Structure function of actin and tropomyosin isoforms and discovery of a unique actin isoform

1. Tropomyosin [TM] was isolated from following sources; shark fast skeletal muscle, which contains a single, partially phosphorylated, alpha type isoform, and salmonid fast, slow and cardiac muscle. At 50mM ionic strength [pH 7.0, room temperature], thin filaments composed of shark phosphorylated TM produce a higher steady-state activation of the myosin-S1MgATPase compared to those containing the unphosphorylated protein. This difference is attributable to the extra negative charge associated with phosphoserine 283 which, under certain conditions [neutral pH, low ionic strength], enhances the interaction of adjoining TM molecule. By comparison, none of the salmonid TMs are unphosphorylated. Of the substitutions, which are distributed throughout the molecule, one occurs within the overlap region [Asn276 in fast, His in slow and cardiac]. This correlates with observed weakening of the end-to-end association of the C-terminal histidine-containing isoforms [cardiac and slow] relative to one present in fast muscle (1). Surprisingly, however, thin filament activation of myosinS1MgATPase increases in the order: fast TM < slow TM < cardiac TM. Since thin filaments composed of cardiac TM generate the greatest amount of activation it is apparent that tightness of the joint site is not the sole determinant for regulation. Thin filament based regulation is also sensitive to substitutions within the internal region of tropomyosin. The results of myosin binding show very little difference in the affinity of myosin for thin filaments containing non-identical isoforms of tropomyosin. Therefore these data suggest that the observed changes in steady state rate are due to a change in a kinetic step in the actomyosin ATPase cycle. -- 2. Unlike other vertebrates, salmonids synthesize a unique isoform of actin in each type of striated muscle [fast, slow and cardiac]. Two are virtually identical to each other but one [slow actin] is divergent and is inferred to contain six or seven non-conservative substitutions depending on the pairing [out of a total of twelve] when compared to the other salmonid actins and rabbit actin. Four of these replacements are predicted to occur in sub-domain 3. The other two or three are in sub-domain 1. Of particular interest is the substitution at residue 360, where a neutrally charged amino acid in a variety of isoactins is replaced by Asp in slow actin. This is consistent with the observation that slow actin migrates ahead of other isoactins when analysed by alkaline urea PAGE. Of all actins tested, the one from slow muscle is the least conformationally stable G-actin [calcium form + ATP], as gauged by a number of techniques including electronic circular dichroism. The midpoint of the change in far-UV ellipticity versus temperature for this isoform is 10°C lower than that of other actins studied [transition temperature, 45°C versus 55°C]. The slow isoform also displays a reduced rate of polymerisation, a faster rate of nucleotide dissociation and a lower level of myosin activation. These data suggest that actin heterogeneity is a source of thin filament diversity in some vertebrate skeletal muscle. -- 3. A survey was conducted to determine the distribution of slow muscle actin throughout the animal kingdom. Skeletal muscle actin extracted from certain species, selected on the basis of their taxonomical position, was characterized. Of the species surveyed, a unique isoform was identified in slow muscle of Chondrichthyes [mako shark] and Osteichthyes [teleosts, salmon, Atlantic herring and tuna] but not amphibians [frog] and avians [chicken]. In the latter two cases, the same isoform is present in both types of skeletal muscles. Further, a slow muscle actin gene is absent from the genomes of puffer fish and human. This suggests that a slow muscle actin gene starts to express randomly and then stops expressing after short period of time. This appearance and disappearance is a unique feature, which has occurred during the course of evolution in other genes as well. According to phylogenetic tree analyses performed using several methods [neighbour-joining, minimum evolution and MrBayes method] for selected amino acid and nucleotide sequences, a close relationship between salmon slow muscle actin and puffer fish cardiac actin which is considered one of the oldest actins, was observed. Finally, an actin clone was isolated from a dogfish shark skeletal muscle cDNA library and sequenced. The nucleotide sequence clusters with that chicken actin, which again underlines the extraordinary degree to which the protein has been conserved