Holroyde M J, Robertson S P, Johnson J D, Solaro R J, Potter J D
J Biol Chem. 1980 Dec 25;255(24):11688-93.
The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.
心肌肌钙蛋白（Tn）复合物由一个钙结合亚基（TnC）、一个抑制亚基（TnI）和一个原肌球蛋白结合亚基（TnT）组成，已从牛心肌中分离出的纯化肌钙蛋白亚基重新组装而成。通过使用乙二醇双四乙酸（EGTA）或乙二胺四乙酸（EDTA）调节游离钙浓度的平衡透析法，测定了心肌Tn的钙结合特性。心肌Tn每摩尔结合3摩尔钙离子，包含两个结合常数为3×10⁸ M⁻¹的钙结合位点和一个结合常数为2×10⁶ M⁻¹的结合位点。在4 mM氯化镁存在下，高亲和力位点的结合常数降至3×10⁷ M⁻¹，而钙离子与低亲和力位点的结合不受影响。心肌Tn的两个高亲和力钙结合位点类似于骨骼肌Tn的两个钙-镁位点，而单个低亲和力位点类似于骨骼肌Tn的两个钙离子特异性位点（波特，J.D.，和杰尔盖利，J.（1975年）《生物化学杂志》250，4625 - 4633）。TnC和TnI（摩尔比1:1）复合物的钙结合特性与Tn相似。心肌TnC每摩尔也结合3摩尔钙离子，包含两个结合常数为1×10⁷ M⁻¹的位点和一个结合常数为2×10⁵ M⁻¹的单个位点。假设镁离子和钙离子竞争TnC和Tn的高亲和力位点，镁离子的结合常数分别为0.7和3.0×10³ M⁻¹。心肌肌原纤维ATP酶活性对钙离子的依赖性与由重组肌钙蛋白复合物调节的肌动球蛋白制剂相似。通过心肌Tn的钙结合特性以及心肌肌原纤维ATP酶活性作为钙离子浓度函数的比较，发现在毫摩尔浓度的镁离子存在下，ATP酶的激活发生在与心肌Tn的钙离子特异性位点结合钙离子相同的钙离子浓度范围内。
