Van Dyke R W, Hornick C A, Belcher J, Scharschmidt B F, Havel R J
J Biol Chem. 1985 Sep 15;260(20):11021-6.
Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.
多囊泡体（MVB）是内吞途径中的前溶酶体细胞器，从经雌激素处理的大鼠肝脏中制备，并检测其是否存在ATP依赖性质子转运。通过吖啶橙荧光猝灭评估的囊泡酸化是ATP依赖性的（ATP远大于GTP、UTP），比匀浆富集25倍，用质子载体或非离子去污剂预处理可消除，pH最适值为7.5，受N-乙基马来酰亚胺（NEM）（IC50约为5 microM）和N，N'-二环己基碳二亚胺（IC50约为5 microM）抑制，对钒酸盐、哇巴因和寡霉素的抑制有抗性。酸化没有特定的阳离子需求；然而，最大酸化速率取决于Cl-的存在（Km约为20 mM）。其他阴离子在支持酸化方面效果较差（Cl-＞Br-＞＞葡萄糖酸盐、NO3-、SO42-和甘露醇），实际上NO3-即使在存在150 mM Cl-的情况下也会抑制酸化。基于以下几点，质子转运机制似乎是电生的：（a）在存在葡萄糖酸钾的情况下，缬氨霉素增强酸化；（b）双（3-苯基-5-氧代异恶唑-4-基）五甲川草酚（一种膜电位敏感阴离子染料）的ATP依赖性荧光猝灭。此外，质子转运机制产生的pH和电势梯度的大小在存在和不存在Cl-的情况下似乎呈反比变化。最后，MVB表现出对哇巴因和寡霉素有抗性的ATP酶活性，但受1 mM NEM抑制32.3%，受200 microM二环己基碳二亚胺抑制33.7%，受KNO3抑制18.7%。因此，在分离的MVB中，NEM敏感的ATP酶活性可能代表质子泵的酶学等效物。这些研究鉴定并表征了大鼠肝脏MVB中一种ATP依赖性电生质子转运过程，该过程具有肝脏和其他组织的网格蛋白包被囊泡、内体、溶酶体、高尔基体和内质网中描述的质子泵许多特性。MVB的酸化在对Br-和NO3-的反应上与大鼠肝脏网格蛋白包被囊泡略有不同，表明这两种细胞器的膜特性可能不同。
