肌钙蛋白上的钙和镁结合位点及其在肌原纤维三磷酸腺苷酶调节中的作用。

The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

作者信息

Potter J D, Gergely J

出版信息

J Biol Chem. 1975 Jun 25;250(12):4628-33.

Abstract

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.

摘要

纯化的肌钙蛋白（Tn）是由钙离子结合亚基（TnC）、抑制亚基（TnI）和原肌球蛋白结合亚基（TnT）组成的复合物，每摩尔能结合4摩尔钙离子。其中两个位点结合钙离子的结合常数为5×10⁻⁸ M⁻¹，另外两个位点的结合常数为5×10⁻⁶ M⁻¹。在2 mM氯化镁存在的情况下，与四个位点的结合可以用5×10⁻⁶ M⁻¹的单一亲和常数来表征。纯化的TnC每摩尔也结合4摩尔钙离子；两个位点的结合常数为2×10⁻⁷ M⁻¹，另外两个位点的结合常数为2×10⁻⁵ M⁻¹。在2 mM氯化镁存在的情况下，高亲和力位点的结合常数降至2×10⁻⁶ M⁻¹，而钙离子与低亲和力位点的结合不受影响。假设镁离子和钙离子在TnC和Tn上的高亲和力位点存在竞争，钙离子结合的变化可以分别用5×10⁻³ M⁻¹和5×10⁻⁴ M⁻¹的KMg值来解释。在没有铯离子的情况下，Tn和TnC每摩尔结合4摩尔镁离子。在2 mM镁离子存在的情况下，当钙离子浓度接近10⁻⁵ M时，每摩尔TnC结合四个钙离子和仅两个镁离子，这一事实进一步支持了镁离子和钙离子在TnC和Tn上的高亲和力钙离子结合位点存在直接竞争的观点。这些结果表明，Tn和TnC包含六个二价阳离子结合位点：两个高亲和力钙离子结合位点，也能竞争性地结合镁离子（钙离子 - 镁离子位点）；两个对钙离子亲和力较低且不结合镁离子的位点（钙离子特异性位点）；以及两个结合镁离子但不结合钙离子的位点（镁离子特异性位点）。TnC和TnI的复合物（1:1摩尔比）具有与Tn相同的结合特性，这表明TnC与TnI相互作用时发生了构象变化。在两种不同的游离镁离子浓度下，对肌原纤维ATP酶活性作为游离钙离子浓度的函数进行的研究表明，钙离子只有在与Tn中的两个钙离子特异性结合位点结合时才会发生完全激活，而不需要钙离子与钙离子 - 镁离子位点结合。