The traditional method for measuring the transport of vacuolar membrane H + is accomplished by in vitro detection with a pH fluorescent dye. However, the high-purity vacuolar membrane separation is complicated in this method, and the sensitivity to the weak H + real-time monitoring is insufficient, which has become a cause of influence on researchers. In recent years, non-invasive micro-testing technology (NMT) has been widely used in electrophysiological research. NMT has the advantages of non-injury, high sensitivity and real-time monitoring of the entire process. So far, although the Ca 2+ flow of beet vacuoles has been measured by NMT, no measurement of the bubble H + flow rate has been reported. Therefore, the establishment of vacuoles and cell membrane system H + flow rate determination method is of great significance for the study of proton movement and membrane protein activity.
In 2011, the Li Yinxin Research Group of the Institute of Botany, Chinese Academy of Sciences, carried out several methodological corrections for the determination of vacuolar H + flow by non-invasive micro-measurement technology (NMT) , which enabled NMT to more accurately measure the bubble H + flow. At the same time, the newly established system was used to determine the difference in vacuolar H + flow rate of transgenic tobacco TS15 and WT tobacco under NaCl stress , and the activity of H-ATPase and PPase on the tonoplast was determined by NMT method. The study first simplified the bubble extraction method based on the characteristics of the NMT visual measurement, omitting the cumbersome ultracentrifugation steps in the conventional detection. Under the stress of 200 mM NaCl, H + outflow increased, but the H + outflow of TS15 was significantly stronger than that of WT, indicating that NHX1 has higher activity in transgenic plants. When ATP and PPi were applied under 200 mM NaCl, the change of vacuolar ΔH + flow rate reflected the activity of H-ATPase and PPase; studies showed that the transgenic tobacco vacuoles showed a larger ΔH + flow rate after ATP application ; The transgenic plants were shown to have higher V-ATPase activity than the WT plants at 200 mM NaCl. At the same time, the accuracy of the NMT assay was confirmed by the traditional method of calculating the H-ATPase and PPase activities. Compared with traditional fluorescence methods, NMT has a greater advantage in terms of operational flow, accuracy of results, and amount of information for conclusions.
This study established and revised a suitable and accurate method for determining the flow rate of vacuolar H + . The difference in ion current can also reflect more physiological characteristics, and it is a new method that can replace the traditional V-ATPase and PPase activity determination methods. This simple method will enable extensive detection and analysis of subcellular and organelle level ion flow rates in the future.
References: Chen XY , et al . Analytical Biochemistry, 2011, 418: 295–297.
Key words: non- invasive micro-test technique, H + flow rate, SeNHX1 , vacuole
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