Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase
Expression, purification, and characterization of subunit E, an essential subunit of the vacuolar ATPase
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DOI：
10.1016/s0006-291x(02)02468-3
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发表时间：
2002-11-01
影响因子：
3.1
通讯作者：
Bailer, SM
中科院分区：
文献类型：
--
作者：
Grüber, G;Godovac-Zimmermann, J;Bailer, SM
重组形式的亚基E（Vma 4p）从酵母液泡ATP酶（V-ATP酶）已在大肠杆菌中过表达，纯化到同质，并通过质谱法进行了探索。通过圆二色光谱对Vma 4p的二级结构进行分析，表明α螺旋含量为32%，β折叠含量为23%。Vma 4p与密切相关的F-1的核苷酸结合亚基α和β形成了杂交复合物。嗜热菌PS3（TF 1）的ATP酶。α（3）β（3）E-杂合复合物具有天然TF 1的ATP酶活性的56%。相比之下，没有Vma 4p的α（3）β（3）-形成显示约24%的总TF 1 ATP酶活性。这是由F-1和V-1亚基组成的具有水解活性的杂交复合物的第一次证明。已分别使用重组Vma 4p、与V-1的α（3）β（3）E-杂合复合物以及来自烟草天蛾的V-1 ATP酶的A（3）B（3）HG-亚复合物探测了V-1中E亚基的排列，表明E亚基在V-1中被屏蔽。(C)2002 Elsevier Science（美国）。All rights reserved.
A recombinant form of subunit E (Vma4p) from yeast vacuolar ATPases (V-ATPases) has been overexpressed in Escherichia coli, purified to homogeneity, and explored by mass spectrometry. Analysis of the secondary structure of Vma4p by circular dichroism spectroscopy indicated 32% alpha-helix and 23% beta-sheet content. Vma4p formed a hybrid-complex with the nucleotide-binding subunits alpha and beta of the closely related F-1. ATPase of the thermophilic bacterium PS3 (TF1). The alpha(3)beta(3)E-hybrid-complex had 56% of the ATPase activity of the native TF1. By comparison, an alpha(3)beta(3)-formation without Vma4p showed about 24% of total TF1 ATPase activity. This is the first demonstration of a hydrolytically active hybrid-complex consisting of F-1 and V-1 subunits. The arrangement of subunit E in V-1 has been probed using the recombinant Vma4p, the alpha(3)beta(3)E-hybrid-complex together with V-1 and an A(3)B(3)HEG-subcomplex of the V-1 ATPase from Manduca sexta, respectively, indicating that subunit E is shielded in V-1. (C) 2002 Elsevier Science (USA). All rights reserved.