Pearlstone J R, Smillie L B
J Biol Chem. 1982 Sep 25;257(18):10587-92.
The binding of rabbit skeletal muscle troponin-T and several of its fragments to various types of tropomyosin immobilized on Sepharose 4B affinity columns equilibrated with 0.1 M NaCl, pH 7.0 buffer has been investigated. With rabbit skeletal muscle alpha-tropomyosin, intact troponin-T was eluted with an NaCl gradient at 0.42 M, while its fragments T1 (residues 1-158) and CB1 (residues 1-151) were eluted at 0.32 M NaCl in either "plus" or "minus" Ca2+ buffer in the presence of troponin-C. Fragment T2 (residues 159-259) was eluted at 0.22 M NaCl in minus Ca2+ buffer in the presence of troponin-C, but in the void volume with troponin-C under plus Ca2+ conditions. With immobilized nonpolymerizable alpha-tropomyosin, T1 was not bound, whereas T2 was eluted at the same NaCl concentration (0.21 M) as with alpha-tropomyosin. This binding was sensitive to Ca2+ in the presence of troponin-C. The results are consistent with a structural interpretation of a two-site model of troponin attachment to alpha-tropomyosin (Mak, A. S., and Smillie, L. B. (1981) J. Mol. Biol. 149, 541-550). With beta-tropomyosin from rabbit skeletal muscle and with tropomyosins from equine platelets and chicken gizzard, the binding of fragment T1 was not observed at 0.1 M NaCl, while that for T2 was the same as for rabbit skeletal alpha-tropomyosin and remained Ca2+-sensitive in the presence of troponin-C. In the case of bovine aorta tropomyosin, neither T1 nor T2 was bound under these conditions.
研究了兔骨骼肌肌钙蛋白 - T及其几个片段与固定在经0.1 M NaCl、pH 7.0缓冲液平衡的琼脂糖4B亲和柱上的各种类型原肌球蛋白的结合情况。对于兔骨骼肌α - 原肌球蛋白，完整的肌钙蛋白 - T在0.42 M NaCl梯度下洗脱，而其片段T1（第1 - 158位氨基酸残基）和CB1（第1 - 151位氨基酸残基）在存在肌钙蛋白 - C的情况下，于“加”或“减”Ca²⁺缓冲液中在0.32 M NaCl处洗脱。片段T2（第159 - 259位氨基酸残基）在存在肌钙蛋白 - C的减Ca²⁺缓冲液中于0.22 M NaCl处洗脱，但在加Ca²⁺条件下与肌钙蛋白 - C一起在空体积处洗脱。对于固定的不可聚合α - 原肌球蛋白，T1不结合，而T2在与α - 原肌球蛋白相同的NaCl浓度（0.21 M）处洗脱。在存在肌钙蛋白 - C的情况下，这种结合对Ca²⁺敏感。结果与肌钙蛋白附着于α - 原肌球蛋白的双位点模型的结构解释一致（Mak, A. S., and Smillie, L. B. (1981) J. Mol. Biol. 149, 541 - 550）。对于来自兔骨骼肌的β - 原肌球蛋白以及来自马血小板和鸡砂囊的原肌球蛋白，在0.1 M NaCl下未观察到片段T1的结合，而T2的结合情况与兔骨骼肌α - 原肌球蛋白相同，并且在存在肌钙蛋白 - C的情况下仍对Ca²⁺敏感。对于牛主动脉原肌球蛋白，在这些条件下T1和T2均不结合。
