A rapid quenching a apparatus permitting to mix for different microvolume solutions

一种快速淬火装置，允许混合不同的微体积溶液

• 批准号: 03558026
• 负责人: TANIGUCHI Kazuya
• 金额: $ 2.94万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03558026/
• 关键词: Na^+, K^+-Pump; H^+, K^+-Pump; rapid quenching; Stopped flow; P-type ATPase; Tyr phosphorylation; H^+ーポンプ; ラビッドクエンチング; H^+-ポンプ; ラピッドクエンチング

A preparation of pig kidney Na^+ K^+-ATPase showed changes in fluorescence energy transfer between probes bound to the alpha-subunit. Excitation of BIPM probe, which was covalently bound to Cys-964, and excitation of FITC probe at Lys-501 gave different FITC fluorescence intensity changes at 520 nm in BIPM-FITC doubly labeled enzyme accompanying formation of reaction intermediates. The data suggest that Fluorescence energy transfer from the BIPM to the FITC probe accompanies the processes of migration of Na^+ and K^+ in the pump molecules. The dynamic fluorescence changes after phosphorylation and dephosphorylation seem to reflect changes in the binding state or the process of migration of these ions.The Lys-480 in the alpha-subunits of Na^+, K^+-ATPase from pig kidneys was specifically modified with PLP or AP2PL probes in the presence of NaCl. The data show that these probes can sense molecular events related to the formation of phosphoenzymes induced by AcP and presumably to the form … More ation of Mg-Na-ATP-enzyme complex. The data also suggest that PLP or AP_2PL probes at Lys-480 in the presence of Na^+ and Mg^<2+> do not affect the transphosphorylation from AcP to Asp-369 to form phosphoenzymes byt that they inhibit the transphosphorylation from the gamma-phosphoryl group of ATP and also ATP binding in the absence of Mg^<2+>.When pig stomach membrane H^+, K^+-ATPase preparations were incubated with [gamma-^<32>P]ATP and Mg^<2+> with vanadate, ^<32>P was incorporated into the alpha-chain of H^+, K^+-ATPase to a steady-state level of approximately 0.7 mol of phosphotyrosine (Tyr (P)) /mol of phosphoenzyme intermediates. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized ^<32>P-containing peptides from the alpha-chain almost completely. A reverse-phase column chromatography of the supernatant gave two peaks of ^<32>P-peptide with similar total radioactivities.The comparison of the recovery of amino acid from each Edman cycle showed that the phosphorylation of Tyr^<10> occurred preceding the phosphorylation of Tyr^7. These data and others suggested the presence of a novel membrane bound enzyme system to participate in reversible phosphorylation of boty Tyr residues in the alpha-chain of H^+, K^+-ATPase. Less

猪肾Na^+ K^+-ATP酶的制备表明，与α亚基结合的探针之间的荧光能量转移发生了变化。激发BIPM探针，这是共价结合到Cys-964，和激发的FITC探针在Lys-501给出了不同的FITC荧光强度的变化，在520 nm处的BIPM-FITC双标记酶伴随着反应中间体的形成。这些数据表明，从BIPM到FITC探针的荧光能量转移伴随着泵分子中Na^+和K^+的迁移过程。磷酸化和去磷酸化后的动态荧光变化似乎反映了这些离子结合状态或迁移过程的变化。在NaCl存在下，用PLP或AP 2 PL探针特异性修饰了猪肾Na^+，K^+-ATP酶α亚基的Lys-480。数据表明，这些探针可以检测到与由AcP诱导的磷酸酶形成相关的分子事件，并且推测这些分子事件与AcP诱导的磷酸酶的形成相关。 ...更多信息 Mg-Na-ATP-酶复合物的反应。这些数据还表明，在Na^+和Mg^&lt;2+&gt;存在的情况下，Lys-480上的PLP或AP_2PL探针不影响AcP转磷酸到Asp-369形成磷酸酶拜特，因为它们抑制ATP的γ-磷酸基的转磷酸化，并且在Mg^&lt;2+&gt;不存在的情况下也抑制ATP的结合。将K^+-ATP酶制备物与[γ-^<32>P]ATP和Mg^&lt;2+&gt;及钒酸盐一起孵育，使^<32>P掺入H^+，K^+-ATP酶的α链，达到约0.7 mol磷酸酪氨酸（Tyr（P））/mol磷酸酶中间体的稳态水平。温和的甲苯磺酰基苯丙氨酰氯甲基酮-胰蛋白酶处理<32>几乎完全溶解了α链上的含β-P的肽。对上清液进行反相柱层析，得到两个<32>总放射性相似的^P-肽峰，比较每个埃德曼循环中氨基酸的回收率，发现Tyr^的磷酸化<10>先于Tyr ^7的磷酸化。这些数据和其他数据表明，存在一种新的膜结合酶系统参与H^+，K^+-ATP酶α链上两个Tyr残基的可逆磷酸化。少

项目成果

K.Taniguchi: "Reversible Changes in the Fluorescence Energy Transfer Accompanying Formation of Reaction Intermediates in Probe-Labeled Na^+,K^+-ATPase" Journal of Biological Chemistry. (1993)

K.Taniguchi：“探针标记的 Na^ ,K^ -ATP 酶中伴随反应中间体形成的荧光能量转移的可逆变化”生物化学杂志。

Eiji Shinoguchi: "The Sodium Pump:Recent Developments" The Rockfeller University Press, 4 (1991)

Eiji Shinoguchi：“钠泵：最新进展”洛克菲勒大学出版社，4 (1991)

Nakamura, Y.: "Different Susceptibility of Phospholipase A_2 Treatment of the Fluorescence Intensity Changes in the Cicinity of Cys-964 and Lys-501 in the alpha-Chain of probe-Labeled Na^+, K^+-ATPase." J.Biochem.115. 454-462 (1994)

Nakamura, Y.：“磷脂酶 A_2 处理对探针标记的 Na+、K+-ATP 酶的 α 链中 Cys-964 和 Lys-501 附近荧光强度变化的不同敏感性”。

Kaya,S.: "Pyridoxal 5'-phosphate Probe at Lys-480 Can Sense the Binding of ATP and the Formation of Phosphoenzymes in Na^+,K^+-ATPase." Journal of Biological Chemistry. 269. 7419-7422 (1994)

Kaya,S.：“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。”

Adachi,Y.: "Na^+,K^+-ATPase Based Bilayer Lipid Membrane Sensor for Adenosine 5'-Triphoshate." Anal.Chem.Acta.281. 577-584 (1993)

Adachi,Y.：“基于 Na^ ,K^ -ATP 酶的双层脂质膜传感器，用于 5-三磷酸腺苷。”