权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

人中性粒细胞超氧化物生成酶-分子基础及激活机制-

基本信息

批准号：
05044181
负责人：
TAMURA Minoru
金额：
$ 2.88万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

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

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系统中活化的酶复合物。在尝试了几种接头和条件后，我们意识到在半重组系统中激活的酶非常不稳定，并且不能通过交联剂有效地稳定，这与含有胞质溶胶的无细胞系统不同。这一结果促使我们寻找细胞质中的稳定因子。因此，我们发现，肌动蛋白在胞质溶胶中可能参与的稳定（并可能在激活）。考虑到这一想法，我们计划修改交联实验以适应该系统，我们意识到这类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Sf 9细胞中自行生产这些重组蛋白。我们已经从兰贝斯博士的团队那里学会了如何培养细胞并让它们产生蛋白质。现在我们有一些库存 ...更多信息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）能激活NADPH氧化酶，而精胺（一种细胞内的多聚胺）能抑制NADPH氧化酶的激活，当加入到透性化的中性粒细胞中时，微摩尔浓度的PA能迅速激活NADPH氧化酶。发现这种激活不依赖于Ca^<2+>、甘油二酯或蛋白激酶c。O_2^-产生速率与生理刺激相似。结果表明，PA可能是激活细胞内NADPH氧化酶的第二信使，而精胺则抑制该氧化酶的无细胞激活（IC_<50>= 18 μ M）。抑制是具体的精胺超过其前体胺。该胺还抑制半重组无细胞系统。动力学研究表明，精胺可能通过与胞浆亚基，特别是p67 phox结合而干扰酶的组装。少