Gusev N B, Friedrich P
Biochim Biophys Acta. 1980 Nov 20;626(1):106-16. doi: 10.1016/0005-2795(80)90202-0.
In an attempt to detect Ca2+-induced conformational changes by crosslinking, rabbit muscle troponin complex was reacted with the bifunctional reagents 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone and dimethyl suberimidate under various conditions and the products were analyzed by dodecyl sulfate gel electrophoresis. In the absence of divalent cations, with the two aromatic reagents at a low reagent/protein ratio, the main cross-link products were troponin T-I and I-C. With dimethyl suberimidate the only major crosslink product was conjugate T-I. Ca2+, alone as well as in the presence of Mg2+, prevented the formation of I-C crosslinks with both aromatic reagents, but it did not affect crosslinking with dimethyl suberimidate. Ca2+ also decreased the number of NH2 groups of troponin that are highly reactive towards 2,4,6-trinitrobenzene sulfonate. Both effects of Ca2+ can be interpreted in terms of a conformational change in the troponin complex elicited by the binding of the specific divalent cation.
为了通过交联检测钙离子诱导的构象变化，将兔肌肉肌钙蛋白复合物在各种条件下与双功能试剂1,3 - 二氟 - 4,6 - 二硝基苯、4,4'-二氟 - 3,3'-二硝基二苯砜和亚胺基二甲酯反应，并通过十二烷基硫酸盐凝胶电泳分析产物。在没有二价阳离子的情况下，使用两种芳香族试剂且试剂/蛋白质比例较低时，主要的交联产物是肌钙蛋白T - I和I - C。使用亚胺基二甲酯时，唯一主要的交联产物是共轭物T - I。单独的钙离子以及在镁离子存在的情况下，都能阻止两种芳香族试剂形成I - C交联，但不影响与亚胺基二甲酯的交联。钙离子还减少了肌钙蛋白中对2,4,6 - 三硝基苯磺酸盐反应性高的氨基数量。钙离子的这两种作用都可以用特异性二价阳离子结合引起的肌钙蛋白复合物构象变化来解释。
