Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium
Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium
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DOI：
10.1016/s0005-2736(02)00506-0
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发表时间：
2002-09-20
影响因子：
3.4
通讯作者：
Schneider, E
中科院分区：
文献类型：
--
作者：
Landmesser, H;Stein, A;Schneider, E
鼠伤寒沙门氏菌的麦芽糖三磷酸腺苷结合盒(ABC)转运体由膜相关复合体(MalFGK(2))和周质底物结合蛋白组成。为了进一步阐明蛋白质亚基之间的相互作用，我们在蛋白脂质体中纯化组分的水平上研究了MalFGK(2)复合体的解离和重组。首先，我们利用新构建的串联定向携带MalK、MalF和MalG基因的表达载体，优化了纯化的复合蛋白的产量。该复合体具有麦芽糖结合蛋白/麦芽糖依赖的ATPase活性，其V-max为1.25摩尔P-I/min/mg，K-m为0.1 mm。ATPase活性对钒酸盐和肠道细菌葡萄糖转运系统中的IIA酶(GLC)敏感。蛋白脂质体具有麦芽糖转运活性，初始速率为61nmol/min/mg。用6.6M尿素处理蛋白脂质体，导致中等暴露的MALK亚基释放，并伴随着ATPase活性的完全丧失。通过在尿素处理的蛋白脂质体中加入更多的纯化的麦芽糖，钒敏感的ATPase活性可以恢复到对照的50%左右。此外，MalKQI40K的表型在溶液中具有ATPase活性，但在与MalFG结合时不具有ATPase活性，通过与Malk缺失的蛋白脂质体重组证实了这一表型。(C)2002 Elsevier Science B.V.保留所有权利。
The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK(2)) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK(2) complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V-max of 1.25 mumol P-i/min/mg and a K-m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA(Glc), a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/ mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQI40K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes. (C) 2002 Elsevier Science B.V. All rights reserved.