Two-dimensional electrophoresis of troponin complex with nonequilibrium pH gradient-sodium dodecyl sulfate polyacrylamide slab gel

A two-dimensional electrophoresis procedure for the separation and analysis of troponin subunits is described in which the protein solution supplemented with 50 mM each of both glutamic and aspartic acids is subjected to nonequilibrium pH gradient electrophoresis in the first dimension. Complete dissolution and gelation of the sample with agarose are essential for analysis of constituent proteins of cardiac myofibrils. Electrophoresis in the first dimension gel is carried out for a relatively short time, 2-3 h. In combination with sodium dodecyl sulfate slab gel electrophoresis (second dimension), three subunits, troponin T, troponin I, and troponin C, of dog cardiac troponin-tropomyosin complex and myofibrils can be simultaneously analyzed quantitatively on a slab gel. The contents of troponin and tropomyosin of cardiac myofibrils were 275 +/- 34 pmol/mg of myofibrillar protein. The molar ratio of troponin T, troponin I, troponin C, and tropomyosin was close to 1 : 1 : 1 : 1 in troponin-tropomyosin complex and myofibrils.     

Troponin-like proteins from muscles of the scallop, Aequipecten irradians.

Ca2+ regulation of molluscan actomyosin adenosine triphosphatase is known to be associated with the myosin molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, however, also suggests the possible presence of troponin, a thin-filament-linked Ca2+-regulatory complex. In the present study, scallop troponin and tropomyosin were prepared and complexed with rabbit actin; the resulting synthetic thin filaments form a Ca2+-dependent actomyosin adenosine triphosphatase with Ca2+-insensitive rabbit myosin, indicating that the troponin in scallops is potentially functional. Scallop troponin I was isolated and mixed with chicken troponin C and troponin T, forming a functional hybrid troponin complex, indicating that scallop and vertebrate troponins may act by a common mechanism. Densitometry of sodium dodecyl sulphate/polyacrylamide gels reveals that in synthetic thin filaments there are larger amounts of troponin than are present in native thin filaments. Amounts present in the intact muscle were not determined.     

Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin.

Tropomodulin is a tropomyosin-binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed-end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol. 127, 1627-1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, approximately 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence-intensity measurements, electron-microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mM KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca-actin than on Mg-actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin- and troponin-bound actin filaments.     

Periodic binding of troponin C.I and troponin I to tropomyosin-actin filaments

We investigated the distribution of troponin C.I and troponin I along tropomyosin-actin filaments by immunoelectron microscopy and found that anti-troponin I antibody formed transverse striations at 38 nm intervals along the bundle of filaments of both troponin C.I-tropomyosin-actin and troponin I-tropomyosin-actin. Since the length of 38 nm corresponds to the repeating period of filamentous tropomyosin along actin double strands, the present study indicates that troponin I is located at a specific region of each tropomyosin, suggesting that a specific interaction between troponin I and tropomyosin is involved in determining the periodic distribution of troponin I along tropomyosin-actin filaments.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site.

We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan. The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited. The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat). The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin. The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin. Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm. While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin. Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Cooperative interactions between troponin molecules bound to the cardiac thin filament

Striated muscle thin filaments contain many troponin molecules, which contact each other indirectly via tropomyosin and actin. Such allosteric interactions between troponin molecules may be responsible for cooperative Ca2+ binding to the regulatory sites of the cardiac thin filament (Tobacman, L. S., and Sawyer, D. S. (1990) J. Biol. Chem. 265, 931-939). To test whether thin filament-bound troponin molecules interact, we studied the competitive binding of troponin and troponin T-troponin I (an inhibitory complex lacking the Ca2+ binding subunit troponin C) to actin-tropomyosin. The relative affinities of these two forms of troponin for the thin filament depended upon their relative concentrations. Under conditions where total binding was saturated, each form binds with greater apparent affinity to sites that have similar neighbors. A theoretical model for competitive binding of two ligands to interacting sites on a linear lattice was developed and fit to the data. Surprisingly, energetically unfavorable interactions occurred between adjacent troponin and troponin T-troponin I molecules not only in the presence of Ca2+, but also in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and/or myosin subfragment 1. Removal of Ca2+ strengthened the affinity of troponin for the thin filament less than 50%. These results suggest that, even in the absence of myosin, long range allosteric interactions occur between troponin molecules. The detailed involvement of tropomyosin and actin in these interactions remains to be established.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.     

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.     

The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils

A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin: myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.     

Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants

Wild-type and mutant thin filaments were isolated directly from "myosinless" Drosophila indirect flight muscles to study the structural basis of muscle regulation genetically. Negatively stained filaments showed tropomyosin with periodically arranged troponin complexes in electron micrographs. Three-dimensional helical reconstruction of wild-type filaments indicated that the positions of tropomyosin on actin in the presence and absence of Ca(2+) were indistinguishable from those in vertebrate striated muscle and consistent with a steric mechanism of regulation by troponin-tropomyosin in Drosophila muscles. Thus, the Drosophila model can be used to study steric regulation. Thin filaments from the Drosophila mutant heldup(2), which possesses a single amino acid conversion in troponin I, were similarly analyzed to assess the Drosophila model genetically. The positions of tropomyosin in the mutant filaments, in both the Ca(2+)-free and the Ca(2+)-induced states, were the same, and identical to that of wild-type filaments in the presence of Ca(2+). Thus, cross-bridge cycling would be expected to proceed uninhibited in these fibers, even in relaxing conditions, and this would account for the dramatic hypercontraction characteristic of these mutant muscles. The interaction of mutant troponin I with Drosophila troponin C is discussed, along with functional differences between troponin C from Drosophila and vertebrates.     

Phosphorylation of troponin in the heart and skeletal muscle by Ca2+-phospholipid-dependent protein kinase

The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo.     

Phosphorylation of troponin and the effects of interactions between the components of the complex

1. The troponin complex from skeletal muscle contains approximately 1 mol of phosphate/80000g of complex, covalently bound to the troponin T component. 2. On prolonged incubation of the troponin complex or troponin T with phosphorylase kinase the phosphate content of troponin T was increased to approx. 3mol/mol. 3. On prolonged incubation of troponin I with phosphorylase kinase up to 1.6mol of phosphate/mol were incorporated. 4. Phosphorylation of troponin I was greatly inhibited by troponin C owing to the strong interaction between these proteins. Thus in the troponin complex troponin T was the main substrate for phosphorylase kinase. The phosphorylation of isolated troponin T was also inhibited by troponin C. 5. Troponin I was phosphorylated when the troponin complex was incubated with a bovine cardiac 3′:5′-cyclic AMP-dependent protein kinase. Troponin T either in its isolated form or in the troponin complex was not phosphorylated by bovine protein kinase to any significant extent under the conditions used. 6. If the troponin complex was dephosphorylated to 0.2mol/mol, or phosphorylated up to 2.5mol/mol there was no significant effect on the ability of normal concentrations to confer Ca²⁺ sensitivity on the adenosine triphosphatase of densensitized actomyosin.     

The effect of various low molecular weight compounds on the cation-binding properties of troponin with the heart

Using models of various complexity (isolated troponin C, troponin C-troponin I complex, troponin complex, troponin-tropomyosin complex, myofibrils), the effects of several low molecular weight organic compounds on the Ca(2+)-binding properties of troponin C were investigated. Trifluoperazine, calmidazolium and substance 48/90 increased the affinity of Ca(2+)-specific sites of troponin C both in the case of isolated troponin and in all the complexes under study. Nicardipine had no effect on the cation-binding activity of isolated troponin C, but increased the affinity of the Ca(2+)-binding sites of troponin C in the complex with troponin I. The cardiotonic drugs APP 201-533 and DPI 201-106 had practically no effect on the cation-binding properties of isolated troponin C or of simple complexes of troponin C. At the same time APP 201-533 increased, whereas DPI, 201-106 decreased the affinity of the Ca(2+)-binding sites of troponin C in myofibrils. It is concluded that the effects of the drugs on the cation-binding properties of troponin C depend on the protein-protein interaction with the filament. Studies of physiological activity of low molecular weight organic compounds require a detailed analysis of their effects on the Ca(2+)-binding activity of troponin C included into protein complexes of different complexity.