Gläser H U, Höfer M
Botanisches Institut der Universität Bonn, F.R.G.
Biochim Biophys Acta. 1987 Dec 11;905(2):287-94. doi: 10.1016/0005-2736(87)90457-3.
Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases.
通过对来自鲁氏梅奇酵母的纯化质膜组分和磷脂酰胆碱（Sigma公司的II-S型）进行冻融，重建了质膜囊泡。在碱金属阳离子存在的情况下，向重建的质膜囊泡中添加ATP后，会产生质子梯度（内部呈酸性）。当质膜囊泡内外均存在K+时，ΔpH的产生效率最高。原钒酸盐可抑制质膜囊泡中的ATP酶活性和质子转运（100μM时抑制50%）。未添加磷脂酰胆碱重建的质膜囊泡除了产生ΔpH外，还产生了电势差Δψ（内部为正）。Δψ的产生不表现出K+特异性。50μM二环己基碳二亚胺完全抑制了Δψ的产生，而K+通道阻滞剂奎宁（5μM）使Δψ增加了8倍。这些试剂对质子梯度的影响要小得多。考虑到鲁氏梅奇酵母质膜ATP酶对K+的依赖性刺激，这些结果进一步支持了ATP酶作为部分生电H+/K+交换器发挥作用的结论，其他酵母质膜ATP酶也有类似情况。
