Laboratoire de Physiologie Végétale Appliquée, Université des Sciences et Techniques du Languedoc, Place Eugéne Bataillon, F-34060, Montpellier-Cédex, France.
Planta. 1983 Jul;157(4):324-30. doi: 10.1007/BF00397403.
The function of the Mg-dependent ATPase of Hevea tonoplast in active proton transport was investigated by using a purified tonoplast fraction containing tightly sealed vesicles. In the used experimental conditions, the uptake of [(14)C]triphenylmethyl-phosphonium ion ([(14)C]TPMP(+)) and [(3)H]tetraphenyl-phosphonium ion ([(3)H]TPP(+)) by the vesicles indicated a transmembrane potential difference, negative inside. In parallel, the uptake of [(14)C]methylamine into the vesicles monitored a transmembrane pH gradient, interior acid. The addition of 5 mM Mg-ATP markedly depolarized the membrane and increased the magnitude of trnasmembrane pH gradient. These ATP-driven events were substrate specific for Mg-ATP. They were strongly inhibited by ATPase inhibitors such as N, N'-dicyclohexylcar-bodiimide. They were completely eliminated by proton conductors such as carbonylcyanide-p-trifluoromethoxy-phenylhydrazone and 5-chloro-3-tert-butyl-2'-chloro-4-nitro-salicylanilide. They depended on the pH of the medium, the maximum being reached at about pH 7.0. These data provide in vitro evidence that the Mg-ATPase localized at tonoplast level is an electrogenic pump. They are consistent with the hypothesis that an electrogenic H(+) pump is catalyzed by the tonoplast ATPase of higher plants.
我们利用含有紧密封闭小泡的纯化质膜部分研究了巴西橡胶树液泡膜 Mg 依赖性 ATP 酶在主动质子转运中的功能。在实验条件下，[(14)C]三苯甲基膦离子([(14)C]TPMP(+))和[(3)H]四苯膦离子([(3)H]TPP(+))的摄取表明小泡存在跨膜电位差，内侧为负。同时，[(14)C]甲胺的摄取表明小泡内存在跨膜 pH 梯度，内侧为酸性。加入 5 mM Mg-ATP 会显著去极化质膜并增加跨膜 pH 梯度的幅度。这些由 ATP 驱动的事件是 Mg-ATP 的底物特异性。它们强烈抑制 ATP 酶抑制剂如 N，N'-二环己基碳二亚胺。它们被质子载体如羰基氰化物-p-三氟甲氧基-对苯腙和 5-氯-3-叔丁基-2'-氯-4-硝基水杨酰苯胺完全消除。它们依赖于介质的 pH 值，最大在约 pH 7.0 时达到。这些数据提供了质膜水平上 Mg-ATP 酶是电致泵的体外证据。它们与质膜 ATP 酶催化的电致 H(+)泵假说一致。
