A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.
A vacuolar-type proton pump energizes K+/H+ antiport in an animal plasma membrane.
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DOI：
10.1016/s0021-9258(18)98621-7
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发表时间：
1991-08
期刊：
影响因子：
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通讯作者：
Helmut Wieczorek;M. Putzenlechner;W. Zeiske;U. Klein
中科院分区：
文献类型：
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作者：
Helmut Wieczorek;M. Putzenlechner;W. Zeiske;U. Klein
在本文中，我们证明了液泡型H（+）-ATP酶激活了昆虫质膜的次级主动运输，从而我们提供了一个替代的经典概念，质膜的Na+/K（+）-ATP酶在动物细胞。我们研究了烟草天蛾（Manducasexta）中肠高纯度K（+）转运杯状细胞顶端膜制备物中ATP依赖性和非依赖性囊泡酸化，用荧光吖啶橙子监测。ATP依赖的质子运输被证明是由一个空泡型ATP酶催化的推断从其敏感性亚微摩尔浓度的巴弗洛霉素A1。ATP-独立的阿米洛利敏感的质子运输到囊泡内部依赖于一个外向的跨囊泡膜的K+梯度。这种依赖于K（+）的质子转运可以解释为K+/H+反向转运，因为它对阿米洛利表现出与空泡型质子泵产生的pH梯度的依赖于K（+）的耗散相同的敏感性和阳离子特异性。空泡型ATP酶是唯一的质子泵，因为它可以酸化囊泡独立的囊泡外K+浓度，只要反向运输被阿米洛利抑制。针对纯化的液泡型ATP酶的多克隆抗体抑制ATP酶活性和ATP依赖的质子转运，但不抑制K+/H+反向转运，这表明反向转运蛋白和ATP酶是两个不同的分子实体。实验中，荧光oxonol V被用作囊泡内部正膜电位的指示剂提供了证据的K+/H+ antiport的产电性，并建议，一个以上的H+交换一个K+在反应周期。钾离子梯度依赖性膜电位的产生和囊泡的酸化都对harvestin敏感，harvestin是Na+依赖性转运过程（包括Na+/H+逆向转运）的典型抑制剂。我们的研究结果导致的假设，积极和产电K+分泌烟草天蛾中肠的结果产电K+/nH+反向运输，这是通电的电动组件产生的质子动力的产电液泡型质子泵。
In this paper we demonstrate that a vacuolar-type H(+)-ATPase energizes secondary active transport in an insect plasma membrane and thus we provide an alternative to the classical concept of plasma membrane energization in animal cells by the Na+/K(+)-ATPase. We investigated ATP-dependent and -independent vesicle acidification, monitored with fluorescent acridine orange, in a highly purified K(+)-transporting goblet cell apical membrane preparation of tobacco hornworm (Manduca sexta) midgut. ATP-dependent proton transport was shown to be catalyzed by a vacuolar-type ATPase as deduced from its sensitivity to submicromolar concentrations of bafilomycin A1. ATP-independent amiloride-sensitive proton transport into the vesicle interior was dependent on an outward-directed K+ gradient across the vesicle membrane. This K(+)-dependent proton transport may be interpreted as K+/H+ antiport because it exhibited the same sensitivity to amiloride and the same cation specificity as the K(+)-dependent dissipation of a pH gradient generated by the vacuolar-type proton pump. The vacuolar-type ATPase is exclusively a proton pump because it could acidify vesicles independent of the extravesicular K+ concentration, provided that the antiport was inhibited by amiloride. Polyclonal antibodies against the purified vacuolar-type ATPase inhibited ATPase activity and ATP-dependent proton transport, but not K+/H+ antiport, suggesting that the antiporter and the ATPase are two different molecular entities. Experiments in which fluorescent oxonol V was used as an indicator of a vesicle-interior positive membrane potential provided evidence for the electrogenicity of K+/H+ antiport and suggested that more than one H+ is exchanged for one K+ during a reaction cycle. Both the generation of the K+ gradient-dependent membrane potential and the vesicle acidification were sensitive to harmaline, a typical inhibitor of Na(+)-dependent transport processes including Na+/H+ antiport. Our results led to the hypothesis that active and electrogenic K+ secretion in the tobacco hornworm midgut results from electrogenic K+/nH+ antiport which is energized by the electrical component of the proton-motive force generated by the electrogenic vacuolar-type proton pump.