，5-二磷酸合成二酰甘油和肌醇 (1，4，5)-三磷酸(Ins(1，4，5)P-3)。这两种产品都可能构成 重要的调控信号或产生 生理反应。甘油二酯是一种生理激活剂 钙激活的磷脂依赖的蛋白激酶(蛋白激酶 c)。INS(1，4，5)P-3促进内质网钙离子释放 由中性粒细胞、巨噬细胞和许多其他类型的细胞组成的网状结构。 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，因此有望发挥重要作用 在钙离子介导的细胞事件中。人们对这些酶知之甚少 来自任何类型的吞噬细胞。 这项提案的具体目标是提纯和描述 磷脂酶C和肌醇(1，4，5)-磷酸单酯酶活性 中性粒细胞，并确定它们的亚细胞位置。特例 重点将放在阐明下列监管机制 调节这些酶的活性(即，物理状态 底物、变构修饰剂、共价键修饰)。技术问题 生物化学(即酶学)和细胞生物学(即超微结构 细胞化学)将被使用。这些研究的长期目标是 加深对发生在细胞上的初始生化事件的洞察 一般的刺激，特别是为了阐明 通过吞噬细胞产生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.