Tanokura M, Tawada Y, Ono A, Ohtsuki I
J Biochem. 1983 Feb;93(2):331-7. doi: 10.1093/oxfordjournals.jbchem.a134185.
The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.
通过亲和层析，以及在14 mM Tris/90 mM甘氨酸中与F-肌动蛋白共沉降和聚丙烯酰胺凝胶电泳，对胰凝乳蛋白酶原肌钙蛋白T亚片段与原肌球蛋白、肌钙蛋白I和肌钙蛋白C的结合进行了半定量检测。测量了亚片段的圆二色光谱，以确认亚片段保留了其构象结构。基于这些结果，阐明了原肌球蛋白、肌钙蛋白I和肌钙蛋白C在肌钙蛋白T序列上的结合位点。原肌球蛋白主要与肌钙蛋白T1区域（第1-158位氨基酸残基）结合，其结合强度与和原始肌钙蛋白T结合时相同。在生理条件下，肌钙蛋白T的C末端区域（第243-259位氨基酸残基）是与原肌球蛋白结合的第二个位点。肌钙蛋白I的结合位点被确定为包含第223-227位氨基酸残基的区域。肌钙蛋白C的结合取决于Ca2+离子浓度。肌钙蛋白T2的C末端区域（第159-259位氨基酸残基）被表明是不依赖Ca2+的肌钙蛋白C结合位点，而肌钙蛋白T2的N末端一侧是依赖Ca2+的位点。
