Maytum Robin, Bathe Friederike, Konrad Manfred, Geeves Michael A
University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom.
J Biol Chem. 2004 Apr 30;279(18):18203-9. doi: 10.1074/jbc.M311636200. Epub 2004 Jan 28.
The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT(1)-tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 microm) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin.Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin.Tm.Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin.Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.
利用重组成纤维细胞肌钙蛋白（Tm）同工型5a和5b，研究了原肌球蛋白（Tm）外显子6b与肌钙蛋白（Tn）相互作用及功能的特异性。这些同工型在内部因外显子6a/6b而不同，并且在末端具有非肌肉外显子1b/9d，因此它们缺乏主要的肌钙蛋白T（1）-原肌球蛋白相互作用，从而能够独立于这一相互作用研究外显子6的交换。我们使用动力学技术测量肌球蛋白S1与肌动蛋白结合的调节，并使用荧光标记的Tm直接测量Tn结合，结果表明Tn与两种同工型的结合相似（0.1 - 0.5微米），并且两者都产生调节良好的系统。钙对Tn与肌动蛋白-Tm复合物的结合影响很小，并且在不存在钙的情况下，两个外显子都会使S1与肌动蛋白-Tm-Tn的结合速率降低3倍。这证实了先前的结果，即外显子6对Tn与肌动蛋白-Tm的亲和力或其完全抑制肌动蛋白-肌球蛋白相互作用的能力影响很小。用Tn和Tm5a或骨骼肌Tm（含有外显子6b）重构的细肌丝显示出几乎相同的肌动蛋白-肌球蛋白调节钙依赖性。然而，Tm5b使钙敏感性显著增加，使激活中点几乎移动了一个数量级。这表明外显子6序列以及该区域的Tm结构对Tn的钙调节有显著影响。这一发现支持了这样的证据，即发生在该区域附近的家族性肥厚性心肌病突变会影响钙调节。
