Mukherjee T, Mandal D, Bhaduri A
Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta 700 032, India.
J Biol Chem. 2001 Feb 23;276(8):5563-9. doi: 10.1074/jbc.M008469200. Epub 2000 Nov 21.
Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.
其他实验室使用杜氏利什曼原虫前鞭毛体细胞进行的实验早些时候表明，该寄生虫的质膜Mg2 + -ATP酶是一种H + 外排泵。利用该生物体质膜的表膜微管结构，我们报告了获得具有确定极性的密封血影和密封外翻囊泡的方法。发现H + 快速流入外翻囊泡依赖于ATP（1 mM）和Mg2 +（1 mM）的同时存在。H + 进入速率与酶活性之间的良好对应清楚地表明Mg2 + -ATP酶是一种真正的H + 泵。SCH28080（IC50 =约40 μM）和奥美拉唑（IC50 =约50 μM）强烈抑制H + 进入外翻囊泡，这两种都是哺乳动物胃H +，K + -ATP酶的特征性抑制剂。H + 流入对哇巴因（250 μM）完全不敏感，哇巴因是Na +，K + -ATP酶的典型抑制剂。Mg2 + -ATP酶活性可用K +（20 mM）部分刺激，而K + 可被SCH28080（50 μM）抑制（> 85％）。用奥美拉唑（150 μM）预孵育和用该酶的抑制剂5,5'-二硫代双（2-硝基苯甲酸）（1 mM）完全抑制预加载囊泡中86Rb + 的ATP依赖性快速外流。假设Rb + 是K + 的真正替代物，则建立了ATP依赖性的H + 和K +（1:1）电中性化学计量交换。当施加人工pH梯度（内部碱性）时，可以观察到[U-（14）C] 2-脱氧葡萄糖在密封血影中快速且活性提高10倍的积累。预加载的外翻囊泡中[U-（14）C] d-葡萄糖的快速外流也可以通过用ATP激活该酶来启动。综上所述，质膜Mg2 + -ATP酶已被鉴定为一种电中性H + / K + 反向转运体，其某些特性让人联想到胃H +，K + -ATP酶。该酶可能通过H + -葡萄糖同向转运系统参与葡萄糖的主动积累和K + 的积累。
