OXIDASE RELATED 48K PHOSPHOPROTEIN OF HUMAN NEUTROPHILS

中性粒细胞的NADPH氧化酶催化氧气和NADPH产生02。这种酶在静止的细胞中处于休眠状态，但当细胞受到多种刺激物中的任何一种刺激时，它就会获得活性。在激活过程中，氧化酶的一个亚基p47PH0X被广泛磷酸化，并从细胞质迁移到膜上，在那里它协助活性氧化酶的组装。我们建议在激酶依赖的无细胞氧化酶激活系统中研究这种磷酸化及其与氧化酶激活的关系。p47PH0X将被多种激酶磷酸化，p47PH0X的磷酸化形式将被测试其支持氧化酶激活的能力。在那些支持氧化酶激活的磷酸化形式中，磷酸盐的位置将被确定。磷酸化靶点之间可能的相互作用将通过位点定向诱变进行研究。磷酸化/去磷酸化在氧化酶失活中的作用将被检查。-SH基团参与氧化酶活化的可能性将被研究。我们将研究p47PH0X与GTP相互作用的意义。最后，对p47PH0X进行结晶，并通过x射线分析确定其结构。

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.