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

为了弄清NADPH氧化酶(产氧酶)的亚基结构，我们尝试在半重组体系中固定交联剂激活的酶复合体。在尝试了几种连接物和条件后，我们意识到在半重组系统中激活的酶非常不稳定，而且不能有效地稳定交联剂，这与含有胞浆的无细胞体系中的不同。这一结果促使我们寻找胞浆中的稳定因子。结果，我们发现胞浆中的肌动蛋白可能参与了细胞的稳定(也可能参与了激活)。考虑到这一想法，我们计划对交联实验进行修改以适应系统的需要，我们意识到这种类型的实验需要大量的重组蛋白。因此，我们已经开始在大肠杆菌或Sf9细胞中自行生产这些重组蛋白。我们从兰贝斯博士的团队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Stoc…在本项目的另一部分中，我们寻找了控制细胞内NADPH氧化酶激活的信号分子，发现磷脂酸(PA)诱导了NADPH氧化酶的激活，而精胺(一种细胞多胺)则抑制了NADPH氧化酶的激活。当添加到渗透性中性粒细胞中时，微摩尔浓度的PA能迅速激发活性。这种激活不受Ca~(2+)、甘油和蛋白激酶c的影响，O_2~(2+)的产生与生理刺激相似。这些结果表明，PA可能是激活细胞内NADPH氧化酶的第二信使。另一方面，精胺可抑制该酶的无细胞激活(IC_&lt；50&gt；=18µM)。这种抑制是精胺对其前体胺的专一性抑制。该胺对半重组无细胞系统也有抑制作用。动力学研究表明，精胺可能通过与胞浆亚基，尤其是p67Phox结合来干扰酶的组装。较少

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