Lipid-protein interactions in cytochrome c oxidase. A comparison of covalently attached phospholipid photo-spin-label with label free to diffuse in the bilayer.
Lipid-protein interactions in cytochrome c oxidase. A comparison of covalently attached phospholipid photo-spin-label with label free to diffuse in the bilayer.
复制标题
细胞色素c氧化酶中的脂质-蛋白质相互作用。
DOI：
10.1021/bi00351a010
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Jost,PC
中科院分区：
文献类型：
--
作者：
Griffith,OH;McMillen,DA;Keana,JF;Jost,PC
O. Hayes Griffith，** Debra A.作者声明：John F. W. Keana和Patricia C. Jost分子生物学研究所和化学系，俄勒冈州大学，尤金，俄勒冈州97403接收1985年8月2日摘要：这项研究的目的是澄清可能的起源的运动限制电子自旋共振（ESR）光谱成分在膜中服务。为此目的，磷脂光自旋标记的合成，表征，并用于研究牛心细胞色素c氧化酶的脂质-蛋白质相互作用。该探针的设计与硝基芳基叠氮化物纳入磷脂头基，和自旋标记的sn-2侧链，并进行放射性标记。所得分子，1-棕榈酰-2-（14-乙酰基[2- 3 H]硬脂酰）-sn-甘油基-3-磷酸-N-（4-叠氮基-3-硝基苯基）乙醇胺，在弱光下和在磷脂双层中重建细胞色素c氧化酶所需的程序期间是稳定的。在光解时，光自旋标记物与蛋白质以高产率（50%附着）反应。没有检测到自旋标签的破坏。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的细胞色素c氧化酶反应后的光自旋标记显示最高水平的连接带I，III，和VII，与一些标记的其他频带。标记模式证明了附着位点的分布，这是自旋标记研究所需的。在25 ℃下附着的标记物的ESR光谱表明运动限制性脂质链的恒定分数，与脂质与蛋白质的比率无关。与此相反，自旋标记的磷脂酰胆碱和prephotolyzed光自旋标记，都自由扩散的双层，表现出的行为与多重平衡结合模型。这些结果，以及在-196 ℃下冷冻的膜获得的数据，显示了如何区分导致运动受限的ESR线形的几种情况。这项研究提供了额外的证据表明，通常incontact与蛋白质，而不是聚集文物的脂质部分，占观察到的运动限制组件的ESR谱重构细胞色素c氧化酶在磷脂双分子层。
O. Hayes Griffith,** Debra A. McMillen, John F. W. Keana, and Patricia C. Jost Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, Oregon 97403 Received August 2, 1985 abstract: The aim of this study was to clarify the possible origins of the motion-restricted electron spin resonance(ESR) spectral componentobserved in membranes. For this purpose, a phospholipid photospin-label was synthesized, characterized, and used to study lipid-protein interactions inbeef heart cytochrome c oxidase. The probe was designed with a nitroaryl azide incorporated in the phospholipid head group, and a spin-label on the sn-2 side chain, and was radiolabeled. The resulting molecule, l-palmitoyl-2-(14-proxyl [2-3H] stearoyl)-s «-glycero-3-phospho-iV-(4-azido-3-nitrophenyl) ethanolamine, was stable under subdued light and during theprocedures required to reconstitute cytochrome c oxidase in phospholipid bilayers. Upon photolysis, the photo-spin-label reacted with the protein in high yields (50% attached). There was no detectable destruction of the spin-label. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cytochrome c oxidase after reaction with the photo-spin-label showed highest levels of attachment to bands I, III, and VII, with some labeling of other bands. The labeling pattern demonstrated a distribution of attachment sites, which was needed for the spin-labeling studies. ESR spectra of the attached labels at 25 C indicated a constant fraction of motion-restricted lipid chains, independent of the lipid to protein ratio. In contrast, a spin-labeled phosphatidylcholine and the prephotolyzed photo-spin-label, both free to diffuse in the bilayer, exhibited behavior in agreement with the multiple equilibria binding model. These results, as well as data obtained with membranes frozen at-196 C, show how severalsituations that lead to a motion-restricted ESR line shape can be distinguished. This study provides additional evidence that the fraction of lipids normally incontact with protein, and not aggregation artifacts, accounts for the observed motion-restricted component of ESR spectra of reconstituted cytochrome c oxidase in phospholipid bilayers.