The phosphorylation of troponin I from cardiac muscle.

1. Troponin I isolated from fresh cardiac muscle by affinity chromatography contains about 1.9 mol of covalently bound phosphate/mol. Similar preparations of white-skeletal-muscle troponin I contain about 0.5 mol of phosphate/mol. 2. A 3':5'-cyclic AMP-dependent protein kinase and a protein phosphatase are associated with troponin isolated from cardiac muscle. 3. Bovine cardiac 3':5'-cyclic AMP-dependent protein kinase catalyses the phosphorylation of cardiac troponin I 30 times faster than white-skeletal-muscle troponin I. 4. Troponin I is the only component of cardiac troponin phosphorylated at a significant rate by the endogenous or a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. 5. Phosphorylase kinase catalyses the phosphorylation of cardiac troponin I at similar or slightly faster rates than white-skeletal-muscle troponin I. 6. Troponin C inhibits the phosphorylation of cardiac and skeletal troponin I catalysed by phosphorylase kinase and the phosphorylation of white skeletal troponin I catalysed by 3':5'-cyclic AMP-dependent protein kinase; the phosphorylation of cardiac troponin I catalysed by the latter enzyme is not inhibited.     

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

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

Phosphorylation of isolated components of the troponin complex of skeletal and cardiac muscle phosphorylase kinase from bird skeletal muscles

Pigeon and chicken skeletal muscle phosphorylase kinase purified to a nearly homogeneous state is able to phosphorylate both cardiac and skeletal troponin I and T. After 1-hr incubation, the enzyme transfers up to 0.35 mole of phosphorus per mole of skeletal troponin I, up to 0.5 mole of cardiac troponin I and up to 0.1 mole of cardiac and skeletal troponin T. Avian muscle phosphorylase kinase does not phosphorylate the first serine residue of cardiac and skeletal troponin T, but catalyzes the phosphate incorporation into the site(s) of troponin T located in the central or C-terminal parts of the protein molecule. The rate of troponin phosphorylation by pigeon muscle phosphorylase kinase is pH-dependent: the 6.8/8.2 ratio for troponin I is close to 0,2, whereas that with troponin T varies in the range of 0.5-0.7. Troponin phosphorylation by avian phosphorylase kinase depends on the presence of Ca2+ in the incubation mixture. In the presence of 3 mM EGTA troponin I phosphorylation is inhibited by 70-90%, whereas that of troponin T--by 50%. The experimental results indicate that the phosphorylation of troponin I and T is catalyzed either by two different active centers or by different conformations of the single center of avian phosphorylase kinase.     

Some properties of cardiac troponin T structure.

Troponin T is eluted in multiple peaks when the whole bovine cardiac troponin complex is subjected to DEAE-cellulose chromatography in the presence of 8 M-urea. The heterogeneity observed is due to the presence of two forms of troponin T, differing in their Mr values, amino acid content, degree of phosphorylation and aggregation. Cardiac troponin T contains up to 0.8 mol of phosphate/mol of protein. Rabbit skeletal-muscle troponin T kinase phosphorylates the single site located in the N-terminal pentapeptide of cardiac troponin T. The composition of this peptide, (Ser,Asx,Glx,Glx)Val, is similar to that of skeletal-muscle troponin T. The single thiol group of cardiac troponin T is located some 50-70 residues from the N-terminus. The C-terminal sequence of cardiac troponin T is Trp-Lys, i.e. as is the case of skeletal-muscle troponin T.     

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 sites of phosphorylation of rabbit cardiac troponin I by adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Effect of interaction with troponin C.

1. Troponin I prepared from rabbit hearts contains 1.0-1.5 mol of P/mol when isolated by affinity chromatography. Most of the covalently bound phosphate is located in residues 1-48 of the molecule. 2. 3':5'-Cyclic AMP-dependent protein kinase catalyses phosphorylation at serine-20 and serine-146. Serine-20 is more rapidly phosphorylated than serine-146. 3. In troponin I prepared from frozen hearts by affinity chromatography about 0.3-0.5 mol of P/mol is associated with serine-20 and 0.8-1.0 mol of P/mol with other site(s) in residues 1-48 of the molecule. 4. Phosphorylation at serine-20 and servine-146 is not significantly inhibited by troponin C. 5. The mechansim of the interaction of troponin C with cardiac troponin I is discussed in the light of these results.     

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.     

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.     

Structure-function relationships in cardiac troponin T

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

Cardiac troponin I, isolated from bovine heart, contains two adjacent phosphoserines. A first example of phosphoserine determination by derivatization to S-ethylcysteine

Bovine cardiac troponin containing approximately 3 mol P/mol protein could be separated into its subunits without loss of phosphate. Troponin I and troponin T each contain about 1.5 mol P/mol protein. In troponin I two phosphorylated serine residues could be localized in the N-terminal region by conversion of phosphoserine to S-ethylcysteine. They are located in adjacent positions in the following sequence: -Arg-Arg-Ser(P)-Ser(P)-Ala-Asn-Tyr-Tyr-Arg-Ala-Tyr-Ala-Thr-Glu-Pro- His-Ala-Lys. This sequence shows that the first phosphoserine residue in bovine cardiac troponin I occupies a homologous position to phosphoserine-20 of rabbit cardiac troponin I.     

Purification and subunit composition of guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung.

The guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung was purified to apparent homogeneity by affinity chromography using 8-2-aminoethylthio-cGMP coupled to Sepharose 4B. The kinase activity was purified approximately 6000-fold with an overall recovery of approximately 20%. The product isolated by affinity chromatography contained both cGMP-binding and cGMP-dependent histone kinase activity, indicating that the enzyme was not dissociated into regulatory and catalytic components by the immobilized cGMP derivative. The enzyme had a molecular weight of approximately 165,000 and a sedimentation coefficient of 7.8 S. The purified kinase displayed several characteristics similar to that of the partially purified enzyme including specificity for cGMP and stimulation by high concentrations of magnesium. On sodium dodecyl sulfate gels, only one major polypeptide chain was present having a molecular weight of approximately 81,000. This subunit bound 1 mol of cGMP and exhibited cGMP-dependent protein kinase activity. It is proposed that the native enzyme consists of two identical subunits (Mr=81,000), each of which binds cGMP and catalyzes protein phosphorylation.     

Sites phosphorylated in bovine cardiac troponin T and I. Characterization by 31P-NMR spectroscopy and phosphorylation by protein kinases

Bovine cardiac troponin isolated in a highly phosphorylated form shows four 31P-NMR signals [Beier, N., Jaquet, K., Schnackerz, K. & Heilmeyer, L.M.G. Jr (1988) Eur. J. Biochem. 176, 327-334]. Troponin I, which contains phosphate covalently linked to serine-23 and/or -24 [Swiderek, K., Jaquet, K., Meyer, H. E. & Heilmeyer, L. M. G. Jr (1988) Eur. J. Biochem. 176, 335-342], shows three resonances. Mg2(+)-saturation of holotroponin shifts these troponin I resonances to higher fields. Direct binding of Mg2+ to the phosphate groups can be excluded. Both these serine residues of troponin I, 23 and 24, are substrates for cAMP- and cGMP-dependent protein kinases as well as for protein kinase C. Isolated bovine cardiac troponin T contains 1.5 mol phosphoserine/mol protein, indicating that minimally two serine residues are phosphorylated. One phosphoserine residue is located at the N-terminus. An additional phosphoserine is located in the C-terminal cyanogen bromide fragment, CN4, which contains covalently bound phosphate. Protein kinase C phosphorylates serine-194, thus demonstrating exposure of this residue on the surface of holotoponin.     

Troponin from the myocardium and skeletal muscles: structure and properties

The literary and experimental data on the structure and properties of cardiac and skeletal muscle troponin are reviewed. The cation--binding sites of cardiac and skeletal muscle troponin C are distinguished by specificity; the sites localized in the C-terminal part of the protein molecule can bind both Ca2+ and Mg2+, whereas the sites localized at the N-end specifically bind Ca2+. The use of bifunctional reagents revealed a number of helical sites within the structure of cardiac troponin C (residues 84-92 and 150-158) and of skeletal muscle troponin C (residues 90-98 and 125-136). A comparison of experimental data with the results of an X-ray analysis testifies to the presence in the central part of the troponin C molecule of a long alpha-helical sequence responsible for troponin C interaction with the inhibiting peptide of troponin I. The efficiency of interaction of troponin components depends on Ca2+ concentration; the integrity of the overall troponin complex is mainly provided for by troponin C interaction with troponin I and by troponin I interaction with troponin T. The interaction between troponins T and C is relatively weak, especially in the case of cardiac troponin components. Both skeletal and cardiac muscles synthesize several troponin T isoforms differing in length and amino acid composition of N-terminal 40-60 member peptides. Troponin T isoforms can undergo phosphorylation by several protein kinases. The single site of troponin T which exists in a phosphorylated state in vivo (residue Ser-1) undergoes phosphorylation by specific protein kinase (troponin T kinase) related to casein kinases II. It was assumed that the phosphorylation of Ser-1 residue of troponin T as well as the synthesis of troponin T isoforms differing in the structure of the N-terminal peptide, provides for the regulation of interaction between two neighbouring tropomyosin molecules.     

Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.     

Affinity chromatography and properties of cGMP-dependent protein kinase from prawn tissues

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios close to 7. The native cGMP-dependent protein kinase is probably made up of two subunits each of which contains a regulatory and a catalytic sites.     

Physical properties of human polynucleotide kinase: hydrodynamic and spectroscopic studies.

Human polynucleotide kinase (hPNK) is a putative DNA repair enzyme in the base excision repair pathway required for processing and rejoining strand-break termini. This study represents the first systematic examination of the physical properties of this enzyme. The protein was produced in Escherichia coli as a His-tagged protein, and the purified recombinant protein exhibited both the kinase and the phosphatase activities. The predicted relative molecular mass (M(r)) of the 521 amino acid polypeptide encoded by the sequenced cDNA for PNK and the additional 21 amino acids of the His tag is 59,538. The M(r) determined by low-speed sedimentation equilibrium under nondenaturing conditions was 59,600 +/- 1000, indicating that the protein exists as a monomer, in contrast to T4 phage PNK, which exists as a homotetramer. The size and shape of hPNK in solution were determined by analytical ultracentrifugation studies. The protein was found to have an intrinsic sedimentation coefficient, s(0)(20,w), of 3.54 S and a Stokes radius, R(s), of 37.5 A. These hydrodynamic data, together with the M(r) of 59 600, suggest that hPNK is a moderately asymmetric protein with an axial ratio of 5.51. Analysis of the secondary structure of hPNK on the basis of circular dichroism spectra, which revealed the presence of two negative dichroic bands located at 218 and 209 nm, with ellipticity values of -7200 +/- 300 and -7800 +/- 300 deg x cm(2) x d(mol(-1), respectively, indicated the presence of approximately 50% beta-structure and 25% alpha-helix. Binding of ATP to the protein induced an increase in beta-structure and perturbed tryptophan, tyrosine, and phenylalanine signals observed by aromatic CD and UV difference spectroscopy.     

Protein-induced inactivation and phosphorylation of rabbit muscle phosphofructokinase.

Several previously untested proteins promote the reversible inactivation of rabbit skeletal muscle phosphofructokinase. Grouped in decreasing order of effectiveness, they include the following: skeletal muscle troponin C greater than troponin, the two smooth muscle myosin light chains, alpha-actinin, and S-100 much greater than parvalbumin and soybean trypsin inhibitor. The efficiency of troponin C in this process may even exceed that previously reported for calmodulin. Sequences near calcium binding site III are apparently involved in the troponin C-phosphofructokinase interaction. Troponin C and calmodulin exert calcium-dependent effects on the physical and chemical properties of muscle phosphofructokinase. When calcium is present, comigration with either protein allows the enzyme to enter the stacking gel during urea-polyacrylamide gel electrophoresis. Both enhance the phosphorylation of phosphofructokinase catalyzed by the cAMP-dependent protein kinase, with phosphate incorporations approaching 2 mol of P/mol of protomer. Reaction occurs at Ser774 and at Ser376--a novel site whose phosphorylation is highly sensitive to troponin C and less so to calmodulin. Maximum phosphorylation has slight effect on the catalytic activity of the enzyme under standard assay conditions. The troponin C induced or calmodulin-induced phosphorylation of phosphofructokinase requires calcium and is strongly inhibited by either fructose 2,6-bisphosphate or fructose 1,6-bisphosphate. Inactivation occurs in the presence or absence of calcium, with generally higher concentrations of effectors required for protection in the latter case. Liver and yeast phosphofructokinases shows little activity loss in the presence of either calmodulin or troponin C. We have developed and tested a general mathematical model for the protein-induced inactivation of phosphofructokinase which may find application to other systems.     

Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen.

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.     

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.     

Amino acid sequence of crayfish troponin I

Troponin I is the actomyosin ATPase inhibitory subunit present in the thin filament regulatory complex. The complete amino acid sequence of crayfish tail muscle troponin I has been determined. The protein is composed of 201 amino acid residues and has a molecular weight of 23,547. The N terminus is blocked, likely by an acetyl group. Crayfish troponin I shows a rather low (20-25%) sequence identity with vertebrate troponin Is as compared to the 60-82% identity within the vertebrate phylum. Similar to vertebrate cardiac troponin I, crayfish troponin I contains a 30-residue-long N-terminal extension. In crayfish troponin I, this segment bears significant sequence homology with the heavy or light chains of particular myosins. The actin-binding domain of crayfish troponin I, which displays 57% sequence homology with vertebrate troponin Is, possesses 2 unusual trimethyllysine residues. The consensus sequence of this domain in five troponin Is is as follows: D-L-R-G-K-F-X-R*-P-X-L-R*-R*-V, where R+ stands for Arg/Lys, R* for Arg/trimethyllysine, and X for any amino acid residue. Troponin I possesses two Ca2+-dependent interactive sites for troponin C; one partly overlaps with the actin binding domain and is highly conserved, and the other, corresponding to the 30-residue-long segment following the N-terminal extension in vertebrate cardiac and crayfish troponin I, is poorly conserved in the different troponin Is. Troponin I also interacts with troponin T. The consensus sequence for the interacting site on troponin I is as follows: h-D- -X-D- -R+-Y-D-h-E-h, where h stands for a hydrophobic residue, D- for Asp/Glu, R+ for Arg/Lys, and X for any residue. The five troponin Is further possess one more 15-residue-long segment of high sequence identity near the C terminus. Its evolutionary conservation suggests that this domain is involved in protein-protein interaction.