Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes.
Functional characterization of the Escherichia coli glycerol facilitator, GlpF, in Xenopus oocytes.
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发表时间：
1994-04
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通讯作者：
Christophe Maurel;J. Reizer;Julian I. Schroeder;M. Chrispeels;M. H. Saier
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作者：
Christophe Maurel;J. Reizer;Julian I. Schroeder;M. Chrispeels;M. H. Saier
大肠杆菌的甘油促进剂GlpF是这种革兰氏阴性细菌内膜中假定的非选择性转运通道。它是跨膜通道蛋白的主要内在蛋白（MIP）家族的成员。由于缺乏可在不存在其他细菌蛋白的情况下检查其活性的异源测试系统，其表征受到阻碍。运输甘油介导的蛋白质的特点是注射后的glpF mRNA到非洲爪蟾卵母细胞。GlpF的性质进行了比较，同源植物水通道蛋白，γ液泡膜内在蛋白（γ TIP），以及非同源爪蟾K+通道，Xsha。GlpF选择性地运输甘油，但不运输水或离子，而γ TIP和Xsha分别特异性地运输水和K+。电压钳实验表明，GlpF不是电压激活的离子转运。通过GlpF的甘油转运证明在高达200 mM时是不饱和的，并显示出低的活化温度（Ea = 4.5 kcal/mol），这与Heller等人的结论一致（Heller，K. B.，Lin，E. C. C.的方法，Wilson，T. H.等人（1980）J. Bacteriol. 144，274-278），GlpF通过孔型机制介导甘油扩散。GlpF介导的甘油转运被汞离子（Hg 2+）阻断，但不被N-乙基马来酰亚胺阻断。Hg ~（2+）的抑制作用可被高浓度甘油部分阻断，巯基乙醇可逆转。这些结果可用于表征E.大肠杆菌甘油促进剂。
The glycerol facilitator of Escherichia coli, GlpF, is a putative nonselective transport channel in the inner membrane of this Gram-negative bacterium. It is a member of the major intrinsic protein (MIP) family of transmembrane channel proteins. Its characterization has been hampered by the lack of a heterologous test system in which its activity can be examined in the absence of other bacterial proteins. Transport of glycerol mediated by this protein was characterized following injection of glpF mRNA into Xenopus laevis oocytes. The properties of GlpF were compared with those of the homologous plant water channel protein, gamma tonoplast intrinsic protein (gamma TIP), as well as the nonhomologous Xenopus K+ channel, Xsha. GlpF selectively transported glycerol but not water or ions, while gamma TIP and Xsha were specific for water and K+, respectively. Voltage clamp experiments showed that GlpF was not voltage-activated for ion transport. Glycerol transport via GlpF proved to be nonsaturable up to 200 mM and exhibited a low temperature of activation (Ea = 4.5 kcal/mol), consistent with the conclusion of Heller et al. (Heller, K. B., Lin, E. C. C., and Wilson, T. H. (1980) J. Bacteriol. 144, 274-278) that GlpF mediates glycerol diffusion via a pore type mechanism. GlpF-mediated transport of glycerol was blocked by mercuric ions (Hg2+) but not N-ethylmaleimide. The inhibitory effect of Hg2+ was partially prevented by inclusion of a high concentration of glycerol and reversed by mercaptoethanol. The results serve to characterize the transport properties of the E. coli glycerol facilitator.