Jackman D M, Waddleton D M, Younghusband B, Heeley D H
Department of Biochemistry, Memorial University of Newfoundland, St John's, Canada.
Eur J Biochem. 1996 Dec 1;242(2):363-71. doi: 10.1111/j.1432-1033.1996.0363r.x.
Separate cDNA libraries were constructed from cardiac muscle and slow myotomal muscle of mature brown trout (Salmo trutta). The complete sequence of tropomyosin (TM) that is specific to these muscles was determined from full-length transcripts isolated from the corresponding library. The identity of the sequences was supported by protein data. When compared to the sequence of Atlantic salmon fast myotomal TM [Heeley, D. H., Bieger, T., Waddleton, D. M., Hong, C., Jackman, D. M., McGowan, C., Davidson, W. S. & Beavis, R. C. (1995) Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish, Eur. J. Biochem. 232, 226-234], the main difference in the N- and C-terminal sequences comprising the site of end-to-end overlap occurs at residue 276 where an asparagine in fast TM is replaced by a histidine in both cardiac and slow TM. Trout cardiac TM exhibited greatest similarity to chicken cardiac TM while trout slow TM exhibited greatest similarity to skeletal alpha-TMs. Thus, none of the three salmonid TM sequences corresponds to a beta-type TM. In calorimetry experiments (0.1 M salt, pH 7.00, t = 10-60 degrees C), in the presence of dithiothreitol, differences were observed in the thermal unfolding profiles of the purified isoforms. A single endotherm (tm = 39.5 degrees C) was noted for cardiac TM. Two endotherms were observed for fast TM [tm = 26.5 degrees C and 39.8 degrees C (main)] and slow TM [tm = 37.4 degrees C and 46.9 degrees C (main)]. Fast TM was cloned and over expressed in the bacterial cell lines JM105 and BL21. Upon cell lysis, recombinant TM (rc TM) made in JM105 was rapidly and quantitatively cleaved between residues 6 and 7. Intact rc TM was produced by using BL21, as shown by Edman-based sequencing, carboxypeptidase digestion and mass analysis. In viscometry assays, performed at low ionic strength (pH 7.00, t = 5 degrees C) the full-length rc TM exhibited markedly lower relative viscosity values than the corresponding wild type.
从成熟褐鳟（Salmo trutta）的心肌和慢肌节肌构建了单独的cDNA文库。从相应文库中分离出的全长转录本确定了这些肌肉特有的原肌球蛋白（TM）的完整序列。序列的一致性得到了蛋白质数据的支持。与大西洋鲑快肌节TM的序列[Heeley, D. H., Bieger, T., Waddleton, D. M., Hong, C., Jackman, D. M., McGowan, C., Davidson, W. S. & Beavis, R. C. (1995) 鲑科鱼类快、慢和心肌原肌球蛋白的特性研究，欧洲生物化学杂志232, 226 - 234]相比，构成端对端重叠位点的N端和C端序列的主要差异出现在第276位残基处，快肌TM中的天冬酰胺在心肌和慢肌TM中均被组氨酸取代。鳟鱼心肌TM与鸡心肌TM表现出最大的相似性，而鳟鱼慢肌TM与骨骼肌α-TM表现出最大的相似性。因此，三种鲑科TM序列均不对应于β型TM。在量热实验（0.1 M盐，pH 7.00，t = 10 - 60℃）中，在二硫苏糖醇存在下，观察到纯化的同工型的热解折叠曲线存在差异。心肌TM观察到一个单一的吸热峰（tm = 39.5℃）。快肌TM观察到两个吸热峰[tm = 26.5℃和39.8℃（主要）]，慢肌TM观察到两个吸热峰[tm = 37.4℃和46.9℃（主要）]。快肌TM在细菌细胞系JM105和BL21中进行克隆和过量表达。细胞裂解后，JM105中产生的重组TM（rc TM）在第6和第7位残基之间迅速且定量地被切割。如基于埃德曼测序、羧肽酶消化和质谱分析所示，使用BL21产生了完整的rc TM。在低离子强度（pH 7.00，t = 5℃）下进行的粘度测定中，全长rc TM的相对粘度值明显低于相应的野生型。
