Maytum R, Lehrer S S, Geeves M A
Max Planck Institut für Moleculare Physiologie, Dortmund, Germany.
Biochemistry. 1999 Jan 19;38(3):1102-10. doi: 10.1021/bi981603e.
Thin filament regulation is mediated by the presence of tropomyosin (Tm) and troponin (Tn) on the actin filament. Binding of Tm alone induces two states, closed and open (with the equilibrium between them defined by KT), which differ in their affinity for myosin subfragment 1 (S1). Cooperative switching between the states results in characteristic sigmoidal myosin S1 binding curves. In the presence of Tn and absence of Ca2+, a third state, blocked, has previously been kinetically shown to be present, leading to the three state model of McKillop and Geeves [(1993) Biophys. J. 65, 693-701]. We have measured equilibrium binding of S1 to phalloidin-stabilized pyrene-actin filaments by monitoring the pyrene fluorescence at 50 nM, a concentration 10-fold lower than previously possible. In combination with kinetic studies, we show that the data can be fitted to a modified version of the three-state model with an additional term allowing for a varying apparent cooperative unit size (n). Our results show that the apparent cooperative unit size (n) is dependent upon both the presence of Tn and of Ca2+. Also in the absence of Ca2+, the occupancy of the blocked state (defined by KB) is accompanied by a 2-3-fold reduction in KT. These results are discussed in comparison to the Hill model [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186-3190] and a flexible model of thin filament regulation based upon that of Lehrer et al. [(1997) Biochemistry 36, 13449-13455].
细肌丝调节由肌动蛋白丝上原肌球蛋白（Tm）和肌钙蛋白（Tn）的存在介导。单独结合Tm会诱导两种状态，即关闭状态和开放状态（它们之间的平衡由KT定义），这两种状态对肌球蛋白亚片段1（S1）的亲和力不同。状态之间的协同转换会产生特征性的S形肌球蛋白S1结合曲线。在有Tn且无Ca2+的情况下，先前已通过动力学证明存在第三种状态，即阻断状态，从而产生了McKillop和Geeves的三态模型[（1993年）《生物物理学杂志》65卷，693 - 701页]。我们通过监测50 nM浓度下芘的荧光来测量S1与鬼笔环肽稳定的芘标记肌动蛋白丝的平衡结合，该浓度比之前可能达到的浓度低10倍。结合动力学研究，我们表明数据可以拟合到三态模型的一个修改版本，该版本有一个额外的项允许表观协同单位大小（n）变化。我们的结果表明，表观协同单位大小（n）既取决于Tn的存在，也取决于Ca2+的存在。同样在无Ca2+的情况下，阻断状态的占有率（由KB定义）伴随着KT降低2 - 3倍。将这些结果与希尔模型[（1980年）《美国国家科学院院刊》77卷，3186 - 3190页]以及基于Lehrer等人[（1997年）《生物化学》36卷，13449 - 13455页]的细肌丝调节灵活模型进行了比较讨论。
