Heterogeneity of Atlantic salmon troponin-I.

Three non-identical, full length troponin-I (Tn-I) clones were isolated from an Atlantic salmon myotomal (trunk) muscle cDNA library. The primary structures, which are predicted to range from 172 to 180 amino acids in length, exhibit similar percent identity scores when compared with fast, slow and cardiac specific Tn-Is from higher vertebrates. When the sequence data are considered along with the results of Western blotting it is evident that Tn-I is more heterogeneous in Atlantic salmon than has been previously shown in higher vertebrates.

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 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.

Characterisation of troponin-T from salmonid fish.

Five major troponin-T isoforms were isolated from the myotomal muscles of Atlantic salmon: three from fast muscle (Tn-T1F, Tn-T2F and Tn-T3F) and two from slow muscle (Tn-T1S and Tn-T2S). In addition to their presence in troponin preparations, these proteins were also recognised to be Tn-T on the basis of immunoreaction with anti-troponin-T antibodies and partial amino acid sequence. The electrophoretic mobility in the presence of SDS of the various Tn-Ts increases in the order: 1S < 1F < 2S < 2F < or = 3F. Compositional analysis shows that the higher M(r) forms (1F and 1S) contain considerably more proline, glutamic acid and alanine than the lower-M(r) forms (2F, 3F and 2S). Every isoform lacks cysteine and phosphoserine is present only in isoforms 2F and 3F. All of the Tn-Ts, with the exception of isoform 1F, are N-terminally blocked. CNBr fragments from same cell type Tn-Ts yield identical sequences over at least fifteen Edman cycles. Two full-length cDNA sequences, presumed to represent 1S and 3F, or isoforms that are highly similar, are reported. As documented for higher vertebrate Tn-Ts, the predicted primary structures display a non-uniform distribution of charged amino acids and greater divergence at each end than in the central section. The most striking difference between the two salmonid proteins is the presence of a N-terminal (proline-, glutamic acid- and alanine-rich) extension of about fifty amino acids in Tn-T1s (278 amino acids) that is missing from the fast muscle Tn-T (223 amino acids). The sequences also differ in that 1S lacks the known phosphorylation site while the fast-type isoform contains serine next to the initiating methionine. Of the two, the slow isoform has accumulated the greater number of substitutions.

Characterisation of fast, slow and cardiac muscle tropomyosins from salmonid fish.

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.