Lukacs G L, Rotstein O D, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Canada.
J Biol Chem. 1990 Dec 5;265(34):21099-107.
The mechanism underlying phagosomal acidification was studied in thioglycolate-elicited murine macrophages. The pH of the phagosomal compartment (pHp) was measured fluorimetrically in macrophage suspensions following ingestion of fluorescein isothiocyanate-labeled Staphylococcus aureus. At 37 degrees C, pHp decreased rapidly, reaching a steady state value of 5.8-6.1, while the cytoplasmic pH remained near neutrality, pH 7.1. The phagosome to cytosol pH gradient could be collapsed by addition of nigericin, monensin, or weak bases. The substrate dependence and inhibitor sensitivity profile of phagosomal acidification were investigated in intact and permeabilized cells. Phagosomal acidification was inhibited when ATP was depleted using metabolic inhibitors or permeabilizing the plasma membrane by electroporation. In permeabilized cells, acidification could be initiated by readdition of both Mg2+ and ATP. Neither adenosine 5'-(beta,gamma-imido)triphosphate nor adenosine 5'-(gamma-thio)triphosphate supported phagosomal acidification. Inhibitors of F1F0-type H(+)-ATPase such as oligomycin and azide, and the E1E2-type H(+)-ATPase inhibitor vanadate had no effect on phagosomal acidification. In contrast, the rate of phagosomal acidification was reduced by micromolar concentrations of N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide. In permeabilized cells, nitrate inhibited the acidification with an apparent Ki of 25 mM. Phagosomal acidification was also effectively blocked by the macrolide antibiotic bafilomycin A1, with an apparent Ki of approximately 3 mM in both intact and electroporated cells. In this concentration range, bafilomycin A1 selectively inhibits vacuolar H(+)-ATPases. The substrate requirement and inhibitor susceptibility profile of phagosomal acidification strongly suggest that proton translocation across the phagosomal membrane is mediated by a vacuolar-type H(+)-ATPase.
在巯基乙酸盐诱导的小鼠巨噬细胞中研究了吞噬体酸化的机制。在摄取异硫氰酸荧光素标记的金黄色葡萄球菌后，通过荧光法测量巨噬细胞悬液中吞噬体区室的pH值（pHp）。在37℃时，pHp迅速下降，达到5.8 - 6.1的稳态值，而细胞质pH值保持在接近中性，pH为7.1。加入尼日利亚菌素、莫能菌素或弱碱可使吞噬体与细胞质的pH梯度消失。在完整细胞和通透细胞中研究了吞噬体酸化的底物依赖性和抑制剂敏感性特征。当使用代谢抑制剂耗尽ATP或通过电穿孔使质膜通透时，吞噬体酸化受到抑制。在通透细胞中，重新加入Mg2+和ATP可启动酸化。腺苷5'-（β，γ-亚氨基）三磷酸和腺苷5'-（γ-硫代）三磷酸均不支持吞噬体酸化。F1F0型H(+)-ATP酶抑制剂如寡霉素和叠氮化物以及E1E2型H(+)-ATP酶抑制剂钒酸盐对吞噬体酸化无影响。相反，微摩尔浓度的N-乙基马来酰亚胺和N，N'-二环己基碳二亚胺可降低吞噬体酸化的速率。在通透细胞中，硝酸盐抑制酸化，表观Ki为25 mM。大环内酯类抗生素巴弗洛霉素A1也能有效阻断吞噬体酸化，在完整细胞和电穿孔细胞中的表观Ki约为3 mM。在此浓度范围内，巴弗洛霉素A1选择性抑制液泡H(+)-ATP酶。吞噬体酸化的底物需求和抑制剂敏感性特征强烈表明，质子跨吞噬体膜的转运是由液泡型H(+)-ATP酶介导的。
