Further Characterisation of Fast, Slow and Cardiac Muscle Tropomyosins from Salmonid Fish

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 α‐TMs. Thus, none of the three salmonid TM sequences corresponds to a β‐type TM. In calorimetry experiments (0.1 M salt, pH 7.00, t = 10–60°C), in the presence of dithiothreitol, differences were observed in the thermal unfolding profiles of the purified isoforms. A single endotherm [ t m = 39.5°C) was noted for cardiac TM. Two endotherms were observed for fast TM [ t m = 26.5°C and 39.8°C (main)] and slow TM [ t m = 37.4°C and 46.9°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°C) the full‐length rc TM exhibited markedly lower relative viscosity values than the corresponding wild type.