von Wirén N, Gibrat R, Briat J F
Biochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (URA 2133), Institut National de la Recherche Agronomique et Ecole Nationale Supérieure d'Agronomie, Place Viala, F-34060 Montpellier, France.
Biochim Biophys Acta. 1998 Apr 22;1371(1):143-55. doi: 10.1016/s0005-2736(98)00022-4.
As an attempt to characterize iron(III)-phytosiderophore transport across plant membranes in vitro, a rapid filtration approach was set up in which plasma membrane vesicles from maize roots were incubated with 55Fe-labelled deoxymugineic acid (DMA). Fe-DMA, and not Fe-EDTA, could associate with plasma membrane vesicles. The rate of Fe-DMA association decreased with a half time of 15 min. The initial Fe-DMA association rate, estimated from the amount of Fe-DMA associated after 10 min incubation, exhibited a saturation curve as a function of Fe-DMA concentration. This curve could be satisfactorily fitted to the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein). The association rate of Fe-DMA with control liposomes remained negligible and constant in a pH range from 4 to 8, whereas it strongly increased at acidic pH with plasma membrane vesicles. However, the specific association of Fe-DMA to root plasma membrane could not be explained by a vesicle-filling process because: (i) lowering the vesicle volume by decreasing the osmotic potential of the assay medium with sorbitol did not decrease 55(Fe) labelling of the vesicles, (ii) creating inside-out vesicles by a Brij-58 treatment had almost no effect on Fe-DMA association to vesicles, (iii) 55(Fe) labelling is reversible by EDTA and excess free DMA, and (iv) 55(Fe) labelling was the same using plasmalemma vesicles prepared either from wild type maize or from the ys1 maize mutant deficient in iron-phytosiderophore transport. A model is proposed to account for the observed Fe-DMA association as the result of very slow binding kinetics onto membrane proteins. This model was validated by its ability to describe quantitatively both Fe-DMA association as a function of time and of substrate concentration. A prediction of the model was that association of Fe-DMA to plasma membranes might overcome a high activation energy barrier. Indeed, the Arrhenius plot for the association rate constant was linear with an activation energy of 64 kJ mol-1.
作为在体外表征铁（III）-植物铁载体跨植物膜转运的一种尝试，建立了一种快速过滤方法，其中将来自玉米根的质膜囊泡与55Fe标记的脱氧 mugineic 酸（DMA）一起孵育。Fe-DMA而非Fe-EDTA能够与质膜囊泡结合。Fe-DMA的结合速率以15分钟的半衰期下降。根据孵育10分钟后结合的Fe-DMA量估算的初始Fe-DMA结合速率，呈现出作为Fe-DMA浓度函数的饱和曲线。该曲线可以很好地拟合米氏模型（KM = 600 nM，Vmax = 2 nmol min-1 mg-1蛋白质）。在pH值4至8的范围内，Fe-DMA与对照脂质体的结合速率仍然可以忽略不计且保持恒定，而在酸性pH条件下，与质膜囊泡的结合速率则大幅增加。然而，Fe-DMA与根质膜的特异性结合不能用囊泡填充过程来解释，原因如下：（i）通过用山梨醇降低测定介质的渗透压来降低囊泡体积，并不会降低囊泡的55（Fe）标记；（ii）用Brij-58处理制备外翻囊泡对Fe-DMA与囊泡的结合几乎没有影响；（iii）55（Fe）标记可被EDTA和过量的游离DMA逆转；（iv）使用从野生型玉米或缺乏铁-植物铁载体转运的ys1玉米突变体制备的质膜囊泡，55（Fe）标记是相同的。提出了一个模型来解释观察到的Fe-DMA结合现象，认为这是由于与膜蛋白的结合动力学非常缓慢所致。该模型通过其定量描述Fe-DMA结合随时间和底物浓度变化的能力得到了验证。该模型的一个预测是，Fe-DMA与质膜的结合可能会克服一个高活化能屏障。事实上，结合速率常数的阿伦尼乌斯图呈线性，活化能为64 kJ mol-1。
