The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase.
The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase.
复制标题
DOI：
10.1016/s0021-9258(19)70187-2
复制
发表时间：
1980-12
期刊：
影响因子：
--
通讯作者：
M. Holroyde;S. P. Robertson;J. Johnson;R. Solaro;J. D. Potter
中科院分区：
文献类型：
--
作者：
M. Holroyde;S. P. Robertson;J. Johnson;R. Solaro;J. D. Potter
心肌肌钙蛋白 (Tn) 复合物由 Ca2+ 结合亚基 (TnC)、抑制性亚基 (TnI) 和原肌球蛋白结合亚基 (TnT) 组成，由从牛心肌分离的纯化肌钙蛋白亚基重建。心脏 Tn 的 Ca2+ 结合特性通过使用 EGTA 或 EDTA 调节游离 Ca2+ 浓度的平衡透析来测定。心脏 Tn 结合 3 mol Ca2+/mol，并含有 2 个结合常数为 3 X 10(8) M-1 的 Ca2+ 结合位点和 1 个结合常数为 2 X 10(6) M-1 的结合位点。在 4 mM MgCl2 存在下，较高亲和力位点的结合常数降低至 3 X 10(7) M-1，而 Ca2+ 与较低亲和力位点的结合不受影响。心脏 Tn 的两个高亲和力 Ca2+ 结合位点类似于骨骼 Tn 的两个 Ca2+-Mg2+ 位点，而单个低亲和力位点类似于骨骼 Tn 的两个 Ca2+ 特异性位点（Potter, J. D. 和 Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633）。 TnC 和 TnI（1:1 摩尔比）复合物的 Ca2+ 结合特性与 Tn 相似。心脏 TnC 还结合 3 mol Ca2+/mol，并包含两个结合常数为 1 X 10(7) M-1 的位点和一个结合常数为 2 X 10(5) M-1 的位点。假设 Mg2+ 和 Ca2+ 竞争 TnC 和 Tn 的高亲和力位点，Mg2+ 的结合常数分别为 0.7 和 3.0 X 10(3) M-1。心肌原纤维 ATP 酶活性的 Ca2+ 依赖性与受重构肌钙蛋白复合物调节的肌动球蛋白制剂相似。通过比较心脏 Tn 的 Ca2+ 结合特性和心脏肌原纤维 ATP 酶活性作为 [Ca2+] 和毫摩尔 [Mg2+] 的函数，表明 ATP 酶的激活发生在相同的 [Ca2+] 范围内，其中心脏 Tn 的 Ca2+ 特异性位点结合 Ca2+。
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+.