Determination of ferrichrome binding to the FhuA outer membrane transport protein, periplasmic accumulation of ferrichrome, or transport of ferrichrome into cells using a three-layer oil technique
Determination of ferrichrome binding to the FhuA outer membrane transport protein, periplasmic accumulation of ferrichrome, or transport of ferrichrome into cells using a three-layer oil technique
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DOI：
10.1016/s0003-2697(02)00285-3
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发表时间：
2002-11-01
影响因子：
2.9
通讯作者：
Gestwa, G
中科院分区：
文献类型：
--
作者：
Killmann, H;Gestwa, G
开发了一种新方法，用于测定铁红蛋白与大肠杆菌外膜中 FhuA 转运蛋白的结合、铁红蛋白在周质空间中的积累以及铁红蛋白转运到细胞质中。通过在含有三层不同密度的不可混合溶液的微试管中离心，将细胞与残留的、可溶的、放射性标记的铁铬分离。上水层中的细胞穿过中间的硅油层，但没有进入下面的NaI层，从而聚集在NaI层的顶部；可溶性化合物保留在上层水层中。然后通过离心轻松回收细胞，并通过液体闪烁计数测定放射性。无需任何洗涤步骤即可获得所有测试应用的可重复结果。通过测定各种FhuA突变体的受体结合和铁红转运来测试该方法，与它们的转运活性相反，显示铁红与FhuA仅弱结合，并与常用的硝酸纤维素过滤方法进行比较。两种方法获得了类似的转运速率，但铁红与突变的 FhuA 蛋白的结合以及铁红在周质中的积累只能用新方法测量。 (C) 2002 年爱思唯尔科学（美国）。版权所有。
A new method for the determination of ferrichrome binding to the FhuA transporter in the Escherichia coli outer membrane, ferrichrome accumulation in the periplasmic space, and ferrichrome transport into the cytoplasm was developed. Cells were separated from residual, soluble, radiolabeled ferrichrome by centrifugation in a micro-test tube containing three layers of nonmixable solutions of different densities. Cells in the upper aqueous layer passed through the middle silicone oil layer, but did not enter the underlying NaI layer, thereby accumulating on top of the NaI layer; soluble compounds remained in the upper aqueous layer. Cells were then easily recovered by centrifugation, and radioactivity was determined by liquid scintillation counting. Reproducible results for all applications tested were obtained without the need for any washing steps. The method was tested by determination of receptor binding and transport of ferrichrome with various FhuA mutants which, in contrast to their transport activity, showed only a weak binding of ferrichrome to FhuA and compared with the commonly used cellulose nitrate filter method. Similar transport rates were obtained with the two methods, but binding of ferrichrome to the mutated FhuA proteins and accumulation of ferrichrome in the periplasm could be measured only with the new method. (C) 2002 Elsevier Science (USA). All rights reserved.