Van Dyke R W
J Biol Chem. 1986 Dec 5;261(34):15941-8.
Rat liver multivesicular bodies (MVB), as well as other hepatic subcellular organelles, are acidified by an electrogenic ATP-dependent proton pump that requires Cl- for maximal acidification (Van Dyke, R. W., Hornick, C. A., Belcher, J., Scharschmidt, B. F., and Havel, R.J. (1985) J. Biol. Chem. 260, 11021-11026), suggesting that Cl- serves as a permeable charge-compensating anion. However, we have observed that NO3- is unable to substitute for Cl-. This study was undertaken therefore to examine more closely the effects of Cl- on MVB acidification and to determine whether NO3- and other anions interact with the proton pump. ATP-dependent vesicle acidification and membrane potential (psi) were measured using the fluorescent dyes acridine orange and Oxonol V (bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol), respectively. Cl- both stimulated acidification (Km = 23.2 +/- 4.2 mM) and decreased psi (IC50 = 3.4 +/- 0.6 mM) in a concentration-dependent, nonlinear fashion. In the presence of saturating Cl- (100 mM), however, NO3- (shown to be more permeable than Cl-) and the impermeant anions SO4(2-) and PO4(2-), inhibited both ATP-dependent acidification and psi in a concentration-dependent manner. Other anions, including gluconate and HCO3-, had no effect. The inhibitory effect of NO3- was reversible. Neither SO4(2-) nor PO4(2-) appeared to block Cl- movement across the vesicle membrane as assessed by the ability of Cl- to decrease an established psi. In additional experiments, the effects of anions on relaxation of a previously established pH gradient were measured. Compared to Cl- or gluconate, NO3- had no significant effect on pH gradient relaxation, even when MVB were preloaded with NO3-, indicating that rapid cycling of NO3-/HNO3 across the MVB membrane does not occur. The organic nitrate, isosorbide dinitrate, also inhibited both acidification and psi and, similar to NO3-, had no effect on pH gradient relaxation. By contrast, NO2- potently inhibited both MVB acidification and psi but also rapidly relaxed a pre-established pH gradient, suggesting that NO2- increases MVB membrane proton permeability. Finally, MVB exhibited N-ethylmaleimide-sensitive ATPase activity that was inhibited 23.9% by NO3- (100 mM). In conclusion, although MVB are permeable to a variety of anions (Cl-, Br-, NO3-, NO2-), only Cl- and Br- support maximal rates of acidification by the proton pump.(ABSTRACT TRUNCATED AT 400 WORDS)
大鼠肝脏多囊泡体（MVB）以及其他肝亚细胞器，是由一种依赖ATP的电生性质子泵酸化的，该质子泵需要Cl⁻才能实现最大程度的酸化（范·戴克，R.W.，霍尼克，C.A.，贝尔彻，J.，沙施密特，B.F.，以及哈维尔，R.J.（1985年）《生物化学杂志》260卷，11021 - 11026页），这表明Cl⁻作为一种可通透的电荷补偿阴离子。然而，我们观察到NO₃⁻无法替代Cl⁻。因此开展本研究以更深入地研究Cl⁻对MVB酸化的影响，并确定NO₃⁻和其他阴离子是否与质子泵相互作用。分别使用荧光染料吖啶橙和恶嗪V（双（3 - 苯基 - 5 - 氧代异恶唑 - 4 - 基）五甲川恶嗪）测量依赖ATP的囊泡酸化和膜电位（ψ）。Cl⁻以浓度依赖性的非线性方式刺激酸化（Km = 23.2 ± 4.2 mM）并降低ψ（IC50 = 3.4 ± 0.6 mM）。然而，在存在饱和Cl⁻（100 mM）的情况下，NO₃⁻（已证明比Cl⁻更具通透性）以及非通透阴离子SO₄²⁻和PO₄³⁻，以浓度依赖性方式抑制依赖ATP的酸化和ψ。其他阴离子，包括葡萄糖酸盐和HCO₃⁻，没有影响。NO₃⁻的抑制作用是可逆的。通过Cl⁻降低既定ψ的能力评估，SO₄²⁻和PO₄³⁻似乎都不阻断Cl⁻跨囊泡膜的移动。在额外的实验中，测量了阴离子对先前建立的pH梯度弛豫的影响。与Cl⁻或葡萄糖酸盐相比，即使MVB预先加载了NO₃⁻，NO₃⁻对pH梯度弛豫也没有显著影响，这表明NO₃⁻/HNO₃不会在MVB膜上快速循环。有机硝酸盐异山梨醇二硝酸酯也抑制酸化和ψ，并且与NO₃⁻类似，对pH梯度弛豫没有影响。相比之下，NO₂⁻强烈抑制MVB酸化和ψ，但也迅速弛豫预先建立的pH梯度，这表明NO₂⁻增加了MVB膜质子通透性。最后，MVB表现出对N - 乙基马来酰亚胺敏感的ATP酶活性，该活性被100 mM的NO₃⁻抑制了23.9%。总之，尽管MVB对多种阴离子（Cl⁻、Br⁻、NO₃⁻、NO₂⁻）具有通透性，但只有Cl⁻和Br⁻支持质子泵的最大酸化速率。（摘要截断于400字）
