Investigation of reaction mechanism and higher oligomeric structure of Na-pump based on single-molecule observation technique.

基于单分子观察技术研究钠泵的反应机理和高级低聚结构。

• 批准号: 13142201
• 负责人: KAYA Shunji
• 金额: $ 44.74万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13142201/
• 关键词: Sodium Pump; Fluorescent Probe; Single-molecule observation; Proton Pump

(1) Protomeric images of Na/K-ATPase and H/K-ATPase were observed by electron microsoopy(EM) after rotary-shadowing the solubilized enzyme preparation. Without cross-linking the preparation, dimeric and tetrameric molecules were detected by EM, indicating that the pump molecules are assembled in oligomer(2) We have established the method for determining the oligomericity of P-type ATPases by means of single-molecule observation of fluorescently labeled enzyme using the total internal reflection fluorescence microscopy.(3) Tetrameric structure of P-type ATPases has been determined not only Na/K-ATPase but also H/K-ATPase. In case ofH/K-ATPase, functional unit of the enzyme was shown to be tetraprotomer based on the relationship between the enzyme activity and the oligomeric structure.(4) Analysis of the single mutations ofATP-binding region of Na/K-ATPase suggested that the ATPase molecule has one ATP binding site, which changes the affinity for ATP depending on the ligands present and the ATPase molecules form oligomer to maintain the interaction between them. We have estimated the third Na+ binding region of Na/K-ATPase at the first time.

(1)利用电镜（EM）观察Na/K-ATPase和H/K-ATPase的原聚体图像。在未交联的情况下，通过电子显微镜检测到二聚体和四聚体分子，表明泵分子以低聚体的形式组装。(2)我们建立了利用全内反射荧光显微镜对荧光标记酶进行单分子观察来测定p型atp酶寡聚性的方法。(3) p型atpase的四聚体结构不仅确定了Na/K-ATPase，还确定了H/K-ATPase。以h / k - atp酶为例，根据酶活性与寡聚体结构的关系，表明酶的功能单元为四原体。(4)对Na/K-ATPase ATP结合区的单突变分析表明，ATPase分子具有一个ATP结合位点，根据存在的配体改变对ATP的亲和力，ATPase分子形成寡聚体维持它们之间的相互作用。我们首次估计了Na/ k - atp酶的第三Na+结合区。

项目成果

Evaluation of an ELISA for detection of anti-parietal cell antibody

ELISA 检测抗壁细胞抗体的评价

• 发表时间: 2006
• 期刊: Hepatogastroenterology.
• 作者: Cohen AD;Diab A;Perales MA;Wolchok JD;Rizzuto G;Merghoub T;Huggins D;Liu C;Turk MJ;Restifo NP;Sakaguchi S;Houghton AN.;M.Shoji;K.Sugiu
• 通讯作者: K.Sugiu

Gastric H/K-ATPase liberates two moles of Pi from one mole of phosphoenzyme formed from a high-affinity ATP binding site and one mole of enzyme-bound ATP at the low affinity site during cross-talk between catalytic subunits.

在催化亚基之间的串扰过程中，胃 H/K-ATP 酶从高亲和力 ATP 结合位点形成的 1 摩尔磷酸酶和低亲和力位点处的 1 摩尔酶结合 ATP 中释放出 2 摩尔 Pi。

• 发表时间: 2002
• 期刊: Biochemistry 41
• 作者: Watanabe T;Horiuchi T.;K.Abe
• 通讯作者: K.Abe

Thr-774 (Transmembrane Segment M5), Val-920 (M8), and Glu-954 (M9) Are Involved in Na^+ Transport, and GIn-923 (M8) Is Essential for Na,K-ATPase Activity.

Thr-774（跨膜片段 M5）、Val-920 (M8) 和 Glu-954 (M9) 参与 Na^2 转运，GIn-923 (M8) 对于 Na,K-ATP 酶活性至关重要。

• 发表时间: 2005
• 期刊: The Journal of Biological Chemistry 280
• 作者: Kaneko;S.;et al.;M.Shoji;T.Imagawa
• 通讯作者: T.Imagawa

K.Abe: "Correlation between the activities and the oligomeric forms of pig gastric H/K-ATPase."Biochemstry. 42(51). 15132-15138 (2003)

K.Abe：“猪胃 H/K-ATP 酶的活性与寡聚形式之间的相关性。”生物化学。

K.Abe: "Gastric H/K-ATPase liberates two moles of Pi from one mole of phosphoenzyme fromed from a high-affinity ATP binding site and one mole of enzyme-bound ATP at the low affinity site during cross-talk between catalytic subunits"Biochemstry. (2002)

K.Abe：“在催化亚基之间的串扰过程中，胃 H/K-ATP 酶从高亲和力 ATP 结合位点的 1 摩尔磷酸酶和低亲和力位点的 1 摩尔酶结合 ATP 中释放出 2 摩尔 Pi