A novel method to quantify H+-ATPase-dependent Na+ transport across plasma membrane vesicles.
A novel method to quantify H+-ATPase-dependent Na+ transport across plasma membrane vesicles.
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DOI：
10.1016/j.bbamem.2007.06.028
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
Yongqing Yang;Lei Hu;Xuemei Chen;Eric A. Ottow;A. Polle;Xiangning Jiang
中科院分区：
文献类型：
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作者：
Yongqing Yang;Lei Hu;Xuemei Chen;Eric A. Ottow;A. Polle;Xiangning Jiang
为了防止植物中的钠毒害，Na+通过Na+/H+反向转运蛋白从细胞质中被排除到质外体或液泡中。质膜H+-ATP酶水解ATP并将H+泵过质膜，从而驱动Na+逆其电化学梯度向质外体的次级主动运输。目前测定Na+通量的方法依赖于使用Na-同位素（22 Na），这需要特殊的工作许可或复杂的设备，或者依赖于通过pH敏感探针在存在或不存在Na+的情况下估计H+-ATP酶引起的H+梯度变化的间接方法。迄今为止，还没有方法可以直接定量质膜囊泡中H+-ATP酶依赖的Na+转运。我们开发了一种方法来测量双向H+-ATP酶依赖的钠离子转运在隔离膜囊泡系统使用原子吸收光谱法（AAS）。利用双水相分离法从毛白杨叶片中分离出富含质膜的囊泡进行实验。由于大多数质膜囊泡在反复的水两相分配后具有密封的右侧向外的取向，H+-ATP酶的ATP结合位点暴露于内侧。在渗漏囊泡中预先装入Na+，用于研究H+-ATP酶依赖的Na+转运。我们的数据表明，Na+穿过囊泡膜的运动高度依赖于需要ATP和Mg 2+的H+-ATP酶活性，并在pH 6.5和25 °C下显示出2.50 μM Na+mg− 1膜蛋白min− 1的最佳速率。在这项研究中，第一次，我们建立了新的协议，制备密封的预装右侧出囊泡的研究H+-ATP酶依赖的Na+运输。结果表明，原子吸收光谱法可直接定量测定不同类型质膜囊泡的Na+含量，且与荧光探针法测定结果一致。该方法是一个方便的系统，用于研究双向H+-ATP酶依赖的Na+转运与膜囊泡。
To prevent sodium toxicity in plants, Na+is excluded from the cytosol to the apoplast or the vacuole by Na+/H+antiporters. The secondary active transport of Na+to apoplast against its electrochemical gradient is driven by plasma membrane H+-ATPases that hydrolyze ATP and pump H+across the plasma membrane. Current methods to determine Na+flux rely either on the use of Na-isotopes (22Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H+gradient due to H+-ATPase in the presence or absence of Na+by pH-sensitive probes. To date, there are no methods that can directly quantify H+-ATPase-dependent Na+transport in plasma membrane vesicles. We developed a method to measure bidirectional H+-ATPase-dependent Na+transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H+-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na+sealed for the study of H+-ATPase-dependent Na+transport. Our data implicate that Na+movement across vesicle membranes is highly dependent on H+-ATPase activity requiring ATP and Mg2+and displays optimum rates of 2.50 μM Na+mg−1membrane protein min−1at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H+-ATPase-dependent Na+transport. The results demonstrate that the Na+content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H+-ATPase-dependent Na+transport with membrane vesicles.