Moyes C D, Borgford T, LeBlanc L, Tibbits G F
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Biochemistry. 1996 Sep 10;35(36):11756-62. doi: 10.1021/bi9607057.
Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.
心肌肌动球蛋白ATP酶的激活需要占据肌钙蛋白C（cTnC）的单个低亲和力Ca(2+)结合位点。此前，我们已证明哺乳动物和冷水鲑科鱼类的心脏制剂在Ca2+敏感性方面存在显著差异，尤其是在与温度的关系方面[Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62 - R70]。在本研究中，我们探究了cTnC结构在多大程度上能够解释观察到的肌原纤维Ca2+敏感性差异。克隆、测序了鲑科鱼类（虹鳟）的cTnC，并将其作为麦芽糖结合蛋白融合体在大肠杆菌中表达。编码区与人类cTnC cDNA具有87%的同源性，与人类/牛/猪同工型的161个氨基酸残基中的13个不同。对应于单个调节性Ca(2+)结合位点II的序列与哺乳动物的完全同源。所表达的蛋白质表现出与从鲑科鱼类（大西洋鲑）和牛心室中纯化的cTnC相似的光学性质（圆二色性、内在荧光）。在无活性的Ca(2+)结合位点I中引入了一个色氨酸残基（ScTnC - FW27）以促进Ca2+滴定。Ca(2+)结合常数（K1/2 = 5.33 pCa单位）在报道的哺乳动物cTnC低亲和力位点的范围内。尽管TnC一级结构存在显著差异，但完整心肌肌原纤维的Ca2+亲和力可能受与其他肌钙蛋白相互作用的影响。
