Müller M L, Taiz L
Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA.
J Membr Biol. 2002 Feb 1;185(3):209-20. doi: 10.1007/s00232-001-0124-z. Epub 2002 Feb 13.
The lemon-fruit V-ATPase can exist in two forms: nitrate-sensitive and nitrate-insensitive. Here we report the results of measurements of H+ /ATP stoichiometries using two kinetic methods: one based on steady-state DpH and one based on initial rates of H+-pumping. Our findings indicate that the nitrate-insensitive fruit V-ATPase has an H+ /ATP stoichiometry of ~1, while both the nitrate-sensitive fruit V-ATPase and the epicotyl V-ATPase have stoichiometries of 2, under zero-load conditions. As DpH increases, the stoichiometry of the nitrate-sensitive fruit V-ATPase decreases to 1. Under similar conditions, the stoichiometry of the epicotyl enzyme remains 2. Thus, the pH-dependent variable stoichiometry of the lemon-fruit V-ATPase may represent a key factor in juice sac vacuolar hyperacidification. On the other hand, the H+ /ATP stoichiometry of the epicotyl V-ATPase can decrease from 2 to 1 in the presence of a membrane potential. The low pH of the fruit vacuole is not due solely to the lower H+/ATP stoichiometry of its pump. We show that lumenal citrate and malate improve the coupling of both the epicotyl and fruit V-ATPases and enhance their ability to generate a pH gradient. Since citrate accumulation is restricted to fruit vacuoles, it may be another important determinant of vacuolar pH.
柠檬果实V-ATP酶可以以两种形式存在：对硝酸盐敏感型和对硝酸盐不敏感型。在此，我们报告了使用两种动力学方法测量H⁺/ATP化学计量比的结果：一种基于稳态DpH，另一种基于H⁺泵浦的初始速率。我们的研究结果表明，在零负载条件下，对硝酸盐不敏感的果实V-ATP酶的H⁺/ATP化学计量比约为1，而对硝酸盐敏感的果实V-ATP酶和上胚轴V-ATP酶的化学计量比均为2。随着DpH的增加，对硝酸盐敏感的果实V-ATP酶的化学计量比降至1。在类似条件下，上胚轴酶的化学计量比保持为2。因此，柠檬果实V-ATP酶的pH依赖性可变化学计量比可能是汁囊液泡过度酸化的关键因素。另一方面，在上胚轴V-ATP酶存在膜电位的情况下，其H⁺/ATP化学计量比可从2降至1。果实液泡的低pH值并非仅仅归因于其泵的较低H⁺/ATP化学计量比。我们表明，腔中的柠檬酸和苹果酸改善了上胚轴和果实V-ATP酶的偶联，并增强了它们产生pH梯度的能力。由于柠檬酸积累仅限于果实液泡，它可能是液泡pH值的另一个重要决定因素。
