Biochemical Studies on <H^+> -ATPase and <Ca^(2+)> / <H^+> antiporter on the Yeast Vacuolar Membranes

酵母液泡膜<H^>-ATP酶和<Ca^(2)>/<H^>逆向转运蛋白的生化研究

• 批准号: 60480498
• 负责人: ANRAKU Yasuhiro
• 金额: $ 1.09万
• 依托单位: Faculty of Science, University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1985
• 资助国家: 日本
• 起止时间: 1985 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-60480498/
• 关键词: Vacuole; <H^+> -ATPase; Yeast; Saccharomyces; 輸送蛋白質

(1) Subunit a (Mr=89K) of the vacuolar membrane <H^+> -ATPase of the yeast S. cerevisiae was found to bind 8-azide-[ <(alpha)-^(32)> ]ATP. Labeling by this photo-sensitive ATP derivative was saturable with an apparent dissociation constant of <10^(-6)> M to <10^(-5)> M and decreased in the presence of ATP and ADP.(2) The enzyme was inactivated by NBD-Cl, about 1 <micro> M causing half maximal inactivation in the neutral pH range. This inactivation was prevented by the presence of ATP, ADP, or AMP-PNP. The original activity was restored by treating the inactivated enzyme with 2-mercaptoethanol. Kinetic and chemical studies of the inactivation showed that the activity was lost on chemical modification of a single tyrosine residue per molecule of the enzyme. When the enzyme was inactivated with [ <^(14)C> ]NBD-Cl, subunit a was specifically labeled and this labeling was completely prevented by the presence of ATP, GTP, ADP, or AMP-PNP. It was concluded that subunit a of the yeast vacuolar … More <H^+> -ATPase has a catalytic site that contains a single, essential tyrosine residue.(3) The kinetics of non-steady state hydrolysis of [ <gamma> - <^(32)P> ]ATP indicated the formation of an enzyme-ATP complex and subsequent hydrolysis of bound ATP to ADP and Pi at the NBD-Cl-sensitive catalytic site. NBD-Cl inactivated the single catalytic site and inhibited the formation of an enzyme-ATP complex. A reaction mechanism for ATP hydrolysis with catalytic site cooperativity by the yeast vacuolar <H^+> -translocating ATPase was proposed.(4) Monoclonal antibodies against the vacuolar membrane and purified vacuolar membrane ATPase were systematically obtained. Now, we started to isolate genes of various vacuolar membrane proteins.(5) To elucidate the mechanism for generating the electrochemical potential difference across the vacuolar membrane, we analyzed the conductivities of vacuolar membrane for various ions. We found a novel <Cl^-> transport system and potential dependent ion channel on the vacuolar membrane. Less

(1)酿酒酵母液泡膜<H^+>-ATP酶的亚基a(Mr=89K)被发现与8-叠氮-[<(α)-^(32)>]ATP结合。这种光敏 ATP 衍生物的标记在 <10^(-6)> M 至 <10^(-5)> M 的表观解离常数下达到饱和，并且在 ATP 和 ADP 存在下降低。(2) 该酶被 NBD-Cl 灭活，大约 1 <μM> M 在中性 pH 范围内导致最大失活的一半。 ATP、ADP 或 AMP-PNP 的存在可以防止这种失活。用2-巯基乙醇处理失活的酶即可恢复其原始活性。失活的动力学和化学研究表明，每个酶分子的单个酪氨酸残基的化学修饰导致活性丧失。当用[<^(14)C>]NBD-Cl灭活酶时，亚基a被特异性标记，并且这种标记被ATP、GTP、ADP或AMP-PNP的存在完全阻止。结论是，酵母液泡的亚基 a ... More <H^+> -ATPase 具有一个包含单个必需酪氨酸残基的催化位点。(3) [ <gamma> - <^(32)P> ]ATP 的非稳态水解动力学表明酶-ATP 复合物的形成以及随后结合的 ATP 水解为 ADP 和 Pi NBD-Cl 敏感催化位点。 NBD-Cl 使单个催化位点失活并抑制酶-ATP 复合物的形成。提出了酵母液泡<H^+>转位ATP酶催化位点协同水解ATP的反应机理。(4)系统地获得了抗液泡膜单克隆抗体和纯化的液泡膜ATP酶。现在，我们开始分离各种液泡膜蛋白的基因。(5)为了阐明液泡膜上电化学电位差产生的机制，我们分析了液泡膜对各种离子的电导率。我们在液泡膜上发现了一种新型的<Cl^->传输系统和电位依赖性离子通道。较少的

项目成果

Uchida,E.: J.Biol.Chem.Submitted.

Uchida,E.：J.Biol.Chem.已提交。

Anraku,Y.,Ed.B.P.Marin: "Plant Vacuoles" Plenum Press,New York, (1987)

Anraku,Y.，Ed.B.P.Marin：“植物液泡”全会出版社，纽约，（1987 年）

Ohsumi,Y.,Ed.B.P.Marin: "Biochemistry and Function of Vacuolar Adenosine Triphosphtase in Fungi and Plants" Springer-Verlag,Berlin, 259 (1986)

Ohsumi，Y.，Ed.B.P.Marin：“真菌和植物中液泡腺苷三磷酸酶的生物化学和功能”Springer-Verlag，柏林，259（1986）

Ohsumi, Y., Uchida, E., and Anraku, Y.: "The <H^+> -Translocating ATPase in Vacuolar membranes of Saccharomyces cerevisiae" Biochimistry and Function of Vacuolar Adenosine Triphosphatase (ed. B.P. Marin) Springer-Verlag. 144-150 (1985)

Ohsumi, Y.、Uchida, E. 和 Anraku, Y.：“酿酒酵母液泡膜中的 <H^>-易位 ATP 酶”，液泡腺苷三磷酸酶的生物化学和功能（B.P. Marin 编辑）Springer-Verlag。

Uchida,E., Ohsumi,Y. and Anraku, Y.: "Characterization and Function of a Catalytic Subunit a of <H^+> -translocating Adenosine Triphosphatase from Vacuolar Membrane of Saccharomyces cerevisiae" J. Biol. Chem.

内田，E.，大隅良典，Y.