Separation and characterization of the troponin components from bovine cardiac muscle.

The three major components of bovine cardiac troponin were separated by successive chromatography on sulfopropyl-Sephadex and DEAE-Sephadex columns in the presence of 6 M urea. All three of the bovine cardiac troponin subunits were necessary to restore full troponin activity in both skeletal and cardiac actomyosin ATPase assay systems. The 38,000-dalton subunit bound tropomyosin, and the 20,000-dalton subunit bound calcium, like skeletal TN-T and TN-C, respectively. The 28,000 component, although presumably analogous to skeletal TN-I, gave very little inhibition of actomyosin ATPase activity. Differences between cardiac and skeletal troponin subunits were also found in the elution patterns from ion exchange columns and in amino acid composition, thus demonstrating a significant muscle-type specificity.     

Troponin I: Inhibitor or facilitator

TN-I occurs as a homologous group of proteins which form part of the regulatory system of vertebrate and invertebrate striated muscle. These proteins are present in vertebrate muscle as isoforms, Mr 21000-24000, that are specific for the muscle type and under individual genetic control. TN-I occupies a central position in the chain of events starting with the binding of calcium to troponin C and ending with activation of the Ca2+ stimulated MgATPase of the actomyosin filament in muscle. The ability of TN-I to inhibit the MgATPase of actomyosin in a manner that is accentuated by tropomyosin is fundamental to its role but the molecular mechanism involved is not yet completely understood. For the actomyosin ATPase to be regulated the interaction of TN-I with actin, TN-C and TN-T must undergo changes as the calcium concentration in the muscle cell rises, which result in the loss of its inhibitory activity. A variety of techniques have enabled the sites of interaction to be defined in terms of regions of the polypeptide chain that must be intact to preserve the biological properties of TN-I. There is also evidence for conformational changes that occur when the complex with TN-C binds calcium. Nevertheless a detailed high resolution structure of the troponin complex and its relation to actin/tropomyosin is not yet available. TN-I induces changes in those proteins with which it interacts, that are essential for their function. In the special case of cardiac TN-I its effect on the calcium binding properties of TN-C is modulated by phosphorylation. It has yet to be determined whether TN-I acts directly as an inhibitor or indirectly by interacting with associated proteins to facilitate their role in the regulatory system.     

Troponin and its components from ascidian smooth muscle

Troponin was isolated from the thin filaments of ascidian smooth muscle and separated into three components by ion-exchange chromatography, the molecular weights of which were 33,000, 24,000, and 18,000, respectively. The three components were designated as troponin t (TN-T), troponin I (TN-I), and troponin C (TN-C) in order of molecular weight, since each component had properties similar to those of the respective components of vertebrate skeletal-muscle troponin. The ascidian troponin or the mixture of the three components conferred Ca2+-sensitivity on reconstituted rabbit actomyosin in the presence of tropomyosin. One of the characteristics of the ascidian troponin was Ca2+-dependent activation of actin-myosin interaction in collaboration with tropomyosin, whereas its inhibitory action on the actomyosin ATPase in the absence of Ca2+ was less remarkable. From this, it is concluded that in the ascidian smooth muscle actin-myosin interaction is regulated by an actin-linked troponin-tropomyosin system, but the ascidian troponin acts as a Ca2+-dependent activator of an actomyosin system.     

Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C.

When stoichiometric amounts of tropomyosin (TM) are bound to F-actin in the presence of 2 mM ATP, the MG2+-activated acto-heavy meromyosin (HMM) ATPase is inhibited by about 60% in 5 mM MgCl2-30 mM KCl. If the concentration of MgCl2 is reduced to 1 mM, the inhibition disappears because TM no longer binds to F-actin. Increasing the concentration of KCl to 100 mM restores both the binding and the inhibition. Thus, the binding of TM alone to F-actin causes significant inhibition of the ATPase provided that the HMM is saturated with ATP. (When the HMM is not saturated, TM activates the ATPase). When TM alone can bind stoichiometrically to F-actin, addition of troponin I (TN-I) increases the inhibition from 60% to about 85%, but the TM binding to F-actin is not affected. Under conditions such that TM alone neither inhibits the acto-HMM ATPase nor binds to F-actin, the inhibition caused by TN-I plus TM still approaches 100%. Direct binding studies under these conditions show that TN-I induces binding between TM and F-actin. A dual role for TN-I is proposed: first, TN-I can induce TM to bind to F-actin, causing inhibition of the ATPase; and second, TN-I can itself enhance the inhibition of the ATPase in a cooperative manner. The addition of TN-C in the absence of CA2+ has only a limited effect on the first role, but seems to be able to block completely the cooperative inhibition caused by TN-I such that the residual inhibition is a function only of the TM which remains bound.     

Phosphorylation of cardiac troponin by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

The purpose of this investigation was to characterize the phosphorylation of bovine cardiac troponin by cyclic AMP-dependent protein kinase. The purified troponin-tropomyosin complex from beef heart contained 0.78 +/- 0.15 mol of phosphate per mol of protein. Analysis of the isolated protein components indicated that the endogenous phosphate was predominately in the inhibitory subunit (TN-I) and the tropomyosin-binding subunit (TN-T) of troponin. When cardiac troponin or the troponin-tropomyosin complex was incubated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP, the rate of phosphorylation was stimulated by cyclic AMP and inhibited by the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase. The 32P was incorporated specifically into the TN-I subunit with a maximal incorporation of 1 mol of phosphate per mol of protein. The maximal amount of phosphate incorporated did not vary significantly between troponin preparations that contained low or high amounts of endogenous phosphate. The Vmax of the initial rates of phosphorylation with troponin or troponin-tropomyosin as substrates was 3.5-fold greater than the value obtained with unfractionated histones. The rate or extent of phosphorylation was not altered by actin in the presence or absence of Ca2+. The maximal rate of phosphorylation occurred between pH 8.5 and 9.0. At pH 6.0 and 7.0 the maximal rates of phosphorylation were 13 and 45% of that observed at pH 8.5, respectively. These results indicate that cyclic AMP formation in cardiac muscle may be associated with the rapid and specific phosphorylation of the TN-I subunit of troponin. The presence of endogenous phosphate in TN-T and TN-I suggests that kinases other than cyclic AMP-dependent protein kinase may also phosphorylate troponin in vivo.     

Purified cyclic GMP-dependent protein kinase catalyzes the phosphorylation of cardiac troponin inhibitory subunit (TN-1).

Cyclic AMP- and cGMP-dependent protein kinases catalyze the phosphorylation of cardiac troponin inhibitory subunit (TN-I). Unlike many substrates utilized by both kinases, TN-I is rapidly phosphorylated using relatively low concentrations of the cGMP-dependent protein kinase (0.01 to 0.1 micrometer). At low concentrations of cAMP- and cGMP-dependent protein kinases, approximately twice as much total phosphate is incorporated into TN-I using the cAMP-dependent enzyme. At higher enzyme concentrations, 1 mol of phosphate/mol of TN-I is found using either enzyme. Maximal levels of cAMP- and CGMP-dependent protein kinases do not catalyze additive phosphorylation, suggesting that the two enzymes catalyze the phosphorylation of the same site on TN-I. The results support the concept of overlapping substrate specificity for cAMP- and cGMP-dependent protein kinases, but suggest that cardiac troponin contains additional specificity determinants for the cGMP-dependent protein kinase not found in several other protein substrates.     

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.     

Calcium-induced conformational changes and mutual interactions of troponin components as studied by spin labeling.

The three troponin components, TN-C, TN-I, and TN-T, were spin-labeled with two different derivatives of the nitroxide radical, a maleimide and an imidazole reagent. The ESR spectra of various combinations of labeled and unlabeled components were measured both in the presence and absence of calcium. Conformational changes due to the binding of the components and also due to the binding of calcium were sensitively detected in many combinations as large changes in the spectrum. The conformation of TN-C was modified by both TN-T and TN-I. The effects were larger in the presence of calcium than in its absence. In the presence of calcium, TN-T and TN-I both showed large effects with the maleimide label, while TN-I showed a larger effect than TN-T with the imidazole label. In the absence of calcium, the effect of TN-I was larger than that of TN-T. The senstivitiy of TN-C to calcium was magnified by component binding, since the conformation of TN-C itself was not greatly affected by calcium. The conformation of TN-I was greatly altered only in the presence of both TN-C and calcium. This indicates that the calcium-induced conformational change in TN-C is transmitted to the adjacent TN-I. In reconstituted troponin, the conformation of TN-C was more influenced by TN-I in the presence of calcium and by TN-T in its absence as indicated by the imidazole label. With the maleimide label, TN-I was more influential in the absence of calcium. The effect of calcium on the troponin complex was to make the local environment of the label more rigid. The half-maximal effect was observed at 2 X 10(-6)M calcium with TN-I in various complexes, while it was 10(-5)M with TN-C in the complexes. In any case the calcium effects became discernible at 10(-6)M and saturated at 10(-4)M.     

Sensitivity of actomyosin ATPase to calcium and strontium ions. Effect of hybrid troponins

1. Hybrid or reconstituted troponins were prepared from troponin components of rabbit skeletal muscle and porcine cardiac muscle and their effect on the actomyosin ATPase activity was measured at various concentrations of Ca2+ or Sr2+. The Ca2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing cardiac troponin I was slightly higher than that with troponin containing skeletal troponin I. The Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin containing skeletal troponin C was higher than that with troponin containing cardiac troponin C. 2. Reconstituted cardiac troponin was phosphorylated by cyclic AMP-dependent protein kinase. The Ca2+ sensitivity of actomyosin ATPase with cardiac troponin decreased upon phosphorylation of troponin I; maximum ATPase activity was depressed and the Ca2+ concentration at half-maximum activation increased. On the other hand, phosphorylation of troponin I did not change Sr2+ sensitivity. 3. The inhibitory effect of cardiac troponin I on the actomyosin ATPase activity was neutralized by increasing the amount of brain calmodulin at high Ca2+ and Sr2+ concentrations but not at low concentrations. 4. ATPase activity of actomyosin with a mixture of troponin I and calmodulin was assayed at various concentrations of Ca2+ or Sr2+. The Ca2+ or Sr2+ sensitivity of actomyosin ATPase containing skeletal troponin I was approximately the same as that of actomyosin ATPase containing cardiac troponin I. Phosphorylation of cardiac troponin I did not change the Ca2+ sensitivity of the ATPase. 5. The Ca2+ or Sr2+ concentration required for half-maximum activation of actomyosin ATPase with troponin I-T-calmodulin was higher than that of actomyosin ATPase with the mixture of troponin I and calmodulin. Maximum ATPase activity was lower than that with the mixture of troponin I and calmodulin.     

Interactions of desensitized actomyosin with tropomyosin, troponin A, troponin B, and polyanions

Troponin B is an inhibitor of the Mg⁺⁺-activated ATPase activity of actomyosin. The inhibitory effect, which is observed, however, depends upon whether tropomyosin is also present. In the absence of tropomyosin the inhibition by troponin B is markedly reduced by increasing the ionic strength from 0.03 to 0.07, but is not affected by calcium up to a concentration of 10^-4 M. Troponin A relieves the inhibition in both the absence and presence of calcium, an effect which is also shown by many polyanions and is illustrated by using RNA. Tropomyosin enhances the inhibitory effect of troponin B and renders it more resistant to increasing ionic strength but it does not make the inhibition calcium-sensitive. However, when troponin A or low concentrations of polyanions are added to troponin B and tropomyosin, the actomyosin ATPase activity becomes calcium-sensitive; i.e., in the presence of tropomyosin, troponin A or polyanions do not relieve the inhibitory action of troponin B in the absence of calcium but only in its presence. In marked contrast to this is the effect of troponin A in the absence of tropomyosin where it neutralizes the effect of troponin B under all conditions. Thus troponin A and the polyanions both confer calcium regulation on the troponin B-tropomyosin system. The similar effects exhibited by troponin A and the polyanions suggest that the addition of net negative charge to troponin B is an important factor in the conferral of calcium sensitivity. It is also clear that tropomyosin is an essential component of the regulatory mechanism.     

Calcium sensitive binding of troponin to actin-tropomyosin: a two-site model for troponin action

Troponin reconstituted from the inhibitory component (troponin-I) and calcium binding protein (troponin-C) binds readily to actin-tropomyosin in 0.1 mm-EGTA but only poorly in 0.01 mm-CaCl₂ or 0.1 mm-Ca-EGTA. Troponin prepared by extraction of myofibrils with mersalyl, an organic mercurial, contains only these two components and also shows this calcium-sensitive binding and is deficient in its ability to bind to tropomyosin. Troponin-I + C is unable to confer calcium sensitivity on the Mg²⁺ activated actomyosin ATPase in concentrations at which native troponin is fully effective and the ATPase activity remains high in the absence of calcium. Addition of the tropomyosin binding component (troponin-T) to the other two components restores their ability to remain associated with actin-tropomyosin in the presence of calcium as does native troponin; calcium sensitivity is also regained. The results of these experiments have been interpreted in terms of a two-site mechanism of troponin action.     

An effect of diet on the activity of phosphofructokinase in rat heart

Phosphorylation of the inhibitory subunit of cardiac troponin (TN-I) occurs $\text{in vivo}$ after catecholamine intervention through adenylcyclase, cyclic AMP and cyclic AMP dependent protein kinase system. Also, TN-I and tropomyosin binding subunit of troponin (TN-T) are specifically hydrolyzed by calcium-activated neutral protease (CANP). In this study, we compared proteolysis of a set of TNs before and after phosphorylation by cyclic AMP dependent protein kinase plus cyclic AMP, using CANP from cardiac muscle. The initial rate of peptide release from both TNs was the same. After prolonged incubation, however, unphosphorylated TN degradation retarded, while phosphorylated TN proteolysis still continued. The amount of peptide release at the latter phase was dependent on the degree of phosphorylation. These results were confirmed by SDS polyacrylamide gel electrophoresis, and they suggest that a conformational change occurred in the whole TN molecule after phosphorylation of TN-I.     

The isolation and characterization of the ATPase inhibitory protein (TN-I) from bovine cardiac muscle.

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated APTase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 +/- 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 +/- 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.     

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.     

Erythrocyte troponin inhibitor-like protein: Isolation and characterization

A protein was isolated from a human erythrocyte lysate with an apparent molecular weight of 23,000–24,000 daltons. This protein was purified by batch DEAE cellulose followed by column DEAE cellulose chromatography and a gradient of NaCl. On sodium dodecyl sulfate acrylamide electrophoresis, the erythrocyte protein comigrated with muscle troponin inhibitor. An isoelectric precipitation (pH 9.25) was used for the separation of muscle troponin inhibitor from a complex with another troponin component. Both the erythrocyte protein and the muscle troponin inhibitor partially inhibited muscle myosin Ca²⁺ and K⁺-EDTA ATPase activity. Furthermore, they inhibited actin-activated Mg²⁺-ATPase of muscle myosin. The inhibitory effects were absent in the presence of muscle troponin calcium-binding component. Muscle troponin inhibitor and the erythrocyte troponin inhibitor-like protein bound to muscle myosin when myosin was precipitated twice at low ionic strength. The presence of a troponin inhibitor-like protein in erythrocytes suggests that it may be a component in the regulation of contractile activity.     

Ca2+ sensitization and potentiation of the maximum level of myofibrillar ATPase activity caused by mutations of troponin T found in familial hypertrophic cardiomyopathy

Human wild-type cardiac troponin T, I, C and five troponin T mutants (I79N, R92Q, F110I, E244D, and R278C) causing familial hypertrophic cardiomyopathy were expressed in Escherichia coli, and then were purified and incorporated into rabbit cardiac myofibrils using a troponin exchange technique. The Ca2+-sensitive ATPase activity of these myofibrillar preparations was measured in order to examine the functional consequences of these troponin mutations. An I79N troponin T mutation was found to cause a definite increase in Ca2+ sensitivity of the myofibrillar ATPase activity without inducing any significant change in the maximum level of ATPase activity. A detailed analysis indicated the inhibitory action of troponin I to be impaired by the I79N troponin T mutation. Two more troponin T mutations (R92Q and R278C) were also found to have a Ca2+-sensitizing effect without inducing any change in maximum ATPase activity. Two other troponin T mutations (F110I and E244D) had no Ca2+-sensitizing effects on the ATPase activity, but remarkably potentiated the maximum level of ATPase activity. These findings indicate that hypertrophic cardiomyopathy-linked troponin T mutations have at least two different effects on the Ca2+-sensitive ATPase activity, Ca2+-sensitization and potentiation of the maximum level of the ATPase activity.     

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.     

Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I

To understand the molecular mechanisms whereby cardiomyopathy-related cardiac troponin I (cTnI) mutations affect myofilament activity, we have investigated the Ca2+ binding properties of various assemblies of the regulatory components that contain one of the cardiomyopahty-related mutant cTnI. Acto-S1 ATPase activities in reconstituted systems were also determined. We investigated R145G and R145W mutations from the inhibitory region and D190H and R192H mutations from the second actin-tropomyosin-binding site. Each of the four mutations sensitized the acto-S1 ATPase to Ca2+. Whereas the mutations from the inhibitory region increased the basal level of ATPase activity, those from the second actin-tropomyosin-binding site did not. The effects on the Ca2+ binding properties of the troponin ternary complex and the troponin-tropomyosin complex with one of four mutations were either desensitization or no effect compared with those with wild-type cTnI. All of the mutations, however, affected the Ca2+ sensitivities of the reconstituted thin filaments in the same direction as the acto-S1 ATPase activity. Also the thin filaments with one of the mutant cTnIs bound Ca2+ with less cooperativity compared with those with wild-type cTnI. These data indicate that the mutations found in the inhibitory region and those from the second actin-tropomyosin site shift the equilibrium of the states of the thin filaments differently. Moreover, the increased Ca2+ bound to myofilaments containing the mutant cTnIs may be an important factor in triggered arrhythmias associated with the cardiomyopathy.     

Distribution of troponin components in the thin filament studied by immunoelectron microscopy.

1. The localization of specific antiboidies against troponin components, i.e., troponin T(TN-T), troponin I(TN-I, and troponin C(TN-C), was studied by the use of an electrom microscope. 2. Every antibody was distributed along the thin filament with a period of 38 nm. 3. Staining with anti-TN-I or anti-TN-C formed narrow striations. The location of the first striation was 26 nm from the free end of the thin filament. 4. The width of individual striations formed by anti-TN-T was 14--20nm The H-band-side end of each striation coincided with the location of anti-TN-I or anti-TN-C.     

Regulatory proteins of lobster striated muscle.

The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.