Youmans S J, Brodsky W A
Biochim Biophys Acta. 1987 Jun 12;900(1):88-102. doi: 10.1016/0005-2736(87)90281-1.
The ATP-dependent proton transport into vesicles of a mixed membrane fraction obtained from turtle bladder epithelial cells consists of at least two kinetically defined moieties: one, which is maximally inhibited by 25% with nanomolar levels of vanadate, but not inhibited at all with equimolar levels of N-ethylmaleimide, and another, which is maximally inhibited by 70% with micromolar levels of N-ethylmaleimide and by 25% with equimolar levels of vanadate. In contrast to the transport function, the associated enzymatic function (the ouabain-resistant ATPase activity) in these membranes, not inhibited by nanomolar levels of vanadate or N-ethylmaleimide, is maximally inhibited by 40% with micromolar levels of vanadate and by 13% with equimolar levels of N-ethylmaleimide. Independent of these kinetic differences between the enzyme and the transport functions, membranes containing the N-ethylmaleimide-sensitive proton transport function are electrophoretically separable from those containing the vanadate-sensitive transport function. For example, the kinetically defined, vanadate-sensitive proton transport function is recovered exclusively and kinetically identified in one of four electrophoretic membrane fractions, EF-II; while the N-ethylmaleimide-sensitive function is recovered in EF-III as well as in EF-II. Membranes of EF-IV, maximally enriched in ouabain-resistant ATPase activity, possess no proton transport function at all, even in the absence of N-ethylmaleimide or vanadate. Additional data under in vivo as well as under in vitro conditions are required to prove that the vanadate-sensitive proton transport in these vesicles is an in vitro manifestation of the mechanism responsible for generating the vanadate-sensitive luminal acidification process under in vivo conditions in the intact turtle bladder.
从龟膀胱上皮细胞获得的混合膜组分的囊泡中，ATP 依赖性质子转运至少由两个动力学定义的部分组成：一部分，在纳摩尔水平的钒酸盐作用下最大抑制率为 25%，但在等摩尔水平的 N - 乙基马来酰亚胺作用下完全不被抑制；另一部分，在微摩尔水平的 N - 乙基马来酰亚胺作用下最大抑制率为 70%，在等摩尔水平的钒酸盐作用下最大抑制率为 25%。与转运功能相反，这些膜中的相关酶功能（哇巴因抗性 ATP 酶活性），不受纳摩尔水平的钒酸盐或 N - 乙基马来酰亚胺抑制，在微摩尔水平的钒酸盐作用下最大抑制率为 40%，在等摩尔水平的 N - 乙基马来酰亚胺作用下最大抑制率为 13%。与酶和转运功能之间的这些动力学差异无关，含有对 N - 乙基马来酰亚胺敏感的质子转运功能的膜在电泳上可与含有对钒酸盐敏感的转运功能的膜分离。例如，动力学定义的、对钒酸盐敏感的质子转运功能仅在四个电泳膜组分之一的 EF - II 中完全恢复并在动力学上得以鉴定；而对 N - 乙基马来酰亚胺敏感的功能在 EF - III 以及 EF - II 中恢复。EF - IV 的膜，最大程度地富集了哇巴因抗性 ATP 酶活性，即使在没有 N - 乙基马来酰亚胺或钒酸盐的情况下也完全没有质子转运功能。需要体内以及体外条件下的更多数据来证明这些囊泡中对钒酸盐敏感的质子转运是完整龟膀胱体内条件下产生对钒酸盐敏感的管腔酸化过程的机制的体外表现。
