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.
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