Ingraham R H, Swenson C A
J Biol Chem. 1984 Aug 10;259(15):9544-8.
The association constants for the formation of the binary complexes of rabbit fast skeletal muscle troponin subunits have been determined for three solution conditions: (a) 1 mM CaCl2, (b) 3 mM MgCl2 and 1 mM EGTA, and (c) 2 mM EDTA. The subunits were labeled with extrinsic fluorescence probes, either 5-(iodoacetamido)eosin (IAE) or dansylaziridine (DANZ), and the binding was detected by enhancement or quenching of the probe fluorescence. The association constant for the TnI X TnT (where TnI and TnT are the inhibitory subunit and the tropomyosin-binding subunit, respectively, of troponin) complex was measured with two different probes, IAE-TnI and IAE-TnT. The measured values were not affected by the presence of Ca2+ or Mg2+, and the mean values for the three buffer conditions are, respectively, 8.0 X 10(6) and 9.0 X 10(6) M-1 for the two probes. The association constant for TnC-TnI (where TnC is the Ca2+-binding subunit of troponin) interaction was measured with three probes, IAE-TnC, DANZ-TnC, and IAE-TnI. Values of 1.7 X 10(9), 1.2 X 10(8), and 1.0 X 10(6) M-1 were obtained, respectively, in the presence of calcium ion, in the presence of magnesium ion (no calcium), and in the absence of divalent metal ions. A mean value of 4.0 X 10(7) M-1 was obtained for the association constant of TnC X TnT using DANZ-TnC and IAE-TnC as probes in the presence of calcium or magnesium ions. A value of 4.5 X 10(6) M-1 was obtained in the absence of divalent metal ions. The results show that the presence of magnesium ion in the Ca2+-Mg2+ sites strengthens the TnC-TnI and the TnC-TnT interactions and suggest that the troponin structure would be stabilized. This likely results from the effect of magnesium ion on the Ca2+-Mg2+ domains of TnC. The presence of calcium ion in the Ca2+-specific sites provides an additional binding free energy for the TnC-TnI interaction which presumably reflects the changes in the subunit interactions required for the calcium regulatory switch.
(a) 1 mM氯化钙；(b) 3 mM氯化镁和1 mM乙二醇双四乙酸（EGTA）；(c) 2 mM乙二胺四乙酸（EDTA）。亚基用外在荧光探针进行标记，即5-（碘乙酰胺基）曙红（IAE）或丹磺酰氮丙啶（DANZ），并通过探针荧光的增强或淬灭来检测结合情况。使用两种不同的探针IAE-TnI和IAE-TnT测量了肌钙蛋白抑制亚基（TnI）与原肌球蛋白结合亚基（TnT）复合物（其中TnI和TnT分别是肌钙蛋白的抑制亚基和原肌球蛋白结合亚基）的缔合常数。所测值不受Ca2+或Mg2+存在的影响，在三种缓冲液条件下，两种探针的平均值分别为8.0×10⁶和9.0×10⁶ M⁻¹。使用三种探针IAE-TnC、DANZ-TnC和IAE-TnI测量了肌钙蛋白Ca2+结合亚基（TnC）与TnI相互作用的缔合常数。在钙离子存在下、镁离子存在下（无钙离子）和无二价金属离子存在下，分别得到1.7×10⁹、1.2×10⁸和1.0×10⁶ M⁻¹的值。在钙离子或镁离子存在下，使用DANZ-TnC和IAE-TnC作为探针，测得肌钙蛋白TnC与TnT缔合常数的平均值为4.0×10⁷ M⁻¹。在无二价金属离子存在下，得到的值为4.5×10⁶ M⁻¹。结果表明，Ca2⁺-Mg2⁺位点中镁离子的存在增强了TnC-TnI和TnC-TnT的相互作用，并表明肌钙蛋白结构将得到稳定。这可能是由于镁离子对TnC的Ca2⁺-Mg2⁺结构域的影响。Ca2⁺特异性位点中钙离子的存在为TnC-TnI相互作用提供了额外的结合自由能，这大概反映了钙调节开关所需的亚基相互作用的变化。
