Study on Activities and Discrimination of Heads of the Muscle Contractile Protein Myosin by Means of Fluorescence and Laser Photolysis Methods

荧光和激光光解法研究肌肉收缩蛋白肌球蛋白头部的活性和识别

• 批准号: 05640646
• 负责人: KOBASHI Harumichi
• 金额: $ 1.41万
• 依托单位: Gunma University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05640646/
• 关键词: Myosin; Fluorescent Probe; Noncovalently Bonding Probe; Covalently bonding Prove; SH-Modification; NH_2-Modification; Pyrene Derivatives; Trinitrophenylation; ATPase活性; ピレンヨードアセトアミド; ピレンメチルヨードアセテイト; ピレンマレイミド; SH基特異的修飾; NH_2基特異的修飾; 筋収縮蛋白質; ピ

The present research project aimed to elucidate fluorescence behavior of pyrene (P) derivatives which are expected as useful probes for obtaining informations concerning microstructure and functions of M,especially in relation to discrimination of two myosin heads.1. Binding constants of P and 1-pyrenesulfonate ion (PS) with M were estimated to be 4.9x10^5 and 1x10^7 M^<-1>, respectively, in aq soln (pH 7.0) at 20ﾟC2. Pyrene molecules of <greater than or equal> 65% fraction were located in inner hydrophobic regions of M where iodide ion as quencher can not quench the fluorescence of P.PS binds with M in a relatively hydrophillic region accessible to the3. .Some fluorescent probes with a pyrene chromophore react specifically with active SH-or NH_2-groups of M.Myosin modified with N- (1-pyrene) iodoacetamide showed a typical curve in pH dependence of Ca-ATPase activity of SH1-modified M.Change in fluorescence characteristics of the probe was observed associated with conformational change of M induced by the addition of ATP.4. Trinitrophenylation and retrinitrophenylation with 2,4,6-trinitrobenzenesulfonic acid revealed that the two heads of chicken skeletal M were different from each other in pH dependence of Ca-ATPase activity and in reactivities in the presence and absence of PPi. These findings suport the hypothesis of non-identical heads.

本研究旨在阐明芘（P）衍生物的荧光行为，这些衍生物有望作为有用的探针用于获得有关M的微观结构和功能的信息，特别是与区分两种肌球蛋白头的关系.在20 ℃的水溶液（pH 7.0）中，P和1-芘磺酸根离子（PS）与M的结合常数估计分别为4.9x10^5和1x10^7 M^<-1>。65%的芘分子<greater than or equal>位于M的内疏水区，碘离子作为猝灭剂不能猝灭P的荧光。PS与M结合在一个相对疏水的区域，该区域靠近3。一些带有芘发色团的荧光探针能特异性地与M的SH-或NH_2-活性基团发生反应。N-（1-芘）碘乙酰胺修饰的肌球蛋白对SH 1修饰的M的Ca-ATP酶活性具有典型的pH依赖性曲线。用2，4，6-三硝基苯磺酸进行三硝基苯化和反三硝基苯化反应，发现鸡骨骼肌M的两个头部的Ca-ATP酶活性对pH的依赖性以及在PPi存在和不存在下的反应性不同。这些发现支持了不同头部的假设。

项目成果

H.Kobashi, etal.: "Influence of Divalent Metal Ions on Fluorescence of ATP" Abstr. No.2A706,67th Annual Meeting of the Chemical Society of Japan, Tokyo, April. 146 (1994)

H.Kobashi 等人：“二价金属离子对 ATP 荧光的影响”摘要。

A.Hasegawa, etal.: "Distribution of Fluorescence Probes, Pyrene and 1-Pyrenesulfonate Ion, on the Muscle Contractile Protein Myosin" Abstr. No.3D206,65th Annual Meeting of the Chemical Society of Japan, Tokyo, March. 292 (1993)

A.Hasekawa 等人：“荧光探针、芘和 1-芘磺酸根离子在肌肉收缩蛋白肌球蛋白上的分布”摘要。

羽田博: "ニワトリ骨格筋ミオシンのトリニトロフェニル(TNP)化とTNP化ミオシンのヒドラジン処理" 日本化学会第67春季年会講演予稿集,3E318. 937- (1994)

Hiroshi Hada：“鸡骨骼肌肌球蛋白的三硝基苯基化（TNP）和 TNP 修饰的肌球蛋白的肼处理”日本化学会第 67 届春季年会论文集，3E318-（1994）。

根岸 信一: "低濃度のメタノールを含むアセトニトリル中におけるフェニルホスホ硫酸エステルのメタノリシスにおよぼすMg(II)の触媒効果" 日本化学会第67春季年会講演予稿集,2D246. 823 (1994)

Shinichi Negishi：“Mg(II) 对含有低浓度甲醇的乙腈中磷酸苯酯的甲醇分解的催化作用”日本化学会第 67 届春季年会论文集，2D246 (1994)。

S.Negishi, etal.: "Effects of Mg (II) on the Methanolysis of Phenyl Phosphatosulfate in Acetnitrile of Low Methanol Content" Abstr. No.2D246,67th Annual Meeting of the Chemical Society of Japan, Tokyo, April. 823 (1994)

S.Negishi 等人：“镁 (II) 对低甲醇含量乙腈中磷酸苯基磷酸酯甲醇分解的影响”摘要。