Human Neutrophil Superoxide-Generating Enzyme-Molecular Basis and Activation Mechanism-

为了阐明NADPH氧化酶（O_2^-生成酶）的亚基结构，我们尝试用交联剂固定在半重组体系中激活的酶复合体。在尝试了几种连接剂和条件后，我们意识到在半重组体系中激活的酶是非常不稳定的，并且不能有效地通过交联剂稳定，这与在含细胞质溶胶的无细胞体系中不同。结果引导我们在胞浆中寻找稳定因子。因此，我们发现细胞质中的肌动蛋白可能参与了稳定（也可能参与了激活）。考虑到这个想法，我们正计划修改交联实验以适应该系统。我们意识到我们需要大量的重组蛋白来进行这种类型的实验。所以我们已经开始在大肠杆菌或Sf9细胞中自己生产这些重组蛋白。我们从兰贝斯博士的小组那里学到了如何培养细胞并让它们产生蛋白质。现在我们有了一些储备…更多的k来做上面描述的实验。在本项目的另一部分，我们在细胞中寻找控制NADPH氧化酶激活的信号分子，发现磷脂酸（PA）诱导氧化酶激活，精胺（一种细胞多胺）抑制氧化酶激活。当加入渗透中性粒细胞时，微摩尔浓度的PA迅速激发活性。O_2^-的激活与Ca^<2+ bb0、二酰基甘油或蛋白激酶c无关，其生成速率与生理刺激相似。这些结果表明，PA可能作为第二信使激活细胞内NADPH氧化酶。另一方面，精胺抑制了氧化酶的无细胞激活（IC_<50>=18muM）。这种抑制作用对精胺的前体胺具有特异性。该胺对半重组无细胞体系也有抑制作用。动力学研究表明，精胺可能通过与胞质亚基结合，特别是与p67phox结合，干扰酶的组装。少

To clarify the subunit structure of NADPH oxidase (O_2^- generating enzyme), we have tried to fix the enzyme complex activated in a semi-recombinant system by crosslinkers. After trying several linkers and conditions, we realized that the enzyme activated in the semirecombinant system is very labile and is not efficiently stabilized by crosslinkers, different from that in the cell-free system containing cytosol. The result lead us to search for the stabilizing factor in cytosol. As a result, we found that actin in cytosol may be involved in the stabilization (and possibly in the activation). Considering the idea, we are planning to modify the cross-linking experiments ot fit the system.We realized that we need a large amount of recombinant proteins for this type of experiment. So we have started to produce these recombinant proteins in E.coli or Sf9 cells by ourselves. We have learned from Dr.Lambeth's group how to grow the cells and let them produce the proteins. Now we have some stoc … More ks for doing experiments described above.In the other part of this project, we have searched for the signaling molecules which control the activation of NADPH oxidase in the cell, and found that phosphatidic acid (PA) elicits the oxidase activation and spermine, a cellular polymine, suppresses it. When added to permeabilized neutrophils, PA at micromolar concentrations elicited the activity quickly. The activation was found independent of Ca^<2+>, diacylglycerol, or protein kinase c. And the rate of O_2^- generation was similar to that by physiological stimuli. These results show that PA may searve as a second messenger to activate NADPH oxidase in the cell.On the other hand, spermine was found to suppress the cell-free activation of the oxidase (IC_<50>=18muM). The inhibition was specific for spermine over its precursor amines. The amine also inhibited semi-recombinant cell-free system. The kinetic sutdies showed that spermine may interfere with the assembly of the enzyme by binding to the cytosolic subunits, especially to p67phox. Less