Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB
Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB
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DOI：
10.1074/jbc.m313510200
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发表时间：
2004-03-26
影响因子：
4.8
通讯作者：
Fu, D
中科院分区：
文献类型：
--
作者：
Chao, Y;Fu, D
ZitB是阳离子扩散促进剂（CDF）家族的成员，其介导锌穿过大肠杆菌的质膜的流出。我们描述了第一个动力学研究的纯化和重建ZitB通过停流测量金属离子的跨膜通量，使用封装在蛋白脂质体中的金属敏感的荧光指示剂。金属离子填充实验表明，Zn 2+流入的初始速率是ZitB与脂质的摩尔比的线性函数，并且与Zn 2+或Cd 2+的浓度通过双曲线相关，Michaelis-Menten常数（Km）分别为104.9 +/- 5.4 μ M和90.1 +/- 3.7 μ M。质子的耗尽停滞Cd 2+运输其扩散梯度，而四乙基铵离子取代K+不影响Cd 2+运输，表明Cd 2+运输耦合到H+，而不是K+。H+运输推断的H+依赖的Cd 2+运输，显示出双曲线的关系与K-m为19.9 nM的H+。施加H+扩散梯度跨膜引起镉离子通量进出的蛋白脂质体对施加的H+梯度。同样地，施加外向膜电位导致Cd 2+流出，表明ZitB运输的产电效应。综上所述，这些结果表明ZitB是催化Zn 2+或Cd 2+与H+的强制性交换的反向转运蛋白。金属离子与质子的交换化学计量比可能为1：1。
ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K-m) of 104.9 +/- 5.4 muM and 90.1 +/- 3.7 muM, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a K-m of 19.9 nM for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H+ gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1: 1.