FURTHER CHARACTERIZATION 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 seq uence of tropomyosin (TM) that is specific to these muscles was determ ined from full-length transcripts isolated from the corresponding libr ary. 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., McGowa n, C., Davidson, W. S. & Beavis, R. C. (1995) Characterisation of fast , slow and cardiac muscle tropomyosins from salmonid fish, Eur J. Bioc hem. 232, 226-234], the main difference in the N- and C-terminal seque nces comprising the site of end-to-end overlap occurs at residue 276 w here an asparagine in fast TM is replaced by a histidine in both cardi ac and slow TM. Trout cardiac TM exhibited greatest similarity to chic ken cardiac TM while trout slow TM exhibited greatest similarity to sk eletal alpha-TMs. Thus, none of the three salmonid TM sequences corres ponds 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, differenc es were observed in the thermal unfolding profiles of the purified iso forms. A single endotherm (t(m) = 39.5 degrees C) was noted for cardia c TM. Two endotherms were observed for fast TM [t(m) = 26.5 degrees C and 39.8 degrees C (main)] and slow TM [t(m) = 37.4 degrees C and 46.9 degrees C (main)]. Fast TM was cloned and over expressed in the bacte rial cell lines JM105 and BL21. Upon cell lysis, recombinant TM (re TM ) made in JM105 was rapidly and quantitatively cleaved between residue s 6 and 7. Intact re TM was produced by using BL21, as shown by Edman- based sequencing, carboxypeptidase digestion and mass analysis. In vis cometry assays, performed at low ionic strength (pH 7.00, t = 5 degree s C) the full-length re TM exhibited markedly lower relative viscosity values than the corresponding wild type.