Studies of Oligomer Interaction in Cation Pumps in Real Time

阳离子泵中低聚物相互作用的实时研究

• 批准号: 07044049
• 负责人: TANIGUCHI Kazuya
• 金额: $ 2.11万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 无数据
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044049/
• 关键词: Na^+, K^+-ATPase; H^+, -K^+-ATPase; Phosphorylation; Oligomer; Conformation; Kinase; Phospatase

The fluorescent probe, (BIPM), was used to monitor Na^+ translocation coupled to the conversion of E1P to EP2 in pig kidney Na, K-ATPase. The addition of 10 muM ATP to BIPM-labeled Na, K-ATPase gave an increase in fluorescence intensity with an apparent rate of 137/s and the phosphorylation with an apparent rate of 119/s. These results show that the BIPM signal and phosphoenzyme formation have similar kinetics and that, consequently, the conversion of E1P to E2P is very fast. At physiological[ATP], Na^+ release from E2P is rate-limited by phosphorylation and/or the conformational transitions involving Na^+ deocclusion.When pig stomach membrane H^+, K^+-ATPase preparations were incubated with[gamma-^<32>P]ATP,Mg^<2+> and Ca^<2+>, ^<32>P were incorporated into Tyr and Ser residues in the alphachain of H^+, K^+-ATPase. The radioactivity incorporated was shown to turn over. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatmentifollwed by a reverse-phase column chromatography gave th … More ree radio active peptide peaks. The first and the second peaks were assigned, respectively, to be the same amino-terminal phospho peptides, containing boty Tyr^<10>(^<32>P) and Tyr^7(^<32>P) and Tyr^<10>(^<32>P), both of which had been also obtained by the trypsin treatment of the preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and vanadate (Togawa K., Ishiguro T., Kaya S., Shimada A., Imagawa T., and Taniguchi K.(1995)J.Biol. Chem. 270,15475-15478). The third peak was also obtained after the trypsin treatement of partially purified H^+, K^+-ATPase preparations incubated with[gamma-^<32>P]ATP,Mg^<2+> and protein kinase-C + Ca^<2+> or protein kinase-A.Addition of endoproteinase ASP-N to each third peak fraction gave a major ^<32>P peptide peak as shown to be Asp^<21>-Met-Ala-Lys-Met-Ser(^<32>P)-Lys-Lys-Lys^<30> in the alpha-chain. These data and others indicate that amino-terminal domain of the alpha-chain of H^+, K^+-ATPase containing Tyr^7, Tyr^<10> and Ser^<27> is a hot spot for protein kinases dependent phosphorylation. The data also suggest participation of Ca^<2+> and cAMP in these phosphorylation reactions. Less

荧光探针（BIPM）用于监测Na^+易位，耦合到猪肾脏Na，K-ATPase中E1P转化为EP2。在BIPM标记的Na中添加了10个MUM ATP，K-ATPase的荧光强度增加，明显的速率为137/s，磷酸化的速度为137/s，明显的速率为119/s。这些结果表明，BIPM信号和磷酸酶的形成具有相似的动力学，因此，E1P转化为E2P非常快。在物理[ATP]下，从E2P释放的Na^+通过磷酸化和/或涉及Na^+去闭合的构象过渡的速率限制。当猪胃膜H^+时，K^+ - ATPase制剂与[gamma---^<32> p] atp，mg^<2+<2+<2+<32 <32 comply un ure孵育。 h^+，k^+ - atpase的字母。放射性掺入显示可以翻转。通过反相色谱法治疗的轻度甲苯基丙基丙酰基酮 - 酮tressin iflwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolwolw。第一个峰和第二个峰分别分别分配为相同的氨基末端磷脂，其中包含boty tyr^<10>（^<32> p）和tyr^7（^<32> p）和tyr^<10>（^<32> p），这两种情况都通过thyperation in the the the the the the the the the the the the the the the [32 <2+]和钒酸盐（Togawa K.，Ishiguro T.，Kaya S.，Shimada A.，Imimawa T.和Taniguchi K.（1995）J.Biol。Chem。270,15475-15478）。在胰蛋白酶处理的部分纯化的H^+，K^+ - ATPase制剂后，还获得了第三个峰ASP^<21> -MET-MET-ALA-LYS-MET-SER（^<32> P）-lys-lys-lys-lys-lys-lys^<30>在alpha-chain中。这些数据和其他数据表明，含有Tyr^7的H^+，K^+ATPase的α-链的氨基末端结构域，Tyr^<10>和Ser^<27>是依赖蛋白激酶的磷酸化的热点。数据还表明Ca^<2+>和CAMP参与这些磷酸化反应。较少的

项目成果

J.P.Froerhic: "Kinetics of the Phosphoenzyme interconversion reaction in BIPM-labeled pig kiney Na, k-ATPase" Biophysical Journal. 70. 327 (1996)

J.P.Froerhic：“BIPM 标记的猪肾 Na、k-ATP 酶中磷酸酶互变反应的动力学”生物物理学杂志。

K.Togawa: "Reversible Phosphorylation of boty Tyr^7 and Tyr^<10> in the α-chain of pig stomach H^+,K^+-ATPase by a membrane-found kinase and phosphatase." Journal of Biological Chemistry. 26. 15475-15478 (1995)

K. Tokawa：“通过膜发现的激酶和磷酸酶对猪胃 H^+,K^+-ATP 酶的 α 链中的 Tyr^7 和 Tyr^<10> 进行可逆磷酸化。” 26. 15475-15478 (1995)

J.P.Froerhic: "Kinetics of the Phosphoenzyme interconversion reaction in BIPM-labeled pig kiney Na^+,K^+-ATPase." Biophysical Journal. 70. 327 (1996)

J.P.Froerhic：“BIPM 标记的猪肾 Na^ ,K^ -ATP 酶中磷酸酶相互转化反应的动力学。”

K.Togawa: "Revresible Phosphorylation of boty Tyr7 and Tyr10 in the alpha-chain of pig stomach H^+, K^+-ATPase by a membrane-found kinase and phosphatase" Journal of Biological Chemistry. 26. 15475-15478 (1995)

K.Tokawa：“通过膜发现的激酶和磷酸酶对猪胃 H^ 、 K^ -ATP 酶的 α 链中的 Tyr7 和 Tyr10 进行可逆磷酸化”《生物化学杂志》。