Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.     

In vitro phosphorylation of tropomyosin by a kinase from chicken embryo

A tropomyosin kinase has been partially purified from the leg muscle of 11-day-old chick embryos by ammonium sulfate precipitation and DEAE and phosphocellulose chromatography. The tropomyosin kinase requires Mg2+ for its activity, but Ca2+ and cyclic AMP are not needed. Increase in KC1 concentration decreased the tropomyosin kinase activity with over 90% inhibition at 0.2 M KC1. The alpha-tropomyosin subunit from rabbit and chicken skeletal muscle was phosphorylated about five times faster than the beta-tropomyosin subunit. Smooth muscle tropomyosin from chicken gizzard was not phosphorylated. The in vitro phosphorylation site in rabbit and chicken skeletal tropomyosins is a single serine residue close to the COOH terminus, a region intimately engaged in the head to tail polymerization of tropomyosin. Since the amino acid sequences of rabbit alpha- and beta-tropomyosin and chicken alpha-tropomyosin in this region are known, their phosphorylation sites can be unambiguously assigned as the penultimate residue, serine 283.     

Tropomyosin isoforms in developing chicken gizzard smooth muscle

In the embryonic smooth muscle of chicken gizzards we found 4 high-Mr-type and 5 low-Mr-type tropomyosin isoforms in addition to alpha- and beta-isoforms reported already. The criteria by which they were identified as tropomyosin isoforms were as follows: 1) anomalous reduction of electrophoretic mobility in the presence of urea, 2) cross reactivity with antisera against tropomyosins, 3) inclusion in a tropomyosin preparation obtained by the usual method for tropomyosin purification, and 4) binding ability to skeletal muscle actin. At the early stages of development, the amounts of these isoforms were larger than those of alpha- and beta-isoforms, but they gradually decreased at later stages and finally disappeared completely after hatching. Our previous study of gizzard smooth muscle showed that the amount ratio of accumulated tropomyosin to gamma-actin was reasonably constant in the development after hatching, while, at the earlier embryonic stages (7-14 d of incubation), it was lower than expected. The isoforms found in this study were present in amounts large enough to bring the ratio at the earlier stages up to the constant amount ratio observed after hatching. Therefore, the coordinate accumulation of actin and tropomyosin was suggested to occur even at the embryonic stages.     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

Altered actin and troponin binding of amino-terminal variants of chicken striated muscle alpha-tropomyosin expressed in Escherichia coli

We have expressed two variants of chicken striated muscle alpha-tropomyosin in Escherichia coli: fusion tropomyosin containing 80 amino acids of a non-structural influenza virus protein (NS1) on the amino terminus and a non-fusion tropomyosin which is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). From our analysis of purified proteins in vitro we suggest that the amino-terminal region, which is highly conserved in muscle tropomyosins, is crucial for all aspects of tropomyosin function. Both forms are altered in tropomyosin activity: neither shows head-to-tail polymerization, with or without troponin. Unacetylated tropomyosin binds weakly to actin, but in the presence of troponin it binds well and can regulate the actomyosin ATPase. Fusion tropomyosin binds well to actin, but binding of troponin is calcium-sensitive and it does not confer effective calcium sensitivity on the actomyosin ATPase. Our results indicate that the local charge at the amino terminus is critical for actin binding but that normal head-to-tail association is not required. The properties of fusion tropomyosin-troponin interaction are indicative of impaired troponin T binding to tropomyosin and provide evidence for its binding to the amino terminus of tropomyosin.     

Differential control of tropomyosin mRNA levels during myogenesis suggests the existence of an isoform competition-autoregulatory compensation control mechanism.

We have isolated tropomyosin cDNAs from human skeletal muscle and nonmuscle cDNA libraries and constructed gene-specific DNA probes for each of the four functional tropomyosin genes. These DNA probes were used to define the regulation of the corresponding mRNAs during the process of myogenesis. Tropomyosin regulation was compared with that of beta- and gamma-actin. No two striated muscle-specific tropomyosin mRNAs are coordinately accumulated during myogenesis nor in adult striated muscles. Similarly, no two nonmuscle tropomyosins are coordinately repressed during myogenesis. However, mRNAs encoding the 248 amino acid nonmuscle tropomyosins and beta- and gamma-actin are more persistent in adult skeletal muscle than those encoding the 284 amino acid nonmuscle tropomyosins. In particular, the nonmuscle tropomyosin Tm4 is expressed at similar levels in adult rat nonmuscle and striated muscle tissues. We conclude that each tropomyosin mRNA has its own unique determinants of accumulation and that the 248 amino acid nonmuscle tropomyosins may have a role in the architecture of the adult myofiber. The variable regulation of nonmuscle isoforms during myogenesis suggests that the different isoforms compete for inclusion into cellular structures and that compensating autoregulation of mRNA levels bring gene expression into alignment with the competitiveness of each individual gene product. Such an isoform competition-autoregulatory compensation mechanism would readily explain the unique regulation of each gene.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins.

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.     

Caldesmon-binding sites on tropomyosin

The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.     

Phosphorylation and dephosphorylation of a light chain of the chicken gizzard myosin molecule.

Chicken gizzard myosin was incubated with ATP and/or "native" tropomyosin (NTM) of gizzard muscle in the presence or absence of calcium ions. One of the two light chains of the myosin molecule was phosphorylated in the presence of Ca, but not in its absence. The phosphorylated gizzard myosin was dephosphorylated by a crude preparation of myosin light-chain phosphatase obtained from gizzard muscle. In a superprecipitation test in the presence of EGTA, actomyosin reconstituted from dephosphorylated gizzard myosin did not superprecipitate, whereas actomyosin reconstituted from phosphorylated gizzard myosin showed superprecipitation activity which was inhibited by skeletal NTM and reactivated by Ca.     

Effect of muscle and non-muscle tropomyosins in reconstituted skeletal muscle actomyosin

Smooth and non-muscle tropomyosins were found to produce a 2-3-fold Ca-insensitive stimulation of the ATPase activity of reconstituted skeletal muscles actomyosin at normal MgATP concentrations and physiological ratios of myosin to actin. Under the same conditions skeletal muscles tropomyosin had no effect. Similar effects of these three tropomyosins were observed for the low myosin/F-actin ratios necessary for kinetic measurements. Since it could be established that this actomyosin system, with or without tropomyosin, obeyed Michaelian kinetics, the tropomyosin effects could be interpreted in terms of their influence on maximal turnover (V) or on the affinity of myosin for actin (Kapp). Accordingly, gizzard tropomyosin had practically no effect on the affinity and reduced only slightly the value of V, compared to pure actin. In contrast to gizzard tropomyosin, brain tropomyosin produced an approximately twofold increase in both Kapp and V; i.e. it increased the turnover rate but decreased the affinity. It is apparent from the data that brain tropomyosin acts as an uncompetitive activator with respect to pure actin, while having the same V as the actin plus gizzard tropomyosin complex. Further studies on these tropomyosins show that only skeletal and smooth muscle tropomyosin have similar functional properties with respect to troponin inhibition and the activation of the ATPase at low ATP concentrations. It is suggested that the noted increases in V by tropomyosin are caused by the acceleration of the dissociation of the myosin head from actin at the end point of the cross bridge movement.