Müller M, Irkens-Kiesecker U, Rubinstein B, Taiz L
Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
J Biol Chem. 1996 Jan 26;271(4):1916-24. doi: 10.1074/jbc.271.4.1916.
Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATP-driven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes. H+ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N'-dicyclohexylcarbodiimide. In addition, the epicotyl H+ pumping activity was inactivated by oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( > or = 100 mM) was correlated with the release of V1 complexes from the membrane. In contrast, H+ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the V-ATPase inhibitors, and resistant to oxidation. Unlike the epicotyl inhibitors, and resistant to oxidation. Unlike the epicotyl H(+)-ATPase, the fruit H(+)-ATPase activity was partially inhibited by 200 microM vanadate. Cold inactivation treatment failed to inhibit H+ pumping activity of the fruit membranes, even though immunoblasts showed that V1 complexes were released from the membrane. However, cold inactivation doubled the percent inhibition by 200 microM vanadate from 30% to 60%. These results suggest the presence of two H(+)-ATPases in the fruit preparation: a V-ATPase and an unidentified vanadate-sensitive H(+)-ATPase. Attempts to separate the two activities in their native membranes on linear sucrose density density gradients were unsuccessful. However, following detergent-solubilization and centrifugation on a glycerol density gradient, the two ATPase activities were resolved: a nitrate-sensitive V-type ATPase that is also partially inhibited by 200 microM vanadate, and an apparently novel vanadate-sensitive ATPase that is also partially inhibited by nitrate.
柠檬果实液泡将其内腔酸化至pH 2.5，比典型的植物液泡低3个pH单位。为了研究过度酸化的机制，比较了来自柠檬果实和上胚轴的液泡膜囊泡由ATP驱动的质子泵浦动力学。果实液泡膜。上胚轴膜的H⁺泵浦是氯离子依赖性的，受硫酸盐刺激，并被经典的液泡ATP酶（V-ATP酶）抑制剂硝酸盐、巴弗洛霉素、N-乙基马来酰亚胺和N,N'-二环己基碳二亚胺抑制。此外，上胚轴H⁺泵浦活性被氧化失活，二硫苏糖醇可使其逆转。硝酸盐（≥100 mM）对上胚轴V-ATP酶的冷失活与V1复合物从膜上的释放相关。相比之下，富含果实液泡膜的H⁺泵浦不依赖氯离子，对V-ATP酶抑制剂基本不敏感，且抗氧化。与上胚轴抑制剂不同，且抗氧化。与上胚轴H⁺-ATP酶不同，果实H⁺-ATP酶活性被200 μM钒酸盐部分抑制。冷失活处理未能抑制果实膜的H⁺泵浦活性，尽管免疫印迹显示V1复合物从膜上释放。然而，冷失活使200 μM钒酸盐的抑制百分比从30%增加到60%。这些结果表明在果实制剂中存在两种H⁺-ATP酶：一种V-ATP酶和一种未鉴定的对钒酸盐敏感的H⁺-ATP酶。尝试在其天然膜上通过线性蔗糖密度梯度分离这两种活性未成功。然而，在去污剂增溶并在甘油密度梯度上离心后，两种ATP酶活性得以分离：一种对硝酸盐敏感的V型ATP酶，它也被200 μM钒酸盐部分抑制，以及一种明显新型的对钒酸盐敏感的ATP酶，它也被硝酸盐部分抑制。
