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

采用荧光探针（BIPM）监测猪肾Na， k - atp酶中Na^+易位与E1P向EP2转化的关系。在bipm标记的Na， k -ATP酶上添加10 muM ATP，荧光强度增加，表观速率为137/s，磷酸化表观速率为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到E2P的转化非常快。在生理[ATP]中，Na^+从E2P释放受到磷酸化和/或涉及Na^+去封闭的构象转变的速率限制。当猪胃膜H^+， K^+-ATP酶制剂与[γ -^<32>P]ATP孵育时，Mg^<2+>和Ca^<2+>, ^<32>P被掺入H^+， K^+-ATP酶α链中的Tyr和Ser残基中。所吸收的放射性物质被证明是可以翻转的。温和的甲苯苯丙酰氯甲基酮-胰蛋白酶处理，然后进行反相柱层析，得到了更多的放射性活性肽峰。第一个和第二个峰分别为相同的氨基末端磷酸肽，含有Tyr^<10>(^<32 b> P)和Tyr^7（^<32 b> P）和Tyr^<10>(^<32 >p)，这两个峰都是由胰蛋白酶处理的[γ -^<32 >p]ATP,Mg^<2+>和钒酸盐（Togawa K., Ishiguro T., Kaya S., Shimada A., Imagawa T., and Taniguchi K.(1995)J.Biol.）。270年化学,15475 - 15478)。用[γ -^<32>P]ATP、Mg^<2+>和蛋白激酶- c + Ca^<2+>或蛋白激酶- a孵育部分纯化的H^+、K^+-ATP酶制剂，经胰蛋白酶处理后也出现第三个峰。将内啡肽Asp - n添加到每个第三个峰段，得到一个主要的^<32>P肽峰，如α链中的Asp^<21>-Met-Ala-Lys-Met-Ser(^<32>P)-Lys-Lys-Lys^<30>。这些数据和其他数据表明，含有Tyr^7， Tyr^<10>和Ser^<27>的H^+， K^+- atp酶α链的氨基末端结构域是蛋白激酶依赖性磷酸化的热点。数据还表明Ca^<2+>和cAMP参与了这些磷酸化反应。少

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》。