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

Abstract

Tropomyosin (TM) has been isolated from the cardiac muscle, and fast and slow trunk (myotomal) muscles of the mature salmonid fish Atlantic salmon (SuZmo suZur) and rainbow trout (Sulmo guirdneri). 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, 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 a-TM. The fast isoform comigrated with rabbit /3-TM. In developing salmon fry, a more acidic (unphosphorylated) variant of TM was present in addition to, and of similar M , 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"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 a-type TMs than /3-type TMs. Overall, salmon fast TM contains 20 (mostly conservative) substitutions compared to rabbit striated muscle a-TM and 40 (mostly conservative) substitutions compared to rabbit striated muscle /3-TM. This demonstrates that electrophoretic mobility is not, in all instances, a suitable method to assess the isomorphic nature of striated muscle TMs.

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

“…Tropomyosins were prepared from adult fish as described in Heeley et al (1995). Atlantic salmon (Salmo salar), Arctic char (Salvelinus alpinus) and rainbow trout, (Salmo gairdneri) were used as sources of fast myotomal muscle.…”

“…Previous work (Heeley and Hong, 1994;Heeley et al, 1995) has demonstrated that the major striated muscles of mature Atlantic salmon, and species of trout, possess a simple content of tropomyosin (TM), both in terms of the number of major isoforms which are present and their tissue distribution. The specific details arising from these studies were that heart and fast contracting myotomal (trunk) muscle each contain a single non-identical isoform while slow contracting myotomal muscle contains two, closely related, isoforms which are distinct from the TMs found in the other two types of muscle (Heeley and Hong, 1994;Heeley et al, 1995).…”

“…The specific details arising from these studies were that heart and fast contracting myotomal (trunk) muscle each contain a single non-identical isoform while slow contracting myotomal muscle contains two, closely related, isoforms which are distinct from the TMs found in the other two types of muscle (Heeley and Hong, 1994;Heeley et al, 1995). Furthermore, the myotomal muscles are anatomically segregated in the trunk (Bone and Marshall, 1982) and can be easily dissected.…”

“…The complete coding sequences of cardiac TM and slow TM from brown trout are reported and are compared to the previously published sequence of Atlantic salmon fast TM (Heeley et al, 1995). The inferred primary structures provide a basis for classifying the isoforms and interpreting differences in polymerisation (Heeley et al, 1995). In view of the fact that salmonids are cold water fish, preliminary thermal melting experiments have been performed to investigate the structural stability of the isolated isoforms.…”

Patterns of tropomyosin and troponin-T isoform expression in jaw-closing muscles of mammals and reptiles that express masticatory myosin

“…Additional isoforms of either Tm or TnT may be revealed with two-dimensional gel electrophoresis (e.g. Heeley et al, 1995). Undetected species-related differences in post-translational modifications (e.g.…”