Yuanbo Cui, Fan Zhang, Jiachang Yue
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Anal Biochem. 2005 Sep 1;344(1):102-7. doi: 10.1016/j.ab.2005.06.027.
N-(Fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (F-DHPE) is a lipid fluorescence dye sensitive to pH changes and is used in this study for detecting proton flux through F0F1-ATPase within chromatophores driven by ATP hydrolysis. F-DHPE is easily labeled to the outer surface of chromatophores. In the range of pH 7.0 to 9.0, fluorescence intensity is sensitive to pH changes. The sensitivity is especially great in the range of pH 8.2 to 9.0, so pH 8.6 was chosen as the appropriate experimental condition. It is shown that added ATP not only acts as a fluorescence quencher but also can be hydrolyzed by F0F1-ATPase to pump protons into chromatophores, resulting in fluorescence restoration. A stimulator (NaSO3) and various types of inhibitors (NaN3, 5'-adenylyl imidodiphosphate [AMP-PNP], and N,N'-dicyclohexylcarbodiimide [DCCD]) of F0F1 confirmed that fluorescence restoration is caused by ATP-driven proton flux. When loaded with one antibody (anti-beta antibody) or two antibodies (anti-beta antibody and sheep to rabbit second antibody), F0F1-ATPase exhibits lower proton pumping activities, as indicated by fluorescence restoration. The possible mechanism of the inhibition of antibodies on proton pumping activity is discussed.
N-(荧光素-5-硫代甲酰基)-1,2-二己酰基-sn-甘油-3-磷酸乙醇胺三乙铵盐（F-DHPE）是一种对pH变化敏感的脂质荧光染料，本研究中用于检测由ATP水解驱动的色素体内通过F0F1-ATP酶的质子通量。F-DHPE易于标记在色素体的外表面。在pH 7.0至9.0范围内，荧光强度对pH变化敏感。在pH 8.2至9.0范围内敏感性尤其高，因此选择pH 8.6作为合适的实验条件。结果表明，添加的ATP不仅作为荧光猝灭剂，还可被F0F1-ATP酶水解，将质子泵入色素体，导致荧光恢复。F0F1的一种刺激剂（NaSO3）和各种类型的抑制剂（NaN3、5'-腺苷亚氨基二磷酸[AMP-PNP]和N,N'-二环己基碳二亚胺[DCCD]）证实，荧光恢复是由ATP驱动的质子通量引起的。当装载一种抗体（抗β抗体）或两种抗体（抗β抗体和羊抗兔二抗）时，F0F1-ATP酶表现出较低的质子泵浦活性，荧光恢复表明了这一点。讨论了抗体抑制质子泵浦活性的可能机制。
