Amino acid sequence of bovine cardiac troponin I (original) (raw)
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Bovine cardiac troponin T: amino acid sequences of the two isoforms
Biochemistry, 1987
Troponin T (TnT) is the tropomyosin-binding subunit of troponin, the thin filament regulatory complex that confers calcium sensitivity to striated muscle contraction and actomyosin ATPase activity. Bovine cardiac muscle contains two isoforms (TnT-1 and TnT-2) of TnT that differ in sequence near their amino termini. Thin filaments containing TnT-2 require less calcium to activate the MgATPase rate of myosin than do thin filaments containing TnT-1. Using whole troponin T purified from adult bovine cardiac muscle, we have determined the complete amino acid sequence of the larger, more abundant isoform TnT-1. We confirmed that sequence differences between TnT-1 and TnT-2 are confined to the amino-terminal regions and found that TnT-1 makes up approximately 75% of the total troponin T isolated. Partial sequencing of the separated isoforms showed that the difference between them is due solely to residues 15-19 (Glu-Ala-Ala-Glu-Glu) of TnT-1 being absent from TnT-2. The deleted segment may correspond to the product of exon 4 of the chicken cardiac TnT gene [Cooper, T. A,, & Ordahl, C. P. (1985) J . Biol. Chem. 260, 11 140-1 11481. Exon 5, which is developmentally regulated in the chicken, is not expressed in either TnT-1 or TnT-2. TnT-1 contains 284 amino acid residues and has a M, of 33 808, while TnT-2 contains 279 amino acid residues and has a Mr of 33 279. Bovine cardiac TnT contains the only known thiol group in any isolated
Amino acid sequence of porcine cardiac muscle troponin C
Journal of biochemistry, 1989
Troponin C is the Ca2+-receptive protein located on the thin filament of striated and cardiac muscle. We have determined the amino acid sequence of troponin C obtained from porcine cardiac muscle by sequencing and aligning the lysyl endopeptidase and Staphylococcus aureus V-8 protease peptides. It was composed of 161 amino acid residues with a blocked N-terminus. The sequence of porcine cardiac troponin C was identical with that of bovine cardiac troponin C.
Isolation and characterization of a highly phosphorylated troponin from bovine heart
European Journal of Biochemistry, 1988
A modified procedure for isolation of troponin from bovine heart is described, which results in a stable and highly phosphorylated protein. 31P-NMR spectra show up to four phosphoserine signals indicating that at least four serine residues of cardiac troponin are phosphorylated in the intact organ. The hydrodynamic parameters of phosphotroponin are almost identical to those previously published. Characteristically cardiac troponin shows a strong tendency to associate that is dependent on protein concentration. Mg2+ may specifically induce an aggregation, which can be observed during sedimentation. This phenomenon seems to be analogous to the Mg2+induced dimerization of cardiac troponin C [Jaquet, K. and Heilmeyer, L. M. G., Jr (1987) Biochem. Biophys. Res. Commun. 145,1390-13961. Upon Mg2+ saturation a shift of one of the four 31P-NMR signals is observed. The affinity of troponin to Ca2+ is reduced when the protein concentration is enhanced only in the presence of Mg2+. This effect of Mg2+ suggests a model for the regulation of the Ca2+-binding affinity of cardiac troponin.
Human cardiac troponin complex. Structure and functions
Biochemistry (Moscow), 2013
Cardiac muscle contraction is a complex process in which cardiomyocyte stimulation, occurring as a result of cell membrane depolarization and subsequent increase in intracellular Ca 2+ level, is coupled with the generation of mechanical force. The key role in the regulation of muscle contraction belongs to one of the components of thin filament-troponin complex. This protein consists of three subunits-troponin C (TnC), which binds Ca 2+ , troponin I (TnI), which inhibits the ATPase activity of actomyosin complex, and troponin T (TnT), which interacts with tropomyosin. Complex changes in troponin structure that occur after binding of Ca 2+ enable the ATPdependent interaction of myosin with actin and development of muscle contraction. Developmental isoform change, alternative splicing, and various posttranslational modifications of troponin complex proteins allow the fine adjustment of cardiac contraction. Troponin complex also has important medical implications. This protein is a target for some cardiotonics used in therapy of heart failure. A number of mutations in troponin genes lead to the development of different types of cardiomyopathies. In addition, for the last 25 years isoforms of TnI and TnT specific for cardiac muscle tissue have been used for diagnostics of pathologies associated with cardiomyocyte necrosis (myocardial infarction, myocardial trauma, and others). This review summarizes the existing data on structure and functioning of troponin complex components and their role in the regulation of cardiac muscle activity. EXPRESSION OF TROPONIN COMPLEX SUBUNITS Troponin I In humans, troponin I is expressed in three isoforms: fast and slow skeletal isoforms and specific cardiac isoform [1-3]. Genes of all three TnI isoforms are arranged in tandems with TnT genes and are paralogs formed by the triplication of the ancestor TnI/TnT pair of genes [4, 5]. During embryonic development the fast and slow skeletal isoforms of TnI are expressed in all types of skele
Journal of Biological Chemistry, 2019
Striated muscle is activated by myosinand actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The Cterminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca 2+ and for limiting its activation at saturating Ca 2+. Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca 2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. Förster Resonance Energy Transfer analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca 2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steadystate isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction. ______________________________________ Activation of striated muscle occurs both through myosin-linked (1,2) and actin-linked processes (3-5). The actin-linked portion of regulation works primarily through changes in
Isolation and Characterization of the Human Cardiac Troponin I Gene (TNNI3
Genomics, 1996
Three isoforms of TnI that are encoded by separate Troponin I (TnI) is a constituent protein of the tropogenes exist in birds and mammals. The fast skeletal nin complex located on the thin filament of striated isoform (TnIf) is expressed in fast twitch skeletal musmuscle that provides a calcium-sensitive switch for cle fibers. The slow skeletal isoform (TnIs) is expressed striated muscle contraction. Unlike other contractile in both cardiac muscle and slow twitch skeletal muscle proteins, the cardiac isoform of troponin I (TnIc) is fibers during development, but is restricted to slow expressed only in cardiac muscle and therefore offers twitch skeletal muscle fibers in the adult. As in other a model for cardiac-specific expression. It is also subspecies, the human cardiac isoform of TnI (TnIc) differs ject to developmental regulation with increased exfrom those found in skeletal muscle by the presence of pression occurring at the time of birth. Here we dean amino-terminal extension that includes the sescribe the isolation and characterization of the human quence RRRSS (Vallins et al., 1990; TnIc gene (HGMW-approved symbol TNNI3) and its 1990). This sequence, which is also conserved in rat promoter. The gene comprises eight exons contained and bovine TnIc, is phosphorylated on both serines by within 6.2 kb of genomic DNA. The proximal promoter cAMP-dependent protein kinase A (PKA) in vitro (Mittand 1.1-kb 5-flanking region were sequenced, and sevmann et al. , 1992). In the perfused rat heart it is these eral putative cis-acting elements that are conserved serines that are phosphorylated in response to b-adrenbetween the human and the mouse TnIc genes were ergic stimulation England, 1976).
European Journal of Biochemistry, 1994
We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG+GCC and (Gly4) GGG+ GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-Itroponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-Sl Mg-ATPase activity ; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.
Biophysical Journal, 2010
Mutations in troponin, an important muscle protein complex, can result in cardiomyopathy by interfering with the normal muscle activity of the heart. Troponin T (TnT) is the largest subunit of troponin and is involved in binding the troponin complex to the thin filament. Investigation of two mutations associated with cardiomyopathy in TnT, I90M and R173Q, showed different physiological characteristics. The TnT I90M mutation was identified as the causative agent of familial hypertrophic cardiomyopathy (FHC) in a large multi-generational Chinese family and at least two family members with this mutation died of sudden cardiac death. Another mutation in TnT, R173Q, was identified as the underlying cause of dilated cardiomyopathy (DCM). Patients with the TnT R173Q mutation experienced prenatal onset DCM and supraventricular tachycardia at a young age. Functional troponin complexes containing wild-type or mutant TnT's demonstrated similar maximal actomyosin ATPase activity. The inhibitory ability of the troponin complexes containing the I90M mutation was significantly reduced relative to wild-type TnT. Most RCM mutations investigated to date showed a reduced ability to inhibit actomyosin ATPase activity but the RCM mutation, R173Q, did not affect the inhibitory ability of troponin. The mutations showed increased (I90M) and decreased (R173Q) calcium sensitivity of actomyosin ATPase activity consistent with what has been observed for most FHC and DCM mutations. The mutations reduced the rate of degradation of these proteins by calpain relative to wild-type TnT. Overall, these results suggest that although calcium sensitivity may be an indicator of the type of cardiomyopathy no clear trends in maximal or minimal ATPase activity exist that can be used to characterize DCM and FHC mutations.