Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer.
Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer.
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从脂质双层的头基区域标记大肠杆菌的 ATP 合酶。
DOI：
10.1021/bi00396a036
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Capaldi,RA
中科院分区：
文献类型：
--
作者：
Aggeler,R;Zhang,YZ;Capaldi,RA
1987年5月18日摘要：大肠杆菌的腺苷三磷酸酶（ECF！和ECHF 0）与两种赖氨酸特异性试剂十六烷基4-[3 H]甲酰基苯基磷酸钠（HFPP）和甲基4-[3 H]甲酰基苯基磷酸钠（MFPP）以及与光反应性试剂1，2-[3 H]二棕榈酰基-s，n-甘油3-[（4-叠氮基-2-硝基苯基）氨基]乙基]-磷酸]（芳基叠氮基PE）反应。HFPP和arylazidoPE是两亲性分子，通过其十六烷基部分（分别为一条和两条链）插入脂质双层中，其中反应性基团插入磷脂头部基团之间。MFPP是HFPP的水溶性类似物。用HFPP和arylazidoPE获得的ECF/Fq的标记模式非常相似;在这两种情况下，F0部分的a和B亚基是复合物中标记最重的多肽。亚基a的模型，排列在六个跨膜螺旋中，将大部分赖氨酸放置在头基区域，可用于与HFPP反应。与a和B亚基相比，ECFi部分的亚基a和0被非常差地标记，在蛋白质质量的基础上，它们一起掺入的HFPP和芳基叠氮基PE仅为两个F0亚基一起的4%和7.5%。胰蛋白酶切割研究将arylazidoPE对a亚基的任何标记定位于该多肽的N-末端15个残基。当使用MFPP时，α和β亚基比F0亚基反应性强得多。这意味着ECFiF 0中α和β亚基的大部分质量在膜上，而不与双层表面接触。ECFb的一个亚基，即8亚基，在游离ECF中被HFPP和MFPP高度标记[但在ECF 1F 0制剂中被屏蔽而不与这两种探针和芳基叠氮基PE反应]。这意味着，8亚基，ECF [和FO之间的链接，被蛋白质-蛋白质相互作用屏蔽，至少在双层的脂质头基区域。大肠杆菌的腺苷5 '-三磷酸（ATP）合酶（EFiFn）被组织成两个主要结构域，即F1 Fn和F1 Fn。部分，外在的膜脂双层和含有催化位点，和F0部分，内在的膜双层连续体和参与质子易位。ECF含有五种不同的亚基，α、β、γ、δ和α，摩尔比为3：3：1：1：1。F0含有摩尔比为1：2：10或12的三种不同多肽a、B和c [综述见Senior和Wise（1983），步行者et al.（1984）和Bragg（1984）]。ATP合酶的Fi部分在所有生物体中非常相似，具有相同数量的亚基，并且具有α和β多肽的相当大的序列保守性（步行者等人，1984年）。Fi分子量为38万，是一个大的球状结构。大鼠肝酶的X射线研究表明，该蛋白质是尺寸约为110 × 80 A的椭圆体（Amzel等人，1982年）。Fi在F^ q复形中的取向尚不清楚。对细菌、线粒体和叶绿体中ATP酶的电子显微镜研究显示F [相关]。RA得到了瑞士国家科学基金会的奖学金支持。
Revised Manuscript Received May 18, 1987 abstract: The isolated and membrane-bound forms of the adenosinetriphosphatase of Escherichia coli (ECF! and ECFiF0, respectively) have been reacted with two lysine-specific reagents, sodium hexadecyl 4-[3H] formylphenyl phosphate (HFPP) and sodium methyl4-[3H] formylphenyl phosphate (MFPP), and with the photoreactive reagent l, 2-[3H] dipalmitoyl-s,«-glycerol 3-[[[(4-azido-2-nitrophenyl) amino] ethyl]-phosphate](arylazidoPE). HFPP and arylazidoPE are amphipathic molecules, inserting by their hexadecyl moieties (one and two chains, respectively) into the lipid bilayer, with the reactive groups intercalated among the phospholipid head groups. MFPP is the water-soluble analogue of HFPP. The labeling patterns of ECF/Fq obtained with HFPP and arylazidoPE were very similar; in both cases the a and b subunits of the F0 part were the most heavily labeled polypeptides of the complex. Models of subunit a, arranged in six transmembrane helices, place most of the lysines in the head-group region, available for reaction with HFPP. Subunits a and 0 of the ECFi part were very poorly labeled in comparison to the a and b subunits, together incorporating only 4% as much HFPP and 7.5% as much arylazidoPE as the two F0 subunits together on a protein mass basis. Trypsin cleavage studies localized any labeling of the a subunit by arylazidoPE to the N-terminal 15 residues of this polypeptide. When MFPP was used, the a and (J subunits were very much more reacted than the F0 subunits. This impliesthat most of the mass of the a and/3 subunits in ECFiF0 is abovethe membrane and not in contact with the bilayer surface. One subunit of ECFb the 8 subunit, was heavily labeled by HFPP and MFPP in free ECF [but was shielded from reaction with both of these probes and with arylazidoPE in ECFiF0 preparations. The implication is that the 8 subunit, a link between ECF [and F0, is shielded by protein-protein interactions, at least in the lipid head-group region of the bilayer. e adenosine 5'-triphosphate (ATP) synthase of Escherichia coli (EFiF „) is organized into two major domains, the F! part, extrinsic to the membrane lipid bilayer and containing the catalytic sites, and an F0 part, intrinsic to the membrane bilayer continuum and involved in proton translocation. ECF, contains fivedifferent subunits, a,@, y, 8, and «, in the molar ratio 3: 3: 1: 1: 1. F0 contains three different polypeptides, a, b, and c, in the molar ratio 1: 2: 10 or 12 [reviewed in Senior and Wise (1983), Walker et al.(1984), and Bragg (1984)]. The Fi part of the ATP synthase is very similar in all organisms, with the same number of subunits and with considerable se-quence conservation of the a and 0 polypeptides (Walker et al., 1984). Fi, with a molecular weight of 380000, is a large globular structure. X-ray studies of the rat liver enzyme indicate that the protein is an ellipsoid with dimensions of around 110 X 80 A (Amzel et al., 1982). How the Fi is oriented in the F^ q complex is not known. Electron microscopy studies of the ATP synthases from bacteria, mitochondria, and chloroplasts show F [associated fThis research was supported byNational Institutes of Health Grant HL-22050. RA was supported by a fellowship from the Swiss National Science Foundation.