OXIDASE RELATED 48K PHOSPHOPROTEIN OF HUMAN NEUTROPHILS

嗜中性粒细胞的NADPH氧化酶催化氧和NADPH产生O2。 这种酶在静息细胞中处于休眠状态，但当细胞被任何一种试剂刺激时就会获得活性。在活化过程中，氧化酶的亚基之一p47 PHOX被广泛磷酸化，并从胞质溶胶迁移到膜上，在膜上它有助于活性氧化酶的组装。 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。 使用多种激酶将p47 PH 0X磷酸化，并测试磷酸化形式的p47 PH 0X支持氧化酶活化的能力。 在那些支持氧化酶活化的磷酸化形式中，将确定磷酸盐的位置。 将通过定点诱变研究磷酸化靶标之间可能的相互作用。 将检查磷酸化/去磷酸化在氧化酶失活中的作用。 将研究-SH基团参与氧化酶活化的可能性。 p47 PH 0X和GTP之间相互作用的意义将被研究。最后，p47 PH 0X将被结晶并通过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.