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

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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.