The stoichiometry and location of troponin I- and troponin C-like proteins in the myofibril of the bay scallop, Aequipecten irradians

Localization and quantification studies were carried out on bay-scallop (Aequipecten irradians) striated-muscle troponin C- and troponin I-like proteins. Indirect immunofluorescence microscopy of scallop myofibrils stained with either rabbit anti-(scallop troponin I) or anti-(scallop troponin C) antibodies shows staining of all I-bands observed. The results of quantification studies using sodium dodecyl sulfate poly-acrylamide-gel electrophoresis of untreated scallop myofibrils, washed scallop myofibrils, and isolated scallop thin filaments indicate an actin/tropomyosin/troponin-C molar rationn of 7:1:1. The molar ratio for troponin I could not be determined in untreated myofibrils because of interfering bands; in washed myofibrils a value of 0.6 mol of troponin I/mol of tropomyosin was found. Purified scallop troponin C binds Ca2+ and interacts with scallop troponin I to relieve troponin I-induced inhibition of actomyosin ATPase. Although scallop troponin C is an acidic protein, it appears to be less acidic than troponin C from higher organisms. A calmodulin-like protein has been isolated from scallop striated muscle that activates bovine brain phosphodiesterase to the same extent as does brain calmodulin. Its amino acid composition and its electrophoretic mobility on alkaline 6 M-urea/polyacrylamide gels differs from that of scallop troponin C, and it appears not to be associated with thin filaments.

Download Full-text

Functional and evolutionary relationships of troponin C

Physiological Genomics ◽

10.1152/physiolgenomics.00197.2007 ◽

2007 ◽

Vol 32 (1) ◽

pp. 16-27 ◽

Cited By ~ 34

Author(s):

Todd E. Gillis ◽

Christian R. Marshall ◽

Glen F. Tibbits

Keyword(s):

Functional Properties ◽

Thin Filament ◽

Troponin I ◽

Striated Muscle ◽

Troponin T ◽

Extensive Study ◽

Troponin C ◽

High Conservation ◽

Cross Bridges ◽

And Function

Striated muscle contraction is initiated when, following membrane depolarization, Ca2+ binds to the low-affinity Ca2+ binding sites of troponin C (TnC). The Ca2+ activation of this protein results in a rearrangement of the components (troponin I, troponin T, and tropomyosin) of the thin filament, resulting in increased interaction between actin and myosin and the formation of cross bridges. The functional properties of this protein are therefore critical in determining the active properties of striated muscle. To date there are 61 known TnCs that have been cloned from 41 vertebrate and invertebrate species. In vertebrate species there are also distinct fast skeletal muscle and cardiac TnC proteins. While there is relatively high conservation of the amino acid sequence of TnC homologs between species and tissue types, there is wide variation in the functional properties of these proteins. To date there has been extensive study of the structure and function of this protein and how differences in these translate into the functional properties of muscles. The purpose of this work is to integrate these studies of TnC with phylogenetic analysis to investigate how changes in the sequence and function of this protein, integrate with the evolution of striated muscle.

Download Full-text

Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain

Biochemical Journal ◽

10.1042/bj2250713 ◽

1985 ◽

Vol 225 (3) ◽

pp. 713-721 ◽

Cited By ~ 10

Author(s):

D Gravotta ◽

H J F Maccioni

Keyword(s):

Rat Brain ◽

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Bovine Brain ◽

Coated Vesicles ◽

Molar Ratio ◽

Coated Vesicle ◽

Synaptic Plasma Membranes ◽

Morphological Criteria

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.

Download Full-text

Polarization of Tryptophan Fluorescence from Single Striated Muscle Fibers

The Journal of General Physiology ◽

10.1085/jgp.59.1.103 ◽

1972 ◽

Vol 59 (1) ◽

pp. 103-120 ◽

Cited By ~ 82

Author(s):

C. G. dos Remedios ◽

R. G. C. Millikan ◽

M. F. Morales

Keyword(s):

Sarcomere Length ◽

Striated Muscle ◽

Muscle Fibers ◽

Tryptophan Fluorescence ◽

Thin Filaments ◽

A Value ◽

Striated Muscle Fibers ◽

Types Of Muscle Fibers ◽

Cross Bridges ◽

Contraction And Relaxation

Instrumentation has been developed to detect rapidly the polarization of tryptophan fluorescence from single muscle fibers in rigor, relaxation, and contraction. The polarization parameter (P⊥) obtained by exiciting the muscle tryptophans with light polarized perpendicular to the long axis of the muscle fiber had a magnitude P⊥ (relaxation) > P⊥ (contraction) > P⊥ (rigor) for the three types of muscle fibers examined (glycerinated rabbit psoas, glycerinated dorsal longitudinal flight muscle of Lethocerus americanus, and live semitendinosus of Rana pipiens). P⊥ from single psoas fibers in rigor was found to increase as the sarcomere length increased but in relaxed fibers P⊥ was independent of sarcomere length. After rigor, pyrophosphate produced little or no change in P⊥, but following an adenosine triphosphate (ATP)-containing solution, pyrophosphate produced a value of P⊥ that fell between the contraction and relaxation values. Sinusoidal or square wave oscillations of the muscle of amplitude 0.5–2.0% of the sarcomere length and frequency 1, 2, or 5 Hz were applied in rigor when the myosin cross-bridges are considered to be firmly attached to the thin filaments. No significant changes in P⊥ were observed in either rigor or relaxation. The preceding results together with our present knowledge of tryptophan distribution in the contractile proteins has led us to the conclusion that the parameter P⊥ is a probe of the contractile state of myosin which is probably sensitive to the orientation of the myosin S1 subfragment.

Download Full-text

Troponin Variants as Markers of Skeletal Muscle Health and Diseases

Frontiers in Physiology ◽

10.3389/fphys.2021.747214 ◽

2021 ◽

Vol 12 ◽

Author(s):

Monica Rasmussen ◽

Jian-Ping Jin

Keyword(s):

Skeletal Muscle ◽

Troponin I ◽

Striated Muscle ◽

Troponin T ◽

Muscle Quality ◽

Striated Muscles ◽

Thin Filaments ◽

Age Related ◽

Development And Aging ◽

Regulation Of Muscle Contraction

Ca2+-regulated contractility is a key determinant of the quality of muscles. The sarcomeric myofilament proteins are essential players in the contraction of striated muscles. The troponin complex in the actin thin filaments plays a central role in the Ca2+-regulation of muscle contraction and relaxation. Among the three subunits of troponin, the Ca2+-binding subunit troponin C (TnC) is a member of the calmodulin super family whereas troponin I (TnI, the inhibitory subunit) and troponin T (TnT, the tropomyosin-binding and thin filament anchoring subunit) are striated muscle-specific regulatory proteins. Muscle type-specific isoforms of troponin subunits are expressed in fast and slow twitch fibers and are regulated during development and aging, and in adaptation to exercise or disuse. TnT also evolved with various alternative splice forms as an added capacity of muscle functional diversity. Mutations of troponin subunits cause myopathies. Owing to their physiological and pathological importance, troponin variants can be used as specific markers to define muscle quality. In this focused review, we will explore the use of troponin variants as markers for the fiber contents, developmental and differentiation states, contractile functions, and physiological or pathophysiological adaptations of skeletal muscle. As protein structure defines function, profile of troponin variants illustrates how changes at the myofilament level confer functional qualities at the fiber level. Moreover, understanding of the role of troponin modifications and mutants in determining muscle contractility in age-related decline of muscle function and in myopathies informs an approach to improve human health.

Download Full-text

Calcium ion-regulated thin filaments from vascular smooth muscle

Biochemical Journal ◽

10.1042/bj1850355 ◽

1980 ◽

Vol 185 (2) ◽

pp. 355-365 ◽

Cited By ~ 79

Author(s):

S B Marston ◽

R M Trevett ◽

M Walters

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Atpase Activity ◽

Thin Filament ◽

Troponin I ◽

Troponin C ◽

Myosin Atpase ◽

Protein G ◽

Thin Filaments ◽

Myosin Atpase Activity

Myosin and actin competition tests indicated the presence of both thin-filament and myosin-linked Ca2+-regulatory systems in pig aorta and turkey gizzard smooth-muscle actomyosin. A thin-filament preparation was obtained from pig aortas. The thin filaments had no significant ATPase activity [1.1 +/- 2.6 nmol/mg per min (mean +/- S.D.)], but they activated skeletal-muscle myosin ATPase up to 25-fold [500 nmol/mg of myosin per min (mean +/- S.D.)] in the presence of 10(-4) M free Ca2+. At 10(-8) M-Ca2+ the thin filaments activated myosin ATPase activity only one-third as much. Thin-filament activation of myosin ATPase activity increased markedly in the range 10(-6)-10(-5) M-Ca2+ and was half maximal at 2.7 × 10(-6) M (pCa2+ 5.6). The skeletal myosin-aorta-thin-filament mixture gave a biphasic ATPase-rate-versus-ATP-concentration curve at 10(-8) M-Ca2+ similar to the curve obtained with skeletal-muscle thin filaments. Thin filaments bound up to 9.5 mumol of Ca2+/g in the presence of MgATP2-. In the range 0.06-27 microM-Ca2+ binding was hyperbolic with an estimated binding constant of (0.56 +/- 0.07) x 10(6) M-1 (mean +/- S.D.) and maximum binding of 8.0 +/- 0.8 mumol/g (mean +/- S.D.). Significantly less Ca2+ bound in the absence of ATP. The thin filaments contained actin, tropomyosin and several other unidentified proteins. 6 M-Urea/polyacrylamide-gel electrophoresis at pH 8.3 showed proteins that behaved like troponin I and troponin C. This was confirmed by forming interspecific complexes between radioactive skeletal-muscle troponin I and troponin C and the aorta thin-filament proteins. The thin filaments contained at least 1.4 mumol of a troponin C-like protein/g and at least 1.1 mumol of a troponin I-like protein/g.

Download Full-text

Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues

Biochemical Journal ◽

10.1042/bj1770521 ◽

1979 ◽

Vol 177 (2) ◽

pp. 521-529 ◽

Cited By ~ 124

Author(s):

R J Grand ◽

S V Perry ◽

R A Weeks

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Troponin I ◽

Smooth Muscles ◽

Bovine Brain ◽

Troponin C ◽

Rabbit Skeletal Muscle ◽

Polyacrylamide Gels ◽

Rabbit Lung ◽

Chicken Gizzard

1. An acidic protein with properties similar to those of troponin C from rabbit skeletal muscle has been shown to be present in bovine and rabbit smooth muscles, chicken gizzard and rabbit liver, kidney and lung. 2. A simple new method involving the use of organic solvents is described for the purification of the troponin C-like proteins from various tissues. 3. The troponin C-like proteins can be distinguished from rabbit skeletal-muscle toponin C by their electrophoretic behaviour on polyacrylamide gels at pH 8.3 in the presence and absence of Ca2+. The troponin C-like proteins have been shown to form complexes with rabbit skeletal-muscle troponin I that migrate on electrophoresis in polyacrylamide gels. 4. Behaviour on electrophoresis, amino acid analysis and the patterns of CNBr digests on polyacrylamide gels indicate that the troponin C-like proteins from bovine uterus and aorta, rabbit uterus, and liver and chicken gizzard are very similar to, if not identical with, bovine brain modulator protein. 5. With bovine cardiac muscle the organic-solvent method yields a preparation consisting of roughly similar amounts of troponin C and troponin C-like protein. 6. By the isotope-dilution technique, troponin C-like protein has been shown to represent 0.42% of the total protein in rabbit uterus. 7. In homogenates of smooth muscle, rabbit lung, kidney and brain, the troponin C-like proteins form a complex with other protein (or proteins) that requires Ca2+ for its formation and that is not dissociated in 9M-urea.

Download Full-text

Association analysis of TNNI1/XbaI polimorphism on carcass quality in hybrid pigs

Acta Agraria Debreceniensis ◽

10.34101/actaagrar/65/1878 ◽

2015 ◽

pp. 59-62

Author(s):

Andrea Nyilasovits ◽

János Posta ◽

Levente Czeglédi ◽

László Babinszky

Keyword(s):

Troponin I ◽

Striated Muscle ◽

Meat Production ◽

Thin Filaments ◽

Potential Marker ◽

Pcr Rflp ◽

Troponin Complex ◽

Myosin Interaction ◽

Polymerase Chain ◽

Regulation Of Muscle Contraction

The contractile protein, which is encoded by troponin I 1 (TNNI1) gene, is located on the thin filaments of slow fibres in striated muscle. TNNI1 protein is a part of the troponin complex which plays an important role in regulation of muscle contraction by preventing actin-myosin interaction in absence of calcium. According to biological role, this gene can be potential marker for meat production related traits. The aim of this study is to define whether the previously reported gene polymorphism (EU743939:g.5174T>C) is connected with the slaughter traits measured in a standard slaughterhouse of the examined four-line European hybrid. The study included data from 404 gilts and barrows from 2 different samples. The polymorphism was detected using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method with XbaI restriction enzyme. In this study the allele frequencies were found as follows: C: 0.84 and 0.808; T: 0.16 and 0.192. Based on result of the present study no significant impact of polymorphisms on production parameters was found.

Download Full-text

Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161)

Biochemical Journal ◽

10.1042/bj2070185 ◽

1982 ◽

Vol 207 (2) ◽

pp. 185-192 ◽

Cited By ~ 5

Author(s):

N V Barskaya ◽

N B Gusev

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Cardiac Muscle ◽

Sodium Dodecyl Sulphate ◽

Binding Sites ◽

Binding Constant ◽

Troponin I ◽

Biological Activities ◽

Sodium Dodecyl ◽

Troponin C

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0×10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.70×10(7) M-1. The corresponding constants for native troponin C are 5.90×10(7) M-1. and 2.90×10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.

Download Full-text