Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori.
Transport kinetics and selectivity of HpUreI, the urea channel from Helicobacter pylori.
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DOI：
10.1021/bi200887a
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发表时间：
2011-10-11
期刊：
影响因子：
2.9
通讯作者：
Khademi S
中科院分区：
文献类型：
--
作者：
Gray LR;Gu SX;Quick M;Khademi S
幽门螺杆菌在胃的酸性环境中定殖和存活的独特能力严重依赖于通过尿素通道HpUreI摄取尿素。因此，HpUreI可能是一个有前途的目标，针对这种人类病原体的特定药物的发展。为了深入了解该通道的结构/功能关系，我们已经开发了稳定的重组HpUreI的高产量表达和纯化的条件，允许其详细的动力学表征溶解的形式和重组成脂质体。洗涤剂溶解的HpUreI形成均三聚体，如通过化学交联所确定的。尿素解离动力学的纯化HpUreI测定的闪烁接近试验（SPA）的装置，而尿素流出HpUreI含脂蛋白体使用停流光谱测定，以确定尿素通道的动力学和选择性。动力学分析表明，尿素在HpUreI的传导是pH敏感和饱和的半饱和浓度（或K0.5）的~163 mM。尿素的结合HpUreI在较低的pH值增加，但是，尿素结合的表观亲和力（~150 mM）是不显着的pH依赖性。溶质选择性分析表明，HpUreI是高度选择性的尿素和羟基脲。去除尿素分子中的任何一个氨基都会降低它们通过HpUreI的渗透性。与尿素传导类似，水通过HpUreI的扩散是pH依赖性的，在中性pH下具有低透水性。
Helicobacter pylori’s unique ability to colonize and survive in the acidic environment of the stomach is critically dependent on uptake of urea through the urea channel, HpUreI. Hence, HpUreI may represent a promising target for the development of specific drugs against this human pathogen. To obtain insight into the structure/function relationship of this channel, we have developed conditions for the high-yield expression and purification of stable recombinant HpUreI that allowed its detailed kinetic characterization in solubilized form and reconstituted into liposomes. Detergent-solubilized HpUreI forms homo-trimer, as determined by chemical cross-linking. Urea dissociation kinetics of purified HpUreI were determined by means of the scintillation proximity assay (SPA), whereas urea efflux was measured in HpUreI-containing proteoliposomes using stopped-flow spectrometry to determine the kinetics and selectivity of the urea channel. The kinetic analyses revealed that urea conduction in HpUreI is pH sensitive and saturable with a half-saturation concentration (or K0.5) of ~163 mM. Binding of urea by HpUreI was increased at lower pH; however, the apparent affinity of urea binding (~150 mM) was not significantly pH dependent. The solute selectivity analysis indicated that HpUreI is highly selective for urea and hydroxyurea. Removing either amino group of urea molecules diminishes their permeability through HpUreI. Similar to urea conduction, water diffusion through HpUreI is pH-dependent with low water permeability at neutral pH.