Francois J M, Sheng Z, Szczesna D, Potter J D
Department of Molecular & Cellular Pharmacology, University of Miami School of Medicine, Florida 33101, USA.
J Biol Chem. 1995 Aug 18;270(33):19287-93. doi: 10.1074/jbc.270.33.19287.
Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1-100) sites I and II; and TH4 (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH1 was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH3 and TH4) or 20% (TH2) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH2, TH3, and TH4 compared to RSTnC OR TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH1 was able to interact with the fibers in a Ca(2+)-independent (Mg(2+)-dependent) manner, indicating that at a minimum, Ca(2+)-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca2+. TH2 and TH4 appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH2 and TH4 was abolished when Ca2+ was removed. TH2 and TH4 binding to the second site required Ca2+ initially but was maintained in the presence of Mg2+. This interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III and IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca(2+)-Mg2+ sites(III and IV) of TnC.
用凝血酶对兔快肌肌钙蛋白C（RSTnC）进行蛋白水解产生四个独立片段，这些片段含有以下钙结合位点：TH1（第1-120位氨基酸残基）包含位点I-III；TH2（第121-159位氨基酸残基）包含位点IV；TH3（第1-100位氨基酸残基）包含位点I和II；以及TH4（第101-120位氨基酸残基）包含位点III。我们研究了这些片段恢复TnC缺失的去皮骨骼肌纤维中稳态等长力的能力。有趣的是，我们发现RSTnC的所有研究片段都具有天然RSTnC的一些特性，但它们中没有一个能像完整的RSTnC那样完全调节纤维中的收缩。TH1在恢复力方面最有效（65%），而较小的片段产生的力约为未提取纤维初始力的50%（TH3和TH4）或20%（TH2）。此外，与RSTnC或TH1相比，TH2、TH3和TH4需要更高的浓度才能产生力，这表明这些片段与纤维中其结合位点的亲和力降低。与完整的RSTnC一样，TH1能够以不依赖钙（依赖镁）的方式与纤维相互作用，这表明至少，这种类型的结合需要钙结合位点III。其他片段与TnC缺失纤维的初始结合仅在有钙离子存在时发生。TH2和TH4似乎与纤维中的两个不同结合位点结合。与其中一个位点的结合导致部分力的恢复。当去除钙离子时，TH2和TH4与该位点的结合被消除。TH2和TH4与第二个位点的结合最初需要钙离子，但在有镁离子存在时得以维持。TH2和TH4的这种相互作用部分阻止了RSTnC与纤维的重新结合。后一结果表明，这些小片段中的位点III和IV，当从母体蛋白的限制中脱离时，可能呈现出一定程度上类似于TnC的调节位点（I和II）以及钙-镁位点（III和IV）的构象，从而发挥功能。
