Tobacman L S, Sawyer D
Department of Internal Medicine, College of Medicine, University of Iowa, Iowa City 52242.
J Biol Chem. 1990 Jan 15;265(2):931-9.
To investigate the relationship between thin filament Ca2+ binding and activation of the MgATPase rate of myosin subfragment 1, native cardiac thin filaments were isolated and characterized. Direct measurements of 45Ca binding to the thin filament were consistent with non-cooperative binding to two high affinity sites (Ka 7.3 +/- 0.8 x 10(6) M-1) and either cooperative or non-cooperative binding to one low affinity site (Ka 4 +/- 2 x 10(5) M-1) per troponin at 25 degrees C, 30 mM ionic strength, pH 7.06. Addition of a low concentration of myosin subfragment 1 to the native thin filaments produced a Ca2+-regulated MgATPase activity with Kapp (2.5 +/- 1.3 x 10(5) M-1), matching the low affinity Ca2+ site. The MgATPase rate was cooperatively activated by Ca2+ (Hill coefficient 1.8). To determine whether Ca2+ binding to the low affinity sites was cooperative, native thin filament troponin was exchanged with troponin labeled on troponin C with 2-(4'-iodoacetamidanilo)naphthalene-6-sulfonic acid. From the Ca2+-sensitive fluorescence of this complex, Ca2+ binding was cooperative with a Hill coefficient of 1.7-2.0. Using the troponin-exchanged thin filaments, myosin subfragment 1 MgATPase rate activation was also cooperative and closely proportional to Ca2+ thin filament binding. Reconstitution of the thin filament from its components raised the Ca2+ affinity by a factor of 2 (compared with native thin filaments) and incorporation of fluorescently modified troponin raised the Ca2+ affinity by another factor of 2. Stoichiometrically reconstituted thin filaments produced non-cooperative MgATPase rate activation, contrasting with cooperative activation with native thin filaments, troponin-exchanged thin filaments and thin filaments reconstituted with a stoichiometric excess of troponin. The Ca2+-induced fluorescence transition of stoichiometrically reconstituted thin filaments was non-cooperative. These results suggest that Ca2+ binds cooperatively to the regulatory sites of the cardiac thin filament, even in the absence of myosin, and even though cardiac troponin C has only one Ca2+-specific binding site. A theoretical model for these observations is described and related to the experimental data. Well-known interactions between neighboring troponin-tropomyosin complexes are the proposed source of cooperativity and also influence the overall Ka. The data indicate that Ca2+ is four times more likely to elongate a sequence of troponin-tropomyosin units already binding Ca2+ than to bind to a site interior to a sequence of units without Ca2+.
为了研究细肌丝Ca2+结合与肌球蛋白亚片段1的MgATPase活性激活之间的关系，我们分离并鉴定了天然心脏细肌丝。在25℃、离子强度30mM、pH7.06条件下，对细肌丝上45Ca结合的直接测量结果表明，其与两个高亲和力位点的结合是非协同性的（Ka 7.3±0.8×10(6) M-1），每个肌钙蛋白与一个低亲和力位点的结合可能是协同性的，也可能是非协同性的（Ka 4±2×10(5) M-1）。向天然细肌丝中添加低浓度的肌球蛋白亚片段1会产生一种Ca2+调节的MgATPase活性，其Kapp为（2.5±1.3×10(5) M-1），与低亲和力Ca2+位点相匹配。MgATPase活性由Ca2+协同激活（希尔系数为1.8）。为了确定Ca2+与低亲和力位点的结合是否具有协同性，将天然细肌丝肌钙蛋白与用2-(4'-碘乙酰胺基苯胺基)萘-6-磺酸标记的肌钙蛋白C进行交换。根据该复合物对Ca2+敏感的荧光可知，Ca2+结合具有协同性，希尔系数为1.7 - 2.0。使用肌钙蛋白交换后的细肌丝，肌球蛋白亚片段1的MgATPase活性激活也是协同性的，并且与Ca2+在细肌丝上的结合密切相关。由其各组分重构的细肌丝使Ca2+亲和力提高了2倍（与天然细肌丝相比），而掺入荧光修饰的肌钙蛋白又使Ca2+亲和力再提高2倍。化学计量重构的细肌丝产生非协同性的MgATPase活性激活，这与天然细肌丝、肌钙蛋白交换后的细肌丝以及用化学计量过量肌钙蛋白重构的细肌丝产生的协同性激活形成对比。化学计量重构细肌丝的Ca2+诱导荧光转变是非协同性的。这些结果表明，即使在没有肌球蛋白的情况下，即使心脏肌钙蛋白C只有一个Ca2+特异性结合位点，Ca2+也会协同结合到心脏细肌丝的调节位点上。本文描述了这些观察结果的理论模型，并将其与实验数据相关联。相邻肌钙蛋白 - 原肌球蛋白复合物之间的已知相互作用是协同性的推测来源，并且也影响总体Ka。数据表明，Ca2+与已经结合Ca2+的肌钙蛋白 - 原肌球蛋白单元序列结合并使其延长的可能性是与没有Ca2+的单元序列内部位点结合的4倍。
