Heeley D H, Bieger T, Waddleton D M, Hong C, Jackman D M, McGowan C, Davidson W S, Beavis R C
Department of Biochemistry, Memorial University of Newfoundland, St. John's, Canada.
Eur J Biochem. 1995 Aug 15;232(1):226-34. doi: 10.1111/j.1432-1033.1995.tb20803.x.
Tropomyosin (TM) has been isolated from the cardiac muscle, and fast and slow trunk (myotomal) muscles of the mature salmonid fish Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri). When examined electrophoretically, isoforms of TM were detected which were specific, and exclusive, to each type of muscle. Cardiac and fast muscles contained single and distinct isoforms, while slow muscle contained two distinct isoforms, closely related in terms of apparent M(r), and pI. There was no detectable difference between the same TM type from either salmon or trout. On a variety of gel systems, the cardiac and slow isoforms migrated in close proximity to each other and to rabbit alpha-TM. The fast isoform comigrated with rabbit beta-TM. In developing salmon fry, a more acidic (unphosphorylated) variant of TM was present in addition to, and of similar M(r) to, the fast adult isoform. This TM declined in steady-state level during maturation and was virtually undetected in adult muscle. All of the isolated TMs contained little or no covalently bound phosphate and were blocked at the N-terminus. The amino acids released by carboxypeptidase A, when ordered to give maximal similarity to other muscle TMs, were consistent with the following sequences: fast (LDNALNDMTSI) and cardiac (LDHALNDMTSL). The C-terminal region of the slow TM contained His but was heterogeneous. In viscosity measurements, performed as a function of increasing protein concentration, at low ionic strength (t = 5 degrees C, pH 7.00), fast TM exhibited the highest relative viscosity values. Lower and equivalent levels of polymerisation occurred with the cardiac and slow TMs. Polymerisation of all three isoforms was temperature-dependent, with cardiac TM being least sensitive and fast TM being most sensitive. Determination of the complete coding sequence of adult fast TM confirmed the findings of the carboxypeptidase analysis, but the remainder of the sequence more closely resembled alpha-type TMs than beta-type TMs. Overall, salmon fast TM contains 20 (mostly conservative) substitutions compared to rabbit striated muscle alpha-TM and 40 (mostly conservative) substitutions compared to rabbit striated muscle beta-TM. This demonstrates that electrophoretic mobility is not, in all instances, a suitable method to assess the isomorphic nature of striated muscle TMs.
原肌球蛋白（TM）已从成熟鲑科鱼类大西洋鲑（Salmo salar）和虹鳟（Salmo gairdneri）的心肌以及快速和慢速躯干（肌节）肌肉中分离出来。通过电泳检测时，发现TM的同工型对每种肌肉类型具有特异性且是唯一的。心肌和快速肌肉含有单一且独特的同工型，而慢速肌肉含有两种不同的同工型，就表观相对分子质量（M(r)）和等电点（pI）而言密切相关。来自鲑鱼或鳟鱼的相同TM类型之间没有可检测到的差异。在各种凝胶系统中，心肌和慢速同工型彼此靠近迁移，并且与兔α-TM靠近迁移。快速同工型与兔β-TM共迁移。在发育中的鲑鱼苗中，除了快速成年同工型外，还存在一种酸性更强（未磷酸化）的TM变体，其M(r)与快速成年同工型相似。这种TM在成熟过程中稳态水平下降，在成年肌肉中几乎检测不到。所有分离的TM含很少或不含共价结合的磷酸盐，并且在N端被封闭。当按顺序排列以与其他肌肉TM具有最大相似性时，羧肽酶A释放的氨基酸与以下序列一致：快速型（LDNALNDMTSI）和心肌型（LDHALNDMTSL）。慢速TM的C端区域含有组氨酸，但具有异质性。在作为蛋白质浓度增加的函数进行的粘度测量中，在低离子强度（t = 5℃，pH 7.00）下，快速TM表现出最高的相对粘度值。心肌TM和慢速TM发生较低且相当水平的聚合。所有三种同工型的聚合都与温度有关，心肌TM最不敏感，快速TM最敏感。成年快速TM完整编码序列的测定证实了羧肽酶分析的结果，但该序列的其余部分与α型TM比与β型TM更相似。总体而言，与兔横纹肌α-TM相比，鲑鱼快速TM含有20个（大多为保守性）取代，与兔横纹肌β-TM相比含有40个（大多为保守性）取代。这表明在所有情况下，电泳迁移率并非评估横纹肌TM同构性质的合适方法。
