Ueno H, Tawada Y, Ooi T
J Biochem. 1976 Aug;80(2):283-90. doi: 10.1093/oxfordjournals.jbchem.a131275.
Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.
用羧肽酶A [EC 3.4.12.2]（CTM）消化的原肌球蛋白在溶液中的粘度低于未消化的蛋白质。根据消化过程中粘度降低与从羧基末端释放的氨基酸量之间的关系，可以推断出从C末端失去三肽-Thr-Ser-Ile是粘度降低的原因。根据圆二色性测量，-TM的二级结构不受消化影响。CTM在甲醇-水混合物中的粘度没有增加，而原肌球蛋白的粘度则显著增加。这些结果表明，从C末端去除一个小肽后，聚合能力丧失，而二级结构没有改变。添加CTM后，观察到原肌球蛋白溶液的粘度降低，表明CTM与完整的原肌球蛋白相互作用。粘度降低对CTM量的依赖性表明，由于端对端相互作用，CTM以一对一的比例结合原肌球蛋白。由于在中性pH值下，在镁离子存在的情况下可以生长出具有400埃重复带结构的副晶体，因此即使聚合能力丧失，CTM分子中的并排相互作用仍然保持完整。圆盘凝胶电泳图谱表明，肌钙蛋白可以与CTM结合，但在溶液中未观察到由于复合物形成而导致的粘度增加。也就是说，原肌球蛋白的C末端部分对于复合物的形成不是必需的。与F-肌动蛋白结合的CTM量不到与未消化的原肌球蛋白结合量的一半，并且可以通过洗涤程序减少到十分之一。然而，在肌钙蛋白存在的情况下，回收的量恢复到通常与F-肌动蛋白结合的原肌球蛋白水平。因此，可以得出结论，肌钙蛋白结合在原肌球蛋白分子的中间，并加强了原肌球蛋白与F-肌动蛋白的结合。
