Structure and Function of Ion Transport Systems

离子传输系统的结构和功能

• 批准号: 62480456
• 负责人: KAWAKITA Masao
• 金额: $ 4.29万
• 依托单位: The 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: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-62480456/
• 关键词: Sarcoplasmic Reticulum; Calcium Transport; ATPase; Active Transport; Na; H Antiporter; Ion Transport; カルシウム輸送ATPア-ゼ; 運動輸送; ATPアーゼ; カルシウム; 親和性標識; Hアンチポーター; カルシウムポンプ

1. The following results were obtained concerning the structure and the reaction mechanism of Ca^<2+>-transporting ATPase of sarcoplasmic reticulum. (1) Thermolytic digestion of sarcoplasmic reticulum membranes followed by HPLC was shown to be effective in identifying target sites of fluorescence- and affinity-labeling reagents. (2) IAEDANS and NEM(ANM) were shown to be specifically attached to Cys674 and Cys344/364, respectively. (3) Timeresolved fluorescence anisotropy measurements revealed independent flexible motion of submolecular domains of ATPase labeled with IAEDANS and ANM. (4) Products of limited tryptic digestion were purified and analyzed. Susceptibility of Lys218, Lys234 and Arg236 was profoundly affected by binding of Ca^<2+> and AMP-P(NH)P to the ATPase molecule due to a conformational change around these residues. This particular region seems to be intimately involved in energy coupling of the Ca^<2+>-transport, and also is supposed to be the site of contact between Ca^ … More <2+>-binding and ATP-binding domains. The present results thus suggest that interactions between submolecular domains may be important for proper coupling between ATP splitting and Ca^<2+>-transport. (5) An affinity labeling reagent, ATP-pyridoxal was utilized to map the ATP binding site. Lys684 and Lys492 were labeled in the absence of Ca^<2+>, but only the former was labeled in its presence, indicating a Ca^<2+>-dependent conformational change in the ATP-binding site. A close vicinity of Asp351, Lys492 and Lys684 was also suggested. (6) Paramagnetic interactions between covalently attached spin labels and Gd^<3+> (a paramagnetic Ca^<2+> analogue) was analyzed and the Ca^<2+>-binding site was located in the intramembranous region of the ATPase molecule, not being very far from the membrane surface.2. Na^+/H^+ antiporter was reconstituted into proteoliposome by cholatedialysis procedure. Partially purified preparation of the antiporter was obtained from the blush border membranes of bovine kidney through gel filtration and DEAE-Sephacel chromatography. Less

1. 对肌浆网Ca^<2+>-转运atp酶的结构和反应机理进行了研究。(1)肌浆网膜的热解消化和高效液相色谱法可以有效地识别荧光和亲和标记试剂的靶位。(2)发现IAEDANS和NEM（ANM）分别特异附着于Cys674和Cys344/364。(3)时间分辨荧光各向异性测量揭示了以IAEDANS和ANM标记的atp酶亚分子结构域的独立柔性运动。(4)对限制性胰蛋白酶消化产物进行纯化和分析。由于Ca^<2+>和AMP-P(NH)P与atp酶分子的结合引起这些残基周围的构象变化，对Lys218、Lys234和Arg236的敏感性产生了深刻的影响。这个特殊的区域似乎与Ca^<2+>-输运的能量耦合密切相关，也被认为是Ca^…More <2+>-结合域和atp结合域之间的接触位点。因此，目前的结果表明，亚分子结构域之间的相互作用对于ATP分裂和Ca^<2+>-运输之间的适当耦合可能是重要的。(5)利用ATP-吡哆醛亲和标记试剂绘制ATP结合位点。Lys684和Lys492在Ca^<2+>不存在的情况下被标记，但只有前者在Ca^<2+>存在的情况下被标记，这表明atp结合位点发生了Ca^<2+>依赖的构象变化。此外，还发现了与Asp351、Lys492和Lys684接近的基因。(6)分析了共价自旋标签与Gd^<3+>（一种顺磁性Ca^<2+>类似物）之间的顺磁相互作用，发现Ca^<2+>结合位点位于atp酶分子的膜内区域，离膜表面不远。通过胆碱透析法将Na^+/H^+反转运蛋白重组为蛋白脂质体。通过凝胶过滤和deae - sepacel层析，从牛肾红缘膜中获得部分纯化的反转运蛋白。少

项目成果

Suzuki,S.: "Independent Flexible Motion of Submolecular Domains of the Ca^<2+>,Mg^<2+>-ATPase of Sarcoplasmic Reticulum Measured by Time-Resolved Fluorescence Depolarization of Site-Specifically Attached Probes." Biochemistry,. 28. 7734-7740 (1989)

Suzuki,S.：“通过位点特异性附着探针的时间分辨荧光去极化测量肌浆网 Ca^2,Mg^2-ATP 酶亚分子域的独立灵活运动。”

Yamamoto, H.: "Ca^<2+>-Dependent Conformational Change of the ATP-Binding Site of Ca^<2+> Transporting ATPase of Sarcoplasmic Reticulum As Revealed by an Alteration of the Target-Site Specificity of Adenosine Triphosphopyridoxal." J. Biochem. 106 1121-112

Yamamoto, H.：“通过三磷酸吡哆醛腺苷靶位点特异性的改变揭示了肌浆网 Ca^2 转运 ATP 酶的 ATP 结合位点的 Ca^2 依赖性构象变化。”

Kawakita,M.: "Chemical Derivatization of Ca^<2+>-Pump Protein from Skeletal Muscle with N-Substituted Maleimides and 5-(2-Iodoacetamidoethyl)amino-naphthalene 1-Sulfonate" Methods in Enzymol.157. 251-261 (1988)

Kawakita，M.：“用N-取代的马来酰亚胺和5-(2-碘乙酰胺乙基)氨基-萘1-磺酸盐从骨骼肌中化学衍生Ca 22 -泵蛋白”Enzymol.157中的方法。

川喜田正夫: "骨格筋小胞体とカルシウムポンプ" 蛋白質核酸酵素. 33. 1915-1926 (1988)

Masao Kawakita：“骨骼肌内质网和钙泵”蛋白质核酸酶33。1915-1926（1988）

Yamamoto,H.: "Ca^<2+>-Dependent Conformational Change of the ATP-Binding Site of Ca^<2+>Transporting ATPase of Sarcoplasmic Reticulum As Revealed by an Alteration of the Target-Site Specificity of Adenosine Triphosphopyridoxal." J.Biochem.106. 1121-1125 (

Yamamoto,H.：“通过三磷酸吡哆醛腺苷靶位点特异性的改变揭示了肌浆网 Ca^2 转运 ATP 酶的 ATP 结合位点的 Ca^2 依赖性构象变化。”