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

本研究旨在阐明芘(P)衍生物的荧光行为，该衍生物有望作为有用的探针，获得有关M的微观结构和功能的信息，特别是与两个肌球蛋白头的区分有关。在pH 7.0的水溶液中，P和1-芘磺酸盐离子（PS）与M的结合常数分别为4.9x10^5和1x10^7 M^<-1>。大于或等于bbb65 %的芘分子位于M的内部疏水区域，其中碘离子作为猝灭剂不能猝灭P.PS与M在相对亲水区域结合的荧光。一些带有芘发色团的荧光探针与M的sh -或nh_2活性基团发生特异性反应，用N-（1-芘）碘乙酰酰胺修饰的myosin对sh -修饰的M的ca - atp酶活性呈典型的pH依赖性曲线，探针的荧光特性的变化与M的构象变化有关。用2,4,6-三硝基苯磺酸进行三硝基苯和后硝基苯化处理，结果表明，两种鸡骨M对ca - atp酶活性的pH依赖性以及在存在和不存在PPi时的反应活性不同。这些发现支持了头像不相同的假设。

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.