Ohsumi Y, Anraku Y
J Biol Chem. 1983 May 10;258(9):5614-7.
Vacuolar membrane vesicles of Saccharomyces cerevisiae accumulate Ca2+ ion in the presence of ATP, not in the presence of ADP or adenyl-5'-yl imidodiphosphate. Calcium transport showed saturation kinetics with a Km value of 0.1 mM and optimal pH of 6.4. Ca2+ ion incorporated in the vesicles was exchangeable and released completely by a protonophore uncoupler, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847), or calcium-specific ionophore, A23187. The transport required Mg2+ ion but was inhibited by Cu2+ or Zn2+ ions, inhibitors of H+-ATPase of the vacuolar membrane. The transport activity was sensitive to the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked Ca2+ uptake completely, but valinomycin stimulated it 1.35-fold. These results indicate that an electrochemical potential difference of protons is a driving force for this Ca2+ transport. The ATP-dependent formation of the deltapH in the vesicles and its partial dissipation by CaCl2 were demonstrated by fluorescence quenching of quinacrine. This Ca2+ uptake by vacuolar membrane vesicles is suggested to be catalyzed by a Ca2+/H+ antiport system.
酿酒酵母的液泡膜囊泡在ATP存在下积累Ca2+离子，而在ADP或腺苷-5'-亚氨基二磷酸存在下则不积累。钙转运呈现饱和动力学，Km值为0.1 mM，最适pH为6.4。囊泡中掺入的Ca2+离子是可交换的，并可被质子载体解偶联剂3,5-二叔丁基-4-羟基亚苄基丙二腈（SF6847）或钙特异性离子载体A23187完全释放。该转运需要Mg2+离子，但受到液泡膜H+-ATPase的抑制剂Cu2+或Zn2+离子的抑制。转运活性对H+-ATPase抑制剂N,N'-二环己基碳二亚胺敏感，但对寡霉素或钒酸钠不敏感。SF6847或尼日利亚菌素完全阻断Ca2+摄取，但缬氨霉素可使其刺激1.35倍。这些结果表明质子的电化学势差是这种Ca2+转运的驱动力。通过喹吖因的荧光猝灭证明了囊泡中ΔpH的ATP依赖性形成及其被CaCl2部分耗散。液泡膜囊泡的这种Ca2+摄取被认为是由Ca2+/H+反向转运系统催化的。
