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At least 19 recordsLinked to original sources
PubMed1978

Studies on the F-actin.tropomyosin.troponin complex. II. Partial reconstitution of thin filament by F-actin, tropomyosin and the tropomyosin binding component of troponin (TN-T).

It was found that thin filaments are reconstituted from F-actin, tropomyosin and the tropomyosin binding component of troponin (TN-T) in vitro according to a self-assembly mechanism. Although a gel structure is formed when the three proteins are mixed in the order F-actin, TN-T and tropomyosin, it is in a metastable state and spontaneously transforms to dispersed filaments in an equilibrium state. The rate of the transformation is largely dependent on temperature. When the mixing order of TN-T and tropomyosin is reversed, large amounts of the complex appear immediately in the dispersed filament form. Dependence on the order of mixing is observed only when the molar ratio of TN-T to tropomyosin is about one, i.e. at the physiological ratio. When the molar ratio of TN-T to tropomyosin is either above or below one, a stable gel form or a stable dispersed filament form, was respectively, obtained independently of the mixing order of the three proteins.

Actins↗
PubMed1975

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells.

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.

Actins↗
PubMed1976

Properties of non-polymerizable tropomyosin obtained by carboxypeptidase A digestion.

Tropomyosin digested with carboxypeptidase A [EC 3.4.12.2] (CTM) shows a lower viscosity than the undigested protein in solution. From the relation between the viscosity decrease and the amount of amino acids liberated from the carboxyl terminus during this digestion, it is inferred that loss of the tri-peptide-Thr-Ser-Ile from the C-terminus is responsible for the decrease in viscosity. The secondary structure of -TM was not affected by the digestion according to circular dichroic measurements. The viscosity of CTM did not increase in methanol-water mixtures, whereas that of tropomyosin increased markedly. These results indicate that polymerizability was lost upon the removal of a small peptide from the C-terminus without change in the secondary structure. A decrease in the viscosity of tropomyosin solutions was observed on the addition of CTM, indicating that CTM interacts with intact tropomyosin. The dependence of the viscosity decrease on the amount of CTM showed that CTM binds tropomyosin in a one-to-one ratio as a result of end-to-end interaction. Since paracrystals having a 400 A repeated band structure could be grown in the presence of Mg ions at neutral pH, side-by-side interactions in CTM molecules remain intact, even though polymerizability is lost. The disc gel electrophoretic pattern showed that troponin could bind to CTM, but no increase in viscosity due to the complex was observed in solution. That is, the C-terminal part of tropomyosin is not required for the formation of the complex. The amount of CTM bound to F-actin was less than half of that bound to undigested tropomyosin, and could be reduced to one-tenth by a washing procedure. In the presence of troponin, however, the amount recovered to the level of tropomyosin normally bound to F-actin. Therefore, it is concluded that troponin is bound in the middle of the tropomyosin molecule and strengthens the binding of tropomyosin to F-actin.

Actins↗
PubMed1975

Non-polymerizable tropomyosin and control of the superprecipitation of actomyosin.

Non-polymerizable tropomyosin was prepared by the digestion of several C-terminal residues of tropomyosin with carboxypeptidase A [EC 3.4.12.2]. The intrinsic viscosity and molecular weight of the non-polymerizable tropomyosin were almost the same as those of untreated tropomyosin. Like untreated tropomyosin, the non-polymerizable tropomyosin in combination with troponin repressed the superprecipitation of actomyosin in the absence of calcium, while this repression was released by addition of calcium. However, the curve representing the superprecipitation rate as a function of pCa was less steep than that found with actomyosin containing untreated tropomyosin: in the former case, the rate increased to a plateau over about 2 pCa units, while in the latter case, it did so over about 1 pCa unit. These experimental results provide evidence that the "co-operation" in the regulation mechanism of skeletal muscle contraction, which is indicated by the steep curve of the contraction versus pCa relation, is mediated by tropomyosin-tropomyosin interaction along the thin filament.

Actomyosin↗
PubMed1976

The conformational stabilities of tropomyosins.

The stability to denaturation by heat and guanidine hydrochloride of seven vertebrate (including skeletal, cardiac and smooth muscle) tropomyosins and three invertebrate tropomyosins was examined. The transition profiles were discontinuous and in many cases distinct plateaux were observed which indicated the presence of unique partially unfolded states at intermediate temperatures and guanidine hydrochloride concentrations. The denaturation by guanidine hydrochloride could be described in the majority of cases by a model in which the native state unfolds to a partially unfolded stable intermediate which then unfolds to the completely denatured state. On this basis it was possible to estimate the free energies of unfolding in water. It was shown that part of the alpha-helical structure of tropomyosin is only marginally stable and the free energy of unfolding in water of this segment is less than values found for globular proteins, whereas another segment (or segments) has a stability comparable to that found for globular proteins. The stepwise unfolding may be explained in terms of the coiled-coil interactions in tropomyosin. Differences in stability were found between tropomyosins from different muscles of the same species as well as between species, no two tropomyosins giving the same denaturation profiles. The invertebrate tropomyosins showed a wider range of stabilities, that from scallop striated muscle being far more easily denatured than all the others. No correlation was found between the stability of tropomyosin and the type of regulatory system of the muscle. A comparison of the results from vertebrate and invertebrate species suggests that there has been no selection for proteins of higher or lower stability during the evolutionary time scale.

Amino Acids↗
PubMed1978

Specific phosphorylation at serine-283 of alpha tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle.

Tropomyosin, extracted from the leg muscle of frogs that had been injected with [32P]orthophosphate, was fractionated into two components, alpha and beta, on a CM-cellulose column. Radioactivity was associated only with the alpha component. A single phosphorylation site was located at serine-283 (pentultimate at the COOH-terminal end) of the frog alpha tropomyosin. The same phosphorylated peptide was recovered in low yields from both rabbit skeletal alpha and cardiac tropomyosin. The presence of covalently bound phosphate in alpha tropomyosin and its absence in the beta component of rabbit skeletal muscle was suggested by 31P NMR spectroscopy. The amino acid sequences around the phosphorylation sites of frog and rabbit tropomyosin are identical. Because this sequence is not similar to any other known phosphorylation site in proteins, this indicates the existence of either specific kinase or phosphatase that can distinguish between alpha and beta tropomyosins. In a model proposed for the head-to-tail overlap of alpha tropomyosin molecules, one O-phosphoserine-283 residue could form a salt linkage with lysine-6 on one side of the overlap region and another with lysine-12 on the other side. This would predict a difference in the stability of polymers of phosphorylated and nonphosphorylated alphaalpha and alphabeta dimers of tropomyosin.

Amino Acid Sequence↗
PubMed1975

Regulation of muscle contraction: bindings of troponin and its components to actin and tropomyosin.

The bindings of troponin components to actin and tropomyosin has been studied by cosedimentation with actin and affinity chromatography. It is shown that troponin binds to actin and tropomyosin in the presence and absence of calcium but the binding to actin is sensitive to ionic strength. Troponin-I + C binds to actin-tropomyosin in the absence of calcium but not to actin or tropomyosin alone. Troponin-I binds to actin and the binding is improved in the presence of tropomyosin even though troponin-I does not bind to tropomyosin alone. Troponin-C does not bind to actin or tropomyosin. The results suggest that the binding of troponin by actin is influenced by tropomyosin. A model of regulation by troponin is proposed.

Actins↗
PubMed1977

Interaction between Acanthamoeba actin and rabbit skeletal muscle tropomyosin.

The binding of 125I-labeled muscle tropomyosin to Acanthamoeba and muscle actin was studied by ultracentrifugation and by the effect of tropomyosin on the actin-activated muscle heavy meromyosin ATPase activity. Binding of muscle tropomyosin to Acanthamoeba actin was much weaker than its binding to muscle actin. For example, at 5 mM MgCl2, 2 mM ATP, and 5 micronM actin, tropomyosin bound strongly to muscle actin but not detectably to Acanthamoeba actin. When the concentration of actin was raised from 5 micronM to 24 micronM in the presence of 80 mM KCl, the binding of tropomyosin to Acanthamoeba actin approached its binding to muscle actin. As with muscle actin, the addition of muscle heavy meromyosin in the absence of ATP induced binding of tropomyosin in Acanthamoeba actin under conditions were binding would otherwise not have occurred. The most striking difference between the interactions of muscle tropomyosin with the two actins, however, was that under conditions where tropomyosin was found to both actins, its stimulated the Acanthamoeba actin-activated heavy meromyosin ATPase but inhibited the muscle actin-activated heavy meromyosin ATPase.

Actins↗
PubMed1978

Existence of common antigenic sites in tropomyosins.

1. Tropomyosins were extracted from vertebrate and invertebrate muscles, and their immunolo;ical characteristics were compared using antisera against tropomyosins from chicken skeletal and cardiac muscles. 2. Antigenic sites common to those of chicken skeletal muscle tropomyosin were found in all the tropomyosins tested, although the reactions of these common antigenic sites in an immunodiffusion test were weak in tropomyosins from phylogenetically distant animals. 3. An immunological difference was found between alpha-tropomyosins from chicken cardiac muscle and rabbit cardiac muscle. Thus they had specific antigenic sites in addition to the common ones. 4. A component was found in a 1 M KCL extract of Tetrahymena pyriformis which reacted with antiserum against chicken skeletal muscle tropomyosin.

Animals↗
PubMed1976

Interaction of tropomyosin with troponin components.

1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1 M KC1 at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules. 2. The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgC1(2) solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-I at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0. 3. The TN-C component does not precipitate tropomyosin in 0.1 M KC1. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgC12.

Animals↗
PubMed1978

Immunological purification of sea urchin egg tropomyosin.

The antiserum against lantern muscle tropomyosin of the sea urchin was prepared, and the presence of tropomyosin in the sea urchin egg was shown by immunodiffusion test between the antiserum and the egg tropomyosin fraction which was prepared according to the purification method for muscle tropomyosin. The sea urchin egg tropomyosin was isolated from the immuno-precipitate formed between the antiserum and the egg tropomyosin fraction. The subunit molecular weight of the egg tropomyosin was calculated to be 29,000.

Animals↗
PubMed1979

Fluorometric studies on conformational changes in tropomyosin associated with depolymerization.

Spectrofluorometric studies on the conformational changes in tropomyosin associated with depolymerization of the molecules were carried out using 1-anilino-8-naphthalene sulfonate (ANS). When ANS-probed tropomyosin was depolymerized to its monomer, the fluorescence intensity markedly increased, with a decrease in fluorescence polarization. On the other hand, the emission maxima of the ANS-tropomyosin complexes of both forms were the same. The temperature dependence of the polarization of the complexes at various KCl concentrations suggested that the segmental motion of a moiety containing the fluorophore was considerably activated by depolymerization of tropomyosin. In the polymerized and oligomeric forms, a thermal transition in the polarization was observed with a transition temperature of 30 degrees C. Titration curves of tropomyosin with ANS showed simple saturation kinetics with both monomer and polymer, and the apparent dissociation constants were estimated to be 9.93 X 10(-5) M (monomer) and 7.43 X 10(-5) M (polymer). On the other hand, the number of the ANS-binding sites increased from 0.5 to 2.0 per tropomyosin monomer on depolymerization of the molecules. Based on these results, the conformational state of tropomyosin in the polymerized form is discussed.

Anilino Naphthalenesulfonates↗
PubMed1977

Polymerizability of rabbit skeletal tropomyosin: effects of enzymic and chemical modifications.

Polymerizability of tropomyosin was unaffected by the removal of the three terminal residues 282, 283, and 284 using carboxypeptidase A. However, when residue 281 was removed, polymerizability was abolished. These results are consistent with a 9-residue molecular head-to-tail overlap in polymerized tropomyosin, in which residue 281 plays a space-filling role at the center of the overlap core. In acetylation studies, loss of polymerizability closely paralleled the extent of acetylation of lysine-7, and this residue was more susceptible to acetylation than any other. The effect of acetylation on polymerizability was probably caused not only by cleavage of salt-bridge between lysine 7 epsilon-NH2 and residue 284 alpha-COOH but also by distortion of the overlap core by the N-acetyl group. Specific modification of methionine in tropomyosin indicated that, in addition to residue 281, methionine-8 is also involved in formation of the overlap core. Modified nonpolymerizable tropomyosins could still bind to F-actin, indicating that the head-to-tail polymerization of tropomyosin is not a prerequisite for actin binding, although the regularity of tropomyosin molecules along the actin helix is presumably disrupted.

Acetates↗
PubMed1975

Purification and characterization of cardiac tropomyosins.

A new procedure was developed to purify tropomyosin. The procedure was an adaptation of that described for purification of myosin. By eliminating troponin before precipitating with (NH4)2 SO4, it was possible to obtain pure tropomyosin from the same preparation from which myosin was purified. When tropomyosin was subjected to isoelectrofocusing two tropomyosins were present, having similar isoelectric points of pH 5.4 and 5.6; two tropomyosin subunits were resolved in the presence of 6 M urea. The two subunits had very similar isoelectric points, pH 4.7 and 5.0. According to Ouchterlony analyses the tropomyosins from canine skeletal and cardiac tissue were immunologically identical when incubated with goat gammaG antitropomyosin (cardiac).

Amino Acids↗
PubMed1978

Tropomyosin fragments obtained by tryptic digestion.

Rabbit skeletal alpha-tropomyosin, separated by hydroxyapatite chromatography, was treated with trypsin (1/100 wt/wt) at 0 degrees C for 24 h. Trypsin-resistant fragments of tropomyosin were separated into the precipitate and supernatant fractions at pH 4.3 in 1 M KCl, and these were subjected to QAE-Sephadex A50 column chromatography for further purification. SDS-gel electrophoresis showed 16,000 and 14,000 dalton bands for the supernatant (s-fragment) and an 11,500 dalton band for the precipitate (p-fragment). We obtained a 13,500 dalton chain (13,500 dalton fragment) in addition to the s- and p-fragments upon treatment with more dilute trypsin (1/500 wt/wt) for 48 h at 0 degrees C. Both the p- and 13,500 dalton fragment had the same C-terminal portion as intact alpha-tropomyosin, and could form an intra-chain disulfide bond on oxidation. Therefore, these two fragments were deduced to be polypeptides from some points on the N-terminal side of Cys 190 to the intact C-terminal. The s-fragment, on the other hand, did not contain any cysteine, Phe, or His residues according to amino acid analysis, suggesting that the fragment is derived from the N-terminal side from Cys 190. Tentative assignment of the fragments was carried out by amino acid analysis, and C- and N-terminal determination. The p-, s-, and 13,500 dalton fragments appear to be in coiled-coil form in solution, having alpha-helical contents of 77,71, and 64%, respectively, and are able to interact with intact tropomyosin to reduce the viscosity of tropomyosin solution. The s-, p-, and 13,500 dalton fragments have little binding capacity individually to troponin, but the mixture, i.e., the s- and p-fragments, the 13,500 dalton fragment and the N-chain, which was obtained by cleavage at Cys 190, showed clear binding with troponin independent of Ca2+ in solution as detected by gel electrophoresis. The p-fragment showed some binding to troponin, since cross-linkage to troponin was possible by treatment with dimethyl suberimidate. From the result, it can be inferred that the troponin binding regions in tropomyosin are located on both sides of Cys 190, where trypsin attacks more easily than at other parts of the molecule, leaving two trypsin-resistant fragments.

Amino Acid Sequence↗
PubMed1975

Interaction of troponin components with F-actin and F-actin-tropomyosin complex.

1. Both TN-T and TN-I components of troponin interact with F-actin, causing its precipitation at 0.1 M KC1 and neutral pH in a form of highly ordered paracrystals, although the ability of TN-I component to precipitate of F-actin is much weaker. 2. F-actin paracrystals obtained in the presence of both TN-T and TN-I components consist of parallel arrays of F-actin filaments, although the fine structure is in each case different. 3. In the presence of tropomyosin in the proportion equal to that in muscle, less TN-T or TN-I component is needed to obtain full precipitation of F-actin. 4. Paracrystals of F-actin-tropomyosin-TN-T component and F-actin-tropomyosin-TN-I component show regular transverse striation spaced at about 380 A intervals. 5. The TN-C component of troponin solubilizes all precipitates of F-actin with TN-T or TN-I components, regardless of the presence of tropomyosin. 6. The results show that both TN-T or TN-I components can bind independently to F-actin-tropomyosin complex with the same periodicity, similar to that of the whole troponin in the living muscle.

Actins↗
PubMed1976

The structure and stability of trypsin-resistant segments from rabbit tropomyosin.

Tropomyosin was found to undergo only limited digestion by trypsin at 0 degrees C and the two segments that accumulated amounted to two-thirds of the original protein. They are referred to as segments A and B. These segments were not resistant to trypsin digestion at 20 degrees C and at the latter temperature no large fragments remained as judged by disc gel electrophoresis. Segments A and B were separated from each other on the basis of solubility differences and were found to have molecular weights of 24600 and 21900 respectively. Each of the segments appeared to retain about 70-75% of the helical conformation as judged by circular dichroism at 20 degrees C. However, the segments did not show any of the inhibitory activity of the parent tropomyosin molecule when mixed with troponin in the Mg2+-actomyosin ATPase system. Amino acid analysis showed that the portion of tropomyosin that was digested by trypsin (EC 3.4.21.4) had a lower content of the helix stabilizing residues Glu and Leu and a higher content of the helix-destabilizing residues Arg and Lys. These differences indicate that the digested portion should be less stable in the helical conformation than the two trypsin-resistant segments. End group determinations along with the results of the amino acid analysis indicated that segment A was probably derived from the central one-third of tropomyosin and segment B from the C-terminal one-third. By the process of elimination the N-terminal third appears to have been more liable region that was digested by trypsin. The segments A and B were shown to differ in their stability to denaturation by guanidine-HCl and elevated temperature. All of these observations indicate that tropomyosin is not a uniform structure and is composed of regions of different stability.

Adenosine Triphosphatases↗