Hiratsuka T
Department of Chemistry, Asahikawa Medical College, Hokkaido, Japan.
J Biol Chem. 1990 Nov 5;265(31):18791-6.
In order to study the conformational changes associated with formation of the stable ternary complex of myosin subfragment-1 (S-1) with ADP and orthovanadate (Vi), S-1 was fluorescently labeled with 9-anthroylnitrile, 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole, and 5-(iodoacetamido) fluorescein at the 23-, 50-, and 20-kDa peptide segments of S-1, respectively (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194; Hiratsuka, T. (1986) J. Biol. Chem. 261, 7294-7299; Takashi, R. (1979) Biochemistry 18, 5164-5169). The extrinsic fluorescence of these S-1 derivatives was sensitive not only to binding of ADP but to formation of the stable ternary complex with ADP and Vi. By using these fluorescent properties, the kinetics of formation of the stable ternary complexes of these S-1 derivatives with ADP and Vi, M. ADP.Vi, were analyzed according to the scheme proposed by Goodno (Goodno, C. C. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2620-2624). [Formula; see text] The values obtained for KVi )0.2-0.4 mM) and k (0.03-0.05 s-1) of these S-1 derivatives were similar regardless of the peptide segments of S-1 where the fluorophore had been covalently labeled. These results suggest that the conformational changes, which are induced by formation of the stable ternary complex of S-1 with ADP and Vi, are transmitted to all three peptide segments of S-1 at a similar rate. The present results also encourage us to confirm that the ATPase site of S-1 resides at or near the region where all three peptide segments of S-1 are contiguous.
为了研究肌球蛋白亚片段-1（S-1）与ADP和原钒酸盐（Vi）形成稳定三元复合物时伴随的构象变化，分别在S-1的23 kDa、50 kDa和20 kDa肽段用9-蒽腈、4-氟-7-硝基苯-2-恶唑-1,3-二氮唑和5-（碘乙酰胺基）荧光素对S-1进行荧光标记（平冢，T.（1989年）《生物化学杂志》264，18188 - 18194；平冢，T.（1986年）《生物化学杂志》261，7294 - 7299；高志，R.（1979年）《生物化学》18，5164 - 5169）。这些S-1衍生物的外在荧光不仅对ADP的结合敏感，而且对与ADP和Vi形成稳定三元复合物敏感。利用这些荧光特性，根据古德诺提出的方案（古德诺，C.C.（1979年）《美国国家科学院院刊》76，2620 - 2624）分析了这些S-1衍生物与ADP和Vi，即M·ADP·Vi形成稳定三元复合物的动力学。[公式；见正文] 这些S-1衍生物的KVi（0.2 - 0.4 mM）和k（0.03 - 0.05 s-1）值相似，无论荧光团共价标记在S-1的哪个肽段。这些结果表明，由S-1与ADP和Vi形成稳定三元复合物诱导的构象变化以相似的速率传递到S-1的所有三个肽段。目前的结果也促使我们确认S-1的ATP酶位点位于S-1所有三个肽段相邻的区域或其附近。
