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.     

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.     

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.     

Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy

The differentiation of troponin (TN) in cardiac and skeletal muscles of chicken embryos was studied by indirect immunofluorescence microscopy. Serial sections of embryos were stained with antibodies specific to TN components (TN-T, -I, and -C) from adult chicken cardiac and skeletal muscles. Cardiac muscle began to be stained with antibodies raised against cardiac TN components in embryos after stage 10 (Hamburger and Hamilton numbering, 1951, J. Morphol. 88:49-92). It reacted also with antiskeletal TN-I from stage 10 to hatching. Skeletal muscle was stained with antibodies raised against skeletal TN components after stage 14. It also reacted with anticardiac TN-T and C from stage C from stage 14 to hatching. It is concluded that, during embryonic development, cardiac muscle synthesizes TN-T and C that possess cardiac- type antigenicity and TN-I that has antigenic determinants similar to those present in cardiac as well as in skeletal muscles. Embryonic skeletal muscle synthesizes TN-I that possesses antigenicity for skeletal muscle and TN-T and C which share the antigenicities for both cardiac and skeletal muscles. Thus, in the development of cardiac and skeletal muscles, a process occurs in which the fiber changes its genomic programming: it ceases synthesis of the TN components that are immunologically indistinguishable from one another and synthesizes only tissue-type specific proteins after hatching.     

Inhibition of mitochondrial adenosine triphosphatase activity by traponin comporent, TN-I

One of the troponin components, TN-I, strongly inhibits the ATPase activity of AS-particles obtained from mitochondria, while troponin and the other components, TN-C and TN-T, do not. The inhibition of the ATPase activity by Component TN-I is effective only in the presence of Mg²⁺ and ATP. Component TN-I digested with trypsin completely loses the inhibitory action of the ATPase activity. Adding tropomyosin does not affect the inhibitory effect of Component TN-I on the ATPase activity.     

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.     

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.     

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.     

Circularly polarized emission of terbium(III) substituted bovine cardiac troponin-C.

Upon substitution of Tb(III) for the most easily replaced Ca(II) from bovine cardiac TN-C, irradiation at 280 nm produces an emission at 545 nm from Tb(III) that is partially circularly polarized. Characteristics of these emission spectra produced by energy transfer from a tyrosyl side chain to a juxtaposed Tb(III) are virtually identical to those found in rabbit skeletal muscle TN-C and carp parvalbumin. A single homologous tyrosyl residue occurs in the two troponins and is in turn homologous to a phenylalanyl residue in parvalbumins. Addition of the other troponin subunits, TN-I and TN-T, to Tb(III)-TN-C weakens the total emission and completely quenches the circularly polarized emission.     

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.     

A fluorescence stopped-flow study on troponin labeled with N-ethyl maleimide and N-(p-(2-benzimidazolyl)phenyl) maleimide

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.     

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.     

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.     

Circular dichroism and difference spectra of members of the troponin-tropomyosin system in cetyltrimethylammonium bromide.

The detergent cetyltrimethylammonium bromide (CTAB) was used as a perturbant to study protein structure. Low concentrations of CTAB induced difference spectra for Ac-Trp-OEt and Ac-Tyr-OEt. The delta epsilonM values at their difference maxima were found to be 1300 at 292 nm for Ac-Trp-OEt and 400 at 287 for Ac-Tyr-OEt. These values were used to determine the number of tyrosine residues exposed in tropomyosin and troponin C, as well as the tyrosine and tryptophan residues exposed in troponin I and troponin T. In tropomyosin and troponin C all of the tyorosine residues were accessible to detergent. For TN-T, three of four tyrosines were free while the tryptophan residues were only partially exposed. In the case of TN-I both tyrosines were fully exposed but again evidence was obtained for a partially buried tryptophan chromophore. The stability of these proteins to CTAB was studies by measuring the far-uv circular dichroism spectra. Tropomyosin was quite sensitive to detergent and suffered a 60% loss in ellipticity at the concentration of CTAB used. The troponins, on the other hand, were affected to a lesser extent.     

Types of troponin components during development of chicken skeletal muscle

Changes in troponin components during development of chicken skeletal muscles have been investigated by using electrophoretic, immunoelectrophoretic, and immunoelectron microscopic methods. Previous reports (S. V. Perry and H. A. Cole, 1974, Biochem. J.141, 733–743; J. M. Wilkinson, 1978, Biochem. J.169, 229–238) pointed out that breast and leg muscles of adult chicken contain different types of troponin-T (TN-T), i.e., breast- and leg-type TN-T, respectively. However, the present paper indicates that the embryonic breast muscle contains leg-type TN-T. As development progresses two types of TN-T, i.e., breast- and leg-type TN-T, are found, and finally breast-type TN-T becomes the only species of TN-T present in the breast muscle. This change is well coordinated with the change of tropomyosin in the breast muscle. In contrast, the leg muscle contains leg-type TN-T through all the developmental stages. Leg-type TN-T is present in myogenic cells in vitro, irrespective of their origin, whether from the breast or leg muscle. The types of troponin-I and troponin-C in both breast and leg muscles do not change during development. The significance of the changes in the types of TN-T is discussed in terms of differential gene expression during development of chicken breast and leg muscles.     

Binding of lys-plasminogen to E-fragment of fibrinogen

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.     

The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin.

The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 +/- 2 000, consistent with a value of 37 000 +/- 1 000 determining by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent alpha-helical content of 43 +/- 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.     

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.     

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.