Ruiz-Cristin J, Briskin D P
Department of Agronomy, University of Illinois, Urbana 61801.
Arch Biochem Biophys. 1991 Feb 15;285(1):74-82. doi: 10.1016/0003-9861(91)90330-l.
The mechanism of NO3- transport was examined in isolated plasma membrane vesicles from maize (Zea mays L., hybrid B73 X LH 51) roots using 36ClO3- as a radiotracer analog for NO3-. When an acid-exterior delta pH was imposed across the vesicle membrane, uptake of 36ClO3- was stimulated and the time course of radiolabel uptake displayed an overshoot phenomenon characteristic of the coupling of one solute gradient ot the movement of another solute. Evidence supporting delta pH as the driving force for 36ClO3- uptake included a dependence of the overshoot peak and initial rate of 36ClO3- uptake on the magnitude of the imposed delta pH, the occurrence of delta pH-driven 36ClO3- uptake in the presence of KSCN/valinomycin, and the ability of an imposed delta pH to drive 36ClO3- uptake when radiolabel was equilibrated across the membrane. When delta pH-driven 36ClO3- transport was examined in the presence of NO3-, radiolabel uptake was inhibited in a competitive manner. This was consistent with the carrier having the capacity to use either ClO3- or NO3- and supports the use of this radiotracer as an analog for NO3- in transport studies. When delta pH-driven 36ClO3- uptake was examined as a function of 36ClO3- concentration and delta pH, saturation kinetics were observed and the magnitude of the imposed delta pH affected the Km but not the Vmax for 36ClO3- uptake. This suggested an ordered binding mechanism where 36ClO3- would bind to the protonated form of the carrier prior to translocation. Radiolabeled 36ClO3- uptake was inhibited by treatment of the vesicles with phenylglyoxal, suggesting the involvement of arginine moieties in the process of transport. Taken together, these results support the presence of a H+/NO3- symport carrier at the plasma membrane which could be involved in mediating energy-dependent NO3- uptake into plant cells.
利用³⁶ClO₃⁻作为NO₃⁻的放射性示踪类似物，对玉米（Zea mays L.，杂交种B73×LH 51）根系分离的质膜囊泡中NO₃⁻的转运机制进行了研究。当在囊泡膜上施加外酸性质子动力势（ΔpH）时，³⁶ClO₃⁻的摄取受到刺激，放射性标记摄取的时间进程呈现出一种溶质梯度与另一种溶质移动偶联的过冲现象。支持ΔpH作为³⁶ClO₃⁻摄取驱动力的证据包括过冲峰值和³⁶ClO₃⁻摄取初始速率对施加的ΔpH大小的依赖性、在KSCN/缬氨霉素存在下发生的ΔpH驱动的³⁶ClO₃⁻摄取，以及当放射性标记在膜上达到平衡时施加的ΔpH驱动³⁶ClO₃⁻摄取的能力。当在NO₃⁻存在下检测ΔpH驱动的³⁶ClO₃⁻转运时，放射性标记摄取受到竞争性抑制。这与载体具有使用ClO₃⁻或NO₃⁻的能力一致，并支持在转运研究中使用这种放射性示踪剂作为NO₃⁻的类似物。当检测ΔpH驱动的³⁶ClO₃⁻摄取作为³⁶ClO₃⁻浓度和ΔpH的函数时，观察到饱和动力学，并且施加的ΔpH大小影响³⁶ClO₃⁻摄取的Km但不影响Vmax。这表明存在一种有序结合机制，其中³⁶ClO₃⁻在转运之前会与载体的质子化形式结合。用苯乙二醛处理囊泡会抑制放射性标记的³⁶ClO₃⁻摄取，这表明精氨酸部分参与了转运过程。综上所述，这些结果支持质膜上存在H⁺/NO₃⁻同向转运载体，其可能参与介导植物细胞中能量依赖的NO₃⁻摄取。
