Crider B P, Xie X S, Stone D K
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
J Biol Chem. 1994 Jul 1;269(26):17379-81.
Vacuolar-type proton-translocating ATPases are complex heterooligomers that are characterized by a specific inhibition by bafilomycin A1. These enzymes have a peripheral ATP hydrolytic domain as well as a transmembranous sector. The transmembranous sector has been isolated by glycerol gradient centrifugation, and this subcomplex is composed of polypeptides of 116, 39, and 17 kDa. Both this sector and native holoenzyme were reconstituted into potassium-loaded (150 mM KCl) liposomes prepared from pure lipids. When diluted into potassium-free buffer, a valinomycin-induced membrane potential did not drive proton uptake, as assessed by acridine orange quenching. In contrast, pretreatment of both the reconstituted proton pump and isolated transmembranous sector at pH 4.2 activated a latent proton conductance. Bafilomycin A1 (1 nM) inhibited ATP-energized proton pumping catalyzed by the proton pump, as well as membrane potential-driven proton flow through both the acid-activated proton pump and the isolated proton pore. Thus bafilomycin A1 inhibits vacuolar proton pumps by blocking proton conduction through the proton pore, which we term VB.
液泡型质子转运ATP酶是复杂的异源寡聚体，其特征是被巴弗洛霉素A1特异性抑制。这些酶有一个外周ATP水解结构域以及一个跨膜区。跨膜区已通过甘油梯度离心分离出来，该亚复合物由116、39和17 kDa的多肽组成。这个区域和天然全酶都被重构到由纯脂质制备的加载钾（150 mM KCl）的脂质体中。当稀释到无钾缓冲液中时，通过吖啶橙猝灭评估，缬氨霉素诱导的膜电位并不能驱动质子摄取。相反，在pH 4.2对重构的质子泵和分离的跨膜区进行预处理会激活潜在的质子传导。巴弗洛霉素A1（1 nM）抑制质子泵催化的ATP驱动的质子泵送，以及膜电位驱动的质子通过酸激活的质子泵和分离的质子孔的流动。因此，巴弗洛霉素A1通过阻断质子通过我们称为VB的质子孔的传导来抑制液泡质子泵。
