Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae.
Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(19)69736-x
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发表时间：
1981-03
期刊：
影响因子：
--
通讯作者：
Y. Ohsumi;Y. Anraku
中科院分区：
文献类型：
--
作者：
Y. Ohsumi;Y. Anraku
本文研究了碱性氨基酸在离体条件下进入酿酒酵母细胞液泡的转运机制。从纯化的液泡制备右侧出的液泡膜小泡。精氨酸仅在ATP存在下被囊泡有效地摄取，而在ADP或AMP-腺苷-5 '-基亚氨二磷酸存在下不被囊泡有效地摄取。它是可交换的，并完全释放的质子载体，3，5-二叔丁基-4-羟基benzilidenemalonitrile（SF 6847）。该转运需要Mg ~（2+）离子，但Cu ~（2+）、Ca ~（2+）或Zn ~（2+）离子抑制该转运。转运活性对ATP酶抑制剂N，N ′-二环己基碳二亚胺（DCCD）敏感，而对寡霉素和钒酸钠不敏感。SF 6847或尼日利亚菌素完全阻断精氨酸摄取，但缬氨霉素没有影响。9-氨基吖啶荧光淬灭显示跨膜囊泡的Δ pH的ATP依赖性形成。这些结果表明，DCCD敏感，镁2 +-ATP酶的液泡膜是必不可少的主动运输的供能系统，质子的电化学电位差是这种基本的氨基酸运输的驱动力。精氨酸转运显示饱和动力学，Km值为0.6 mM，该机制可以很好地解释由H+/精氨酸反向转运。
The mechanism of transport of basic amino acids into vacuoles of cells of the yeast Saccharomyces cerevisiae was investigated in vitro. Right-side-out vacuolar membrane vesicles were prepared from purified vacuoles. Arginine was taken up effectively by the vesicles only in the presence of ATP, not in the presence of ADP or AMP-adenosyl-5‘-yl imidodiphosphate. It was exchangeable and was released completely by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847). The transport required Mg2+ ion but was inhibited by Cu2+, Ca2+, or Zn2+ ions. The transport activity was sensitive to the ATPase inhibitor N,N‘-dicyclohexylcarbodiimide (DCCD), but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked arginine uptake completely, but valinomycin had no effect. ATP-dependent formation of a delta pH across the membrane vesicles was shown by quenching of 9-aminoacridine fluorescence. These results indicate that DCCD-sensitive, Mg2+-ATPase of vacuolar membranes is essential as an energy-donating system for the active transport, and that an electrochemical potential difference of protons is a driving force of this basic amino acid transport. Arginine transport showed saturation kinetics with a Km value of 0.6 mM and the mechanism was well explained by an H+/arginine antiport.