Experimental Triple Mixing Rapid Filtiation Apparatus

实验三重混合快速过滤装置

• 批准号: 07558220
• 负责人: TANIGUCHI Kazuya
• 金额: $ 0.9万
• 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: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558220/
• 关键词: Na, K-ATPase; Rb.Occlusion; 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; ラピッドフィルトレーション; 構造変化; イオン閉塞; 蛍光プローブ

Pig Kidney Na, K-ATPase preparations modified with N- [p- (2-benzimidazolyl) phenyl] maleimide (BIPM-enzyme) were incubated with 0.7muM N- (4-sulfobutyl) -4- [4- (p-dipentyl-aminophenyl) butadienyl] pyridinium, inner salt (RH421). The resulting enzyme preparations changed each relative fluorescence intensity under conditions of accumulation of various reaction intermediates.Addition of ATP to the enzyme preparations in the presence of 2M Na^+ and 4 mM Mg^<2+> induced a rapid decrease in RH421 fluorescence (25/s) follwed by slow phosphoenzyme formation and decrease in BIPM fluorescence (5-6/s). The addition of ATP to the preparation in the presence of 16 mM Na^+ and 1 mM Ca^<2+> induced sequential decreases in fluorescence of RH421 (44/s) and BIPM(26/s), which were followed by slow increases in both BIPM fluorescence and phosphoenzyme (around 6/s) and slower increase in RH421 fluorescence (1.7/s). RH421 seemed to sense ATP dependent molecular events preceding BIPM fluorescence decrease prior ADP sensitive phosphoenzyme formation and following K^+ -sensitive phosphoenzyme formation.

用N- [P-（2-苯并咪唑基）苯酰亚胺（BIPM-酶）修饰的猪肾Na，K-ATPase制剂与0.7MUM N-（4-硫磺丁基）-4- [4- [4-（p-氨基氨基苯基苯基苯基苯基苯基苯基）pyridim pryidinium salt（Rh421）孵育。在各种反应中间体的积累条件下，所得的酶制剂改变了每种相对荧光强度。在16 mm Na^+和1 mm Ca^<2+>存在的制备中，添加ATP在RH421（44/s）和BIPM（26/s）的荧光下诱导的顺序降低，随后，BIPM荧光和磷酸酶（6/s）和rh421荧光（1.7/s）的BIPM荧光和磷酸化酶的增长均缓慢增加。 RH421似乎感觉到BIPM荧光之前的ATP依赖性分子事件会降低先前的ADP敏感磷酸酶形成，并在K^+敏感的磷酸酶形成之后。

项目成果

T.Tsuda: "ATP and Acetyl Phosphate Induce Molecular Events near the ATP binding site and the Membrane Domain Na,K-ATPase:The Terameric Nature of the Enzyme." J.Biol.Chem.(in press).

T.Tsuda：“ATP 和乙酰磷酸在 ATP 结合位点和膜结构域 Na,K-ATP 酶附近诱导分子事件：酶的四聚体性质。”

T.Tsuda: "Fluorescein 5'-Isothiocyanate-Modefied Na,K-ATPase at Lys-501 of the alpha-chain,Accepts ATP Independent of Pyridoxal 5'-Diphospho-5'-Adenosine Modification at Lys-480." J.Biochem.123. 169-174 (1998)

T.Tsuda：“α 链 Lys-501 处的荧光素 5-异硫氰酸酯修饰的 Na,K-ATP 酶，在 Lys-480 处接受独立于吡哆醛 5-二磷酸-5-腺苷修饰的 ATP。”

K.Togawa: "Phosphorylation of Tyr-7,Tyr-10,and Ser-27 of alpha-chain in H,K-ATPase by Intrinsic and Extrinsic kinases." Ann.N.Y.Acad.Sci.834. 582-584 (1997)

K.Tokawa：“通过内在和外在激酶对 H,K-ATP 酶中 α 链的 Tyr-7、Tyr-10 和 Ser-27 进行磷酸化。”

Katsuhiko Togawa: "Phosphorylation of Tyr^7, Tyr^<10> and Ser^<27> of α Chain in H^+, K^+-ATPase by Intrinsic and Extrinsic Kinases" Ann. New York, Acad. Sci.(in press). (1997)

Katsuhiko Tokawa：“H^+、K^+-ATP 酶中 α 链的 Tyr^7、Tyr^<10> 和 Ser^<27> 通过内在和外在激酶的磷酸化”Ann，Acad Sci。 (1997)

T.Imagawa: "Does Binding of Ouabain to Human alpha 1 subunit of Na,K-ATPase Affect the ATPase Activity of Adiacent Rat alpha 1 subunit?" Jpn.J.Pharmacal.(in press).

T.Imakawa：“哇巴因与人类 Na,K-ATP 酶 α 1 亚基的结合是否会影响 Adiacent Rat α 1 亚基的 ATP 酶活性？”