Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer.
Anionic amphiphile and phospholipid-induced conformational changes in human neutrophil flavocytochrome b observed by fluorescence resonance energy transfer.
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通过荧光共振能量转移观察阴离子两亲物和磷脂诱导的人中性粒细胞黄素细胞色素 b 的构象变化。
DOI：
10.1016/j.bbamem.2004.03.009
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Jesaitis,AlgirdasJ
中科院分区：
文献类型：
--
作者：
Taylor,RossM;Foubert,ThomasR;Burritt,JamesB;Baniulis,Danas;McPhail,LindaC;Jesaitis,AlgirdasJ
整合膜蛋白黄细胞色素 b (Cyt b) 包含人类吞噬细胞 NADPH 氧化酶复合物的催化核心，并用于启动参与消除感染因子的活性氧级联。 NADPH 氧化酶复合物产生的超氧化物已被证明受到吞噬细胞激活后脂质第二信使的酶促生成的特异性调节。在本研究中，Cyt b 特异性单克隆抗体 (mAb 44.1) 用 Cascade Blue (CCB) 标记，并用于共振能量转移 (RET) 研究，探讨一组脂质种类对 Cyt b 结构的影响。 CCB-mAb 44.1 与免疫亲和纯化的 Cyt b 的结合具有高度特异性，并导致稳态供体荧光显着猝灭。使用阴离子两亲物十二烷基硫酸锂 (LDS) 滴定 CCB-mAb 44.1:Cyt b 复合物会导致荧光猝灭的饱和弛豫，这是由于在无细胞测定中 Cyt b 产生最大超氧化物速率所需的两亲物浓度下，Cyt b 发生构象变化。对于阴离子两亲物花生四烯酸（AA），观察到类似的结果，尽管对于花生四烯酸甲酯（AA-ME）没有观察到荧光猝灭的松弛。使用阴离子 18:1 磷脂磷脂酸 (DOPA) 和磷脂酰丝氨酸 (DOPS) 也观察到荧光猝灭的饱和弛豫，而在添加相似水平的中性 18:1 脂质磷脂酰胆碱 (DOPC)、磷脂酰乙醇胺 (DOPE) 或二酰基甘油 (DAG) 时未观察到荧光猝灭的饱和弛豫。对多种磷脂酸 (PA) 种类的进一步检查表明，DOPA 不仅能有效诱导 Cyt b 的构象变化，而且比具有较短、饱和酰基链的 PA 种类引起更显着的构象变化。本研究中提供的数据支持这样的假设：第二信使脂质（例如 AA 和 PA）直接与黄素细胞色素 b 结合并调节与超氧化物生成激活相关的构象状态。
The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production.
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DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Qualliotine-Mann,D;Agwu,DE;Ellenburg,MD;McCall,CE;McPhail,LC
通讯作者：
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DOI：
10.1016/s0021-9258(18)68464-9
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1988
期刊：
The Journal of biological chemistry
影响因子：
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影响因子：
2.9
作者：
Whitaker,JE;Haugland,RP;Moore,PL;Hewitt,PC;Reese,M;Haugland,RP
通讯作者：
Haugland,RP
级联蓝作为含血红素蛋白质共振能量转移研究的供体。
影响因子：
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作者：
Ross M. Taylor;B. Lin;T. R. Foubert;J. B. Burritt;J. Sunner;A. J. Jesaitis
通讯作者：
A. J. Jesaitis