The amino acid sequence of troponin I from rabbit skeletal muscle.

The complete amino acid sequence of rabbit skeletal muscle troponin I was determined by the isolation of the cyanogen bromide fragments and the tryptic methionine-containing peptides. Troponin I contains 179 amino acid residues and has a molecular weight of 20864. Its N-terminus is acetylated. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50055 (23 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.     

Energetics of the binding of calcium and troponin I to troponin C from rabbit skeletal muscle.

We determined the free energy of interaction between rabbit skeletal troponin I (TNI) and troponin C (TNC) at 10 degrees and 20 degrees C with fluorescently labeled proteins. The sulfhydryl probe 5-iodoacetamidoeosin (IAE) was attached to cysteine (Cys)-98 of TNC and to Cys-133 of TNI, and each of the labeled proteins was titrated with the other unlabeled protein. The association constant for formation of the complex between labeled TNC (TNC*) and TNI was 6.67 X 10(5) M-1 in 0.3 M KCl, and pH 7.5 at 20 degrees C. In the presence of bound Mg2+, the binding constant increased to 4.58 X 10(7) M-1 and in the presence of excess of Ca2+, the association constant was 5.58 X 10(9) M-1. Very similar association constants were obtained when labeled TNI was titrated with unlabeled TNC. The energetics of Ca2+ binding to TNC* and the complex TNI X TNC* were also determined at 20 degrees C. The two sets of results were used to separately determine the coupling free energy for binding TNI and Mg2+, or Ca2+ to TNC. The results yielded a total coupling free energy of -5.4 kcal. This free energy appeared evenly partitioned into the two species: TNI X TNC(Mg)2 or TNI X TNC(Ca)2, and TNI X TNC(Ca)4. The first two species were each stabilized by -2.6 kcal, with respect to the Ca2+ free TNI X TNC complex, and TNI X TNC(Ca)4 was stabilized by -2.8 kcal, respect to TNI X TNC(Ca)2 or TNI X TNC(Mg)2. The coupling free energy was shown to produce cooperatively complexes formed between TNI and TNC in which the high affinity sites were initially saturated as a function of free Ca2+ to yield TNI X TNC(Ca)4. This saturation occurred in the free Ca2+ concentration range 10(-7) to 10(-5) M. The cooperative strengthening of the linkage between TNI and TNC induced by Ca2+ binding to the Ca2+-specific sites of TNC may have a direct relationship to activation of actomyosin ATPase. The nature of the forces involved in the Ca2+-induced strengthening of the complex is discussed.     

Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C

The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.     

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.     

Purification and properties of troponin T kinase from rabbit skeletal muscle.

A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH4)2SO4 and successive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg2+, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as Km values for ATP and substrate proteins, pH optima, effect of the concentration of Mg2+, substitution of ATP for GTP have also been studied.     

Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.     

The primary structure of actin from rabbit skeletal muscle. Completion and analysis of the amino acid sequence.

Actin is the principal constituent of the thin filaments of muscle, and in order to provide information basic to understanding the molecular basis of actin function we have studied its amino acid sequence. The isolation, compositions, and sequences of cyanogen bromide peptides, ranging in size from 3 to 44 residues, have previously been reported (ELZINGA, M. (1971) Biochemistry 10, 224-229, and other papers in the present series). The peptides have been aligned by isolation and characterization of tryptic peptides that contain methionine. The isolation of one of the CNBr peptides (CB-14) was complicated by the presence of a Met-Thr bond that was only partially split under standard conditions for cyanogen bromide cleavage in formic acid. In this paper conditions are described for increasing the cleavage at this bond. CB-14 is a tetrapeptide, Thr-Gln-Ile-Hse, and this sequence completes the characterization of the actin cyanogen bromide peptides. Finally, the position of CB-14 in the actin sequence as residues 120 to 123 was established by isolation of a chymotryptic overlap peptide. The complete sequence of the 374 residues of actin is presented.     

Synthetic studies on the inhibitory region of rabbit skeletal troponin I. Relationship of amino acid sequence to biological activity

Twelve peptide analogs of the actomyosin ATPase inhibitory region of rabbit skeletal troponin I (Tn-I) have been synthesized by the solid phase method and tested for biological activity in both actomyosin and acto-S1(A1) systems. Acto-S1(A1) is a cleaner and more facile system and we found no important discrepancies in the results for the two systems. These studies indicate that the sequence 105 to 114 is necessary for inhibition and that the inhibition is on the order of that reported for the 21-residue cyanogen bromide fragment of Tn-I (residues 96 to 116). We have shown the importance of lysine 105 and that of a bulky side chain at position 114 to this inhibition. Both the high activity of peptides containing the sequence 105 to 114 compared to Tn-I (45% on a molar basis) and the previously demonstrated tropomyosin specificity of this activity indicate that the relative inhibitory activity of these peptides is applicable to the discussion of the inhibitory activity of Tn-I. We have proven that actin concentration is a significant factor determining the extent of inhibition of both Tn-I and the peptides. We have also established that the charges on alpha-amino and alpha-carboxyl groups of a peptide, which do not appear in the parent protein, can modify the activity of the peptides. This must be taken into account when extrapolating from the activity of a peptide to the activity of the protein.     

Purification and properties of troponin T kinase from rabbit skeletal muscle

A protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) which catalyzes the phosphorylation of troponin T, phosvitin and casein has been purified over 2000 fold from rabbit skeletal muscle. The partial purification of this new enzyme, designated troponin T kinase, involves precipitation of contaminating proteins at pH 6.1, fractionation of the supernatant with (NH₄)₂SO₄ and succesive column chromatographies on DEAE-cellulose, hydroxyapatite and Sepharose 6B. The chromatographic patterns on DEAE-cellulose and hydroxyapatite columns show two peaks of troponin T kinase activity. Gel filtration experiments indicate the existence of multiple, possibly aggregated, forms of the enzyme. The purified enzyme does not catalyze the phosphorylation of phosphorylase b, troponin I, troponin C, tropomyosin, protamine, or myosin light chain 2 nor does it catalyze the interconversion of glycogen synthase I into the D form. Troponin T kinase is not affected by the addition of cyclic nucleotides or AMP to the reaction mixture. Divalent cations (other than Mg²⁺, required for the reaction) do not stimulate the enzyme, and several are inhibitory. Other characteristics of the reaction catalyzed by troponin T kinase, such as K_m values for ATP and substrate proteins, pH optima, effect of the concentration of Mg²⁺, substitution of ATP for GTP have also been studied.     

Complete primary structure of protein phosphatase inhibitor-1 from rabbit skeletal muscle

The complete primary structure of protein phosphatase inhibitor-1 has been determined. The protein consists of a single polypeptide chain of 165 residues, molecular weight 18640. The threonine residue that must be phosphorylated for activation is at position 35 and the active cyanogen bromide peptide, CB-1, comprises residues 2-66. The N-terminal methionine is acetylated and 40% of the inhibitor-1 molecules lack the C-terminal dipeptide Ala-Val. Serine-67 is substantially phosphorylated in vivo, but this phosphoserine residue does not appear to influence the activity of inhibitor-1.     

Chemically skinned mammalian skeletal muscle. I. The structure of skinned rabbit psoas.

We studied the morphology of rabbit psoas muscle fixed at increasing intervals of time in a chemical skinning solution (Wood et al., 1975), or after skinning and storage for times up to 1 week. The storage solution, in which the chemically skinned muscled fibers were kept at -20 degrees C, had the same ionic composition as the skinning solution but was made with 50% (v/v) glycerol. Progressive structural changes occurred in fibers exposed to skinning solution. The structural changes were essentially complete after 24-48 hr in skinning solution and no further changes were detected in fibers stored for periods up to 1 week. Structural changes were: (i) holes or gaps in the plasma membrane; (ii) swelling of mitochondria and disorganization of their internal structure; (iii) slight swelling of the sarcoplasmic reticulum; (iv) disappearance of sarcoplasmic reticulum (SR) feet from triadic gaps. Other changes included loss of glycogen between fibrils and extraction of myoplasm, or the change of its staining properties. All architectural elements of the SR, except "feet", remained during skinning and storage, and the SR remained able to accumulate calcium. The morphology of the myofilaments during chemical skinning and during storage did not differ from control fibers. We conclude that chemical skinning alters the gross structure of the plasma membrane and mitochondria, but produces minimal changes in the sarcoplasmic reticulum and contractile proteins.     

A 26K fragment of troponin T from rabbit skeletal muscle

A 26K fragment of troponin T, which was produced by endogenous proteases in rabbit skeletal muscle, was isolated by SE-Sephadex column chromatography. This fragment sensitized both superprecipitation and ATPase of actomyosin to calcium ions, to the same extent as troponin T. There was no difference in affinity for tropomyosin between this fragment and troponin T as examined by affinity chromatography. Amino acid analysis showed that this fragment consisted of residues Ala-46-Lys-259 of troponin T. The N-terminal 45 residues of troponin T, therefore, are not essential for the physiological action of troponin T. It was also observed that Ca2+-activated neutral protease digested troponin T into the 26K fragment in the native thin filament, while the protease digested troponin T in a different way in the reconstituted thin filament.     

Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the NH2-terminal, regulatory domain of troponin C

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.     

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.