OXIDASE RELATED 48K PHOSPHOPROTEIN OF HUMAN NEUTROPHILS

Scripps Research Institute, La Jolla CA

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Abstract

The NADPH oxidase of neutrophils catalyzes the production of 02 from oxygen and NADPH. The enzyme is dormant in the resting cell, but acquires activity when the cell is stimulated with any of a number of agents. During activation p47PH0X, one of the subunits of the oxidase, is extensively phosphorylated and migrates from the cytosol to the membrane where it assists in the assembly of the active oxidase. We propose to study this phosphorylation and its relationship to the activation of the oxidase in a kinasedependent cell-free oxidase activating system. The p47PH0X will be phosphorylated using a number of kinases and the phosphorylated forms of p47PH0X tested for their ability to support oxidase activation. In those phosphorylated forms that support oxidase activation, the locations of the phosphates will be determined. Possible interactions between the phosphorylation targets will be investigated by site-directed mutagenesis. The role of phosphorylation/dephosphorylation in the deactivation of the oxidase will be examined. The possibility that -SH groups participate in oxidase activation will be investigated. The significance of the interaction between p47PH0X and GTP will be studied. Finally, p47PH0X will be crystallized and its structure determined by x-ray analysis.