Molecular Mechanism of Energy Transduction and regulation of Monovalent cation Pumps

单价阳离子泵能量传导与调控的分子机制

• 批准号: 06454648
• 负责人: TANIGUCHI Kazuya
• 金额: $ 4.67万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454648/
• 关键词: Regulation; Na, K-ATPase; H,K-ATPase; Phosphorylation; キナーゼ; 胃壁H^+ポンプ; ATPase; 脱リン酸化; カルシウムイオン; Na^+ポンプ; P型ポンプ; エネルギー変換; ナトリウムポンプ; H+ポンプ; 構造変化; タンパクキナーゼ; フォスファターゼ; チロシンキナーゼ; Na^+,K^+-ATPase; H^+,K^+-ATPase; イオン輸送ポンプ; プロトンポンプ

When pig stomach membrance H,K-ATPase preparations were incubated with ^^<32>ATP,Mg^<2+> and Ca^<2+>, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal alpha-chain of H,K-ATPase occurred without any detectable phosphorylation in other regions of the chain. Amino acid sequence analysis of purified peptides and others showed a sequential phosphorylation of Tyr-10 and Tyr-7 and sub muM Ca^<2+> activated phosphorylation of Ser-27. The apparent molecular weight of a detergent solubilized Tyr-kinase was estimated to be around 50 kDa by a gel filtration column and the kinase activity detected on a gel band after renaturation. The Ser-kinase present was suggested to be a conventional PKC.H,K-ATPase preparations phosphorylated fusion proteins consised of maltose binding protein and a peptide portion of the alpha-chain from Gly-2 to Gln-111. These data and others suggested that N-terminal cytosolic domain is sufficient for phosphorylation by endogenous Tyr-kinase and PKC in the membrane H,K-ATPase preparations.

当猪胃膜H,K-ATPase制剂与^^<32>ATP、Mg^<2+>和Ca^<2+>一起孵育时，N端α链中特定Tyr和Ser残基的可逆磷酸化H,K-ATP酶的发生在链的其他区域没有任何可检测到的磷酸化。纯化肽和其他肽的氨基酸序列分析显示Tyr-10和Tyr-7的连续磷酸化以及亚μM Ca 2+ 激活Ser-27的磷酸化。通过凝胶过滤柱估计去污剂溶解的酪氨酸激酶的表观分子量约为 50 kDa，并且复性后在凝胶带上检测到激酶活性。存在的 Ser-激酶被认为是由麦芽糖结合蛋白和从 Gly-2 到 Gln-111 的 α 链的肽部分组成的常规 PKC.H,K-ATPase 制剂磷酸化融合蛋白。这些数据和其他数据表明，N 端胞质结构域足以被膜 H,K-ATP 酶制剂中的内源 Tyr 激酶和 PKC 磷酸化。

项目成果

S.Kaya: "Cloning of the Eel Electroplax Na,K-ATPase Subunit." Ann.N.Y.Sci.834. 129-131 (1997)

S.Kaya：“鳗鱼 Electroplax Na,K-ATP 酶亚基的克隆。”

S.Kaya: "Pyridoxal 5'-phosphate probes at Lys-480 can sense the binding of ATP and the formation of phosphoenzymes in Na, K-ATPase." J.Biol.Chem.269. 7419-7422 (1994)

S.Kaya：“Lys-480 处的吡哆醛 5-磷酸探针可以感知 ATP 的结合以及 Na、K-ATP 酶中磷酸酶的形成。”

K.Togawa: "Reversible phosphorylation of both Tyr-10 and Tyr-7 in the alpha-Chain of pig stomach H,K-ATPase by a membrane bound kinase and a phosphatase." J.Biol.Chem.270. 15475-15478 (1995)

K.Tokawa：“猪胃 H,K-ATP 酶的 α 链中的 Tyr-10 和 Tyr-7 通过膜结合激酶和磷酸酶进行可逆磷酸化。”

H.Eguchi: "ATP Induced Dynamic Fluorescence Changes of N- [p- (2benzimidazoly1) pheny1] maleimide Probe at Cys-241 in the alpha-Chain of Pig Stomach H,K-ATPase." J.Biochem.122. 659-665 (1997)

H.Eguchi：“ATP 诱导猪胃 H,K-ATP 酶 α 链中 Cys-241 处的 N-[p-(2​​benzimidazoly1)pheny1]maleimide 探针的动态荧光变化。”

J.P.Froehlich: "Complex Kinetic Behavior in the Na,K- and Ca-ATPases" Ann.N.Y.Sci.834. 280-296 (1997)

J.P.Froehlich：“Na、K-和 Ca-ATP 酶的复杂动力学行为”Ann.N.Y.Sci.834。