ENZYMES MODULATING SECOND MESSENGERS IN NEUTROPHILS

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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.