The components of troponin from chicken fast skeletal muscle. A comparison of troponin T and troponin I from breast and leg muscle.

The three components of troponin were prepared from chicken breast and leg muscle. The troponin I and T components were separated by chromatography on DEAE-cellulose after citraconylation and without the use of urea-containing buffers. The troponin I and C components were similar to their counterparts from rabbit fast skeletal muscle, and a comparison of the troponin I components from breast and leg muscle by amino acid analysis, gel electrophoresis and peptide 'mapping' provides strong evidence for the identity of these proteins. The molecular weights of the troponin T components from breast and leg muscle were 33 500 and 30 500 respectively, determined by gel filtration. A comparison of these two proteins by methods similar to those used for the troponin I components suggested that they differed only in the N-terminal region of the sequence, the breast-muscle troponin T having an extra length of polypeptide chain of approx. 24 residues that is rich in histidine and alanine. The N-terminal hexapeptide sequence, however, is the same in both proteins and is (Ser,Asx,Glx)Thr-Glu-Glu. The genetic implications of these findings are considered.     

The predicted structure of the calcium-binding component of troponin.

The structure prediction of the calcium binding component of troponin (TN-C) incorporates the following assumptions: (1) TN-C contains four regions homologous to the calcium binding "EF hand" of parvalbumin. (2) The four EF hands are arranged in two pairs with overall symmetry, 222. (3) The regions of the calcium binding component of troponin which are not in the four EF hands connect the hands within each pair, one to two and three to four, and connect the pairs, region two to region three. In the resulting model there is a well-defined hydrophobic core made from side chains of all eight helical regions and of the four calcium binding loops. The Ca2+ within pairs are separated by 11 A; while the pairs of Ca2+ are separated from one another by over 30 A. Cys-98 and Tyr-109 are suggested to be sensitive spectroscopic probes. Calcium(1) is suggested to be solvent accessible and most readily replaced by a lanthanide. Because of the overall symmetry of the calcium binding component of troponin, one can anticipate that the inhibitory- and the tropomyosin binding components of troponin are similar to one another.     

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.     

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.     

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.     

Cardiac tropomyosin crystals and their interactions with troponin subunits

Crystals and paracrystals of bovine cardiac tropomyosin and their mixtures with different combinations of troponin subunits were examined in the electron microscope after negative staining. Although the cardiac proteins gave most of the same crystalline and paracrystalline patterns as observed previously with skeletal muscle tropomyosin and troponin, two important differences were noted. Cardiac troponin T was incapable of forming hexagonal networks with either skeletal or cardiac muscle tropomyosins, while skeletal troponin T readily associated in this manner with tropomyosins from either tissue source. Also the characteristic paracrystalline pattern seen with skeletal muscle tropomyosin, troponin T and troponin C only in the presence of calcium was consistently obtained with mixtures of the corresponding cardiac components when calcium was absent.     

Troponin from Akazara scallop striated adductor muscles

Troponin was isolated from striated adductor muscles of the "Akazara" scallop (Chlamys nipponensis akazara), and purified in an active form by DEAE-cellulose (Whatman DE52) column chromatography and subsequent gel filtration on Sephacryl S-300. According to sodium dodecyl sulfate-gel electrophoresis and densitometry, Akazara troponin is composed of three components having molecular weights of 52,000, 40,000, and 20,000 in a molar ratio of 1:1:1. The three components were separated from each other by column chromatography in the presence of 6 M urea and 1 mM EDTA on SP-Sephadex C-50 and DEAE-cellulose. The Mr 20,000 component was regarded as troponin C according to the Ca2+-binding properties, which was found to bind 0.7 mol of Ca2+/mol at 0.1 mM Ca2+. The association constant of Ca2+ to troponin C was estimated to be 5 X 10(5) M-1, and was not affected by the addition of 2 mM MgCl2. The Mr 52,000 component appeared to be troponin I, since it inhibited, together with Akazara tropomyosin, both Mg-ATPase and superprecipitation activities of actomyosin reconstituted from rabbit myosin and actin, and the inhibition of the ATPase activity was diminished by the addition of Akazara troponin C. Finally, the Mr 40,000 component appeared to be troponin T, since it co-precipitated with actin-tropomyosin filament and was indispensable with Akazara troponin C and the Mr 52,000 component (troponin I) for conferring the Ca2+ sensitivity to reconstituted actomyosin.     

A binary complex of troponin-I and troponin-T from Akazara scallop striated adductor muscle.

A binary complex consisting of Mr 19,000 and Mr 40,000 components was co-purified with troponin from a crude troponin fraction of Akazara scallop (Chlamys nipponensis akazara) striated adductor muscle. This complex is incapable of conferring Ca(2+)-sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, rather strongly inhibiting it, but became capable on further complexing with Akazara scallop troponin-C. To examine the effects of the Mr 19,000 and Mr 40,000 components on the ATPase activity, they were separated from each other by CM-Toyopearl column chromatography. The Mr 19,000 component strongly inhibited the Mg-ATPase activity of actomyosin-tropomyosin and the inhibition was reversed by further addition of the Akazara scallop troponin-C. On the other hand, the Mr 40,000 component slightly increased it. On hybridization with the Akazara scallop troponin subunits, the Mr 19,000 and Mr 40,000 components were shown to be able to substitute for troponin-I and troponin-T, respectively. The amino acid compositions of the Mr 40,000 component and troponin-T were almost identical, and those of the Mr 19,000 component and Mr 17,000 C-terminal fragment of the troponin-I resembled each other fairly well. From these results, it may be concluded that the Mr 19,000-40,000 binary complex is the troponin-I-troponin-T complex.     

Troponin from nematode: purification and characterization of troponin from Ascaris body wall muscle

• 1.1. A troponin-like protein was isolated from body wall muscle of Ascaris and separated into three components, the mol. wts of which were approx. 58,000, 36,000 and 20,000 respectively.
• 2.2. The three components were designated as troponin-T (TNT), troponin-I (TNI) and troponin-C (TNC) in order of mol. wt, since each component had properties similar to the respective components of vertebrate skeletal-muscle troponin.
• 3.3. Ascaris troponin were localized on actin filaments with a 44 nm repeat, an approximately 4 nm longer repeat than vertebrate troponin.

     

Troponin: regulatory function and disorders

Study of the molecular biology of the calcium regulation of muscle contraction was initiated by Professor Ebashi’s discovery of a protein factor that sensitized actomyosin to calcium ions. This protein factor was separated into two proteins: tropomyosin and a novel protein named troponin. Troponin is a Ca²⁺-receptive protein for the Ca²⁺-regulation of muscle contraction and, in association with tropomyosin, sensitizes actomyosin to Ca²⁺. Troponin forms an ordered regulatory complex with tropomyosin in the thin filament. Several regulatory properties of troponin, which is composed of three different components, troponins C, I, and T, are discussed in this article. Genetic studies have revealed that many mutations of genes for troponin components, especially troponins T and I, are involved in the three types of inherited cardiomyopathy. Results of functional analyses indicate that changes in the Ca²⁺-sensitivity caused by troponin mutations are the critical functional consequences leading to these disorders. Recent results of this pathophysiological aspect of troponin are also discussed.     

Divalent cation binding properties of slow skeletal muscle troponin in comparison with those of cardiac and fast skeletal muscle troponins

1. New methods of preparing troponins from slow skeletal and cardiac muscle of the chicken have been developed. The electrophoretic mobilities of slow skeletal muscle troponin subunits were different from those of the corresponding fast skeletal muscle subunits. 2. A new method for determining the amount of divalent cations bound to troponin was developed. The principle of the method is to immobilize troponin by conjugating it with Sepharose 4B resin, thus making it readily sedimentable. 3. The numbers of Sr and Ca ions bound to slow muscle troponin at concentrations sufficient to produce maximum contraction were 1.73 and 1.36 mol per mol, respectively, being nearly equal to those of cardiac troponin but half of those of fast muscle troponin. 4. The concentrations of Sr and Ca ions giving half-maximal ion binding to slow muscle troponin (K50%) were 5.5 X 10(-6) M and 4.6 X 10(-7) M, respectively. 5. K50% for Sr of cardiac troponin was significantly higher than that of slow muscle troponin. Although K50% for Sr of cardiac troponin was the same as that of fast muscle troponin, cardiac troponin bound more Sr ions than fast muscle troponin at lower Sr ion concentrations. The mechanism underlying the high sensitivity of cardiac muscle contraction to Sr ions is discussed in comparison with that of slow muscle.     

Troponin: structure, properties, and mechanism of functioning.

This review discusses the structure and properties of the isolated components of troponin, their interaction, and the mechanisms of regulation of contractile activity of skeletal and cardiac muscle. Data on the structure of troponin C in crystals and in solution are presented. The Ca2+-induced conformational changes of troponin C structure are described. The structure of troponin I is analyzed and its interaction with other components of actin filaments is discussed. Data on phosphorylation of troponin I by various protein kinases are presented. The role of troponin I phosphorylation in the regulation of contractile activity of the heart is analyzed. The structural properties of troponin T and its interaction with other components of thin filaments are described. Data on the phosphorylation of troponin T are presented and the effect of troponin T phosphorylation on contractile activity of different muscles is discussed. Modern models of the functioning of troponin are presented and analyzed.     

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.     

Separation of the DNA molecules beyond conventional size limits by gel electrophoresis with sodium dodecyl sulfate.

Electrophoretic mobility of DNA through polyacrylamide as well as agarose gels is greatly increased by sodium dodecyl sulfate (SDS). DNA molecules well beyond the conventionally separable size limits are separated readily and rapidly by gel electrophoresis with SDS in a conventional static electric field. Furthermore in optimal concentration gels DNA molecules of similar molecular sizes are separated better from one another in the presence of SDS than without it. Evidence is presented that SDS may act at least in part by altering conformation of DNA. This simple and readily available means for high resolution separation of hitherto impossible sizes of DNA molecules in polyacrylamide and agarose gels in an ordinary static electric field should find general use in molecular genetic analyses. Structural analyses of DNA-protein complexes are also facilitated by virtue of the simultaneous separation of the DNA and protein components on the same gel lane.     

Effect of calcium ions on the interaction of troponin C with rabbit skeletal muscle troponin I and troponin T immobilized on polyvinyl chloride

Using a new methodological approach based on the binding of 125I-labeled troponin C to troponins I and T immobilized on polyvinylchloride, the Ca2+-dependent interaction of troponin components was investigated. In the absence of Ca2+, two types of sites of troponin C--troponin T interaction were revealed (Kd = 3.6.10(-8) M and 5.10(-7) M). It was found that Ca2+ induced the formation of a troponin I--troponin C complex which was resistant to 5 M urea (Kd = 4.10(-8) M). In the absence of Ca2+, the binary troponin T--troponin C complex also revealed two types of interaction sites (Kd = 7.1.10(-8) M and 2.10(-7) M); however, in the presence of Ca2+ only high affinity sites whose number increased almost 2-fold were revealed. The events that may take place in the whole troponin complex during Ca2+ binding by troponin C are discussed.     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.     

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.     

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.     

Indirect [3H]methyl exchange as a general method for labeling methionine residues: application to calcitonin

Native porcine calcitonin from Armour is known to contain two components. It is shown that these can be separated by cation-exchange chromatography in 8 M urea. The technique of [3H]methyl exchange on the methionine residue was used to prepare each of these in a tritiated form. The reduced components formed by demethylation were found to readily reoxidize at neutral pH, to regenerate the disulfide bridge. Evidence is provided to show that the two forms were partially interconverted during these steps. The reoxidized 3H-labeled products were found to be indistinguishable in chemical, immunological, and biological properties from the equivalent components in native porcine calcitonin and had specific activities of approximately 20 Ci/mmol. It is concluded that this labeling method can be conveniently applied to peptides containing one or more disulfide bridges, to give products of high specific activity in acceptable yield, provided appropriate conditions are used to ensure correct reoxidation occurs.     

Several peculiarities of the interaction of troponin T and troponin C of skeletal and cardiac muscle

Using several independent methods, the interaction between troponin T and troponin C from skeletal and cardiac muscles was studied. It was found that troponin T and troponin C from skeletal muscles form a complex whose stability depends on Ca2+ concentration. Study of interactions between these troponin components demonstrated that both electrostatic and hydrophobic forces are involved in the complex formation. Cardiac troponin T and troponin C weakly interact with each other irrespective of experimental conditions. It was assumed that the weakening of interactions between the components of cardiac troponin is due to structural peculiarities of cardiac troponin T.