Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates
Alternate energy-dependent pathways for the vacuolar uptake of glucose and glutathione conjugates
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DOI：
10.1104/pp.008334
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发表时间：
2002-11-01
期刊：
影响因子：
7.4
通讯作者：
Viitanen, PV
中科院分区：
文献类型：
--
作者：
Bartholomew, DM;Van Dyk, DE;Viitanen, PV
通过开发和应用液相色谱-质谱法测量液泡膜囊对复杂有机分子的转运，表明磺酰脲类除草剂的吸收机制是由配体、葡萄糖或谷胱甘肽决定的，除草剂与配体结合。从红甜菜（Beta vulgaris）储存根中纯化的空泡膜囊泡对糖基化氯磺龙的atp依赖性积累接近Michaelis-Menten动力学，并受到破坏或阻止跨膜H+梯度形成的药物的强烈抑制，但对磷酰过渡态类似物钒酸盐完全不敏感。相反，氯磺隆类似物氯脲酰乙酯的谷胱甘肽偶联物的atp依赖性积累被分散跨膜H+梯度的药物不完全抑制，但被钒酸盐完全消除。然而，在这两种情况下，共轭对于净吸收是必不可少的，因为未共轭的母体化合物在通电或无通电的条件下都不会积累。葡萄糖通过芳香羟基与两种天然存在的苯丙素（对羟基肉桂酸和对羟基苯甲酸）结合，将这些化合物定位为负责氯磺隆-葡萄糖苷运输的转运体的功能等效物，证实了葡萄糖苷摄取的H+梯度依赖性的一般适用性。由此得出结论，H+梯度依赖性、对钒酸盐不敏感的糖苷摄取是由H+反转运蛋白介导的，而钒酸盐敏感的谷胱甘肽缀合物摄取是由atp结合盒转运蛋白介导的。在这样做的过程中，液相色谱-质谱法为研究复杂有机分子的运输提供了一种通用的高灵敏度、高保真度的技术，这些有机分子作为放射性标记衍生物的合成是费力的和/或昂贵的。
Through the development and application of a liquid chromatography-mass spectrometry-based procedure for measuring the transport of complex organic molecules by vacuolar membrane vesicles in vitro, it is shown that the mechanism of uptake of sulfonylurea herbicides is determined by the ligand, glucose, or glutathione, to which the herbicide is conjugated. ATP-dependent accumulation of glucosylated chlorsulfuron by vacuolar membrane vesicles purified from red beet (Beta vulgaris) storage root approximates Michaelis-Menten kinetics and is strongly inhibited by agents that collapse or prevent the formation of a transmembrane H+ gradient, but is completely insensitive to the phosphoryl transition state analog, vanadate. In contrast, ATP-dependent accumulation of the glutathione conjugate of a chlorsulfuron analog, chlorimuronethyl, is incompletely inhibited by agents that dissipate the transmembrane H+ gradient but completely abolished by vanadate. In both cases, however, conjugation is essential for net uptake because neither of the unconjugated parent compounds are accumulated under energized or nonenergized conditions. That the attachment of glucose to two naturally occurring phenylpropanoids, p-hydroxycinnamic acid and p-hydroxybenzoic acid via aromatic hydroxyl groups, targets these compounds to the functional equivalent of the transporter responsible for chlorsulfuron-glucoside transport, confirms the general applicability of the H+ gradient dependence of glucoside uptake. It is concluded that H+ gradient-dependent, vanadate-insensitive glucoside uptake is mediated by an H+ antiporter, whereas vanadate-sensitive glutathione conjugate uptake is mediated by an ATP-binding cassette transporter. In so doing, it is established that liquid chromatography-mass spectrometry affords a versatile high-sensitivity, high-fidelity technique for studies of the transport of complex organic molecules whose synthesis as radiolabeled derivatives is laborious and/or prohibitively expensive.