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At least 451 records · Page 25Linked to original sources
PubMed1999

Cleavage specificity of a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle.

Previously, we reported the purification and characterization of a myofibril-bound serine proteinase (MBP) from carp muscle (Osatomi K, Sasai H, Cao M-J, Hara K, Ishihara T. Comp Biochem Physiol 1997;116B:159-66). In the present study, the N-terminal amino acid sequence of the enzyme was determined, which showed high identity with those of other trypsin-like serine proteases. The cleavage specificity of MBP for dibasic and monobasic residues was investigated using various fluorogenic substrates and peptides. Analyses of the cleaved peptide products showed that the enzyme hydrolyzed peptides both at monobasic and dibasic amino acid residues. Monobasic amino acid residues were hydrolyzed at the carboxyl side; dibasic residues were cleaved either at the carboxyl side of the pair or between the two basic residues and the enzyme showed a cleavage preference for the Arg-Arg pair. Unexpectedly, MBP hydrolyzed lysyl-bradykinin and methionyl-lysyl-bradykinin at the carboxyl side of Gly fairly specifically and efficiently displaying a unique cleavage. Because MBP also degraded protein substrates such as casein and myofibrillar proteins, the substrate specificity of MBP appeared not to be strictly specific.

Amino Acid Sequence↗
PubMed2003

Na+/Ca2+ exchanger-deficient mice have disorganized myofibrils and swollen mitochondria in cardiomyocytes.

The Na(+)/Ca(2+) exchanger (NCX1) plays a key role in maintaining Ca(2+) homeostasis in cardiomyocytes. Disruption of Ncx1 gene in mice results in embryonic lethality between embryonic day 9 and 10, with the mice lacking spontaneous heartbeats. We examined the mechanism of lack of heartbeats in Ncx1-deficient mice. Ultrastructual analysis demonstrated that Ncx1-deficient mice showed severe disorganization of myofibrils, a lack of Z-lines and swelling of mitochondria in cardiomyocytes. However, the expressions of cardiac-specific genes including transcription factor genes and contractile protein genes were not changed in Ncx1-deficient mice. Abnormal Ca(2+) handling itself or the lack of heartbeats due to the inactivation of Ncx1 gene may cause the disorganization of myofibrillogenesis. Although NCX1 protein levels were decreased in heterozygous mice, there were no changes in NCX2 and NCX3 protein levels between wild type and heterozygous mice.

Animals↗
PubMed1994

Inhibition of ATP binding to myofibrils and acto-myosin subfragment 1 by caged ATP.

The inhibitory effect of P3-[1-(2-nitrophenyl)ethyl]adenosine 5'-triphosphate (caged ATP) on the binding of Mg2+-ATP to myofibrils was investigated. The most sensitive method was found to be the monitoring of single turnovers of [gamma-32P] ATP hydrolysis using the quench flow technique. The method was tested using ADP, which was found to have an inhibition constant of 145 microM, in agreement with previous reports. Caged ATP behaved as a simple competitive inhibitor of ATP binding with an inhibition constant of 1.6 mM. The inhibitory effect of these ligands on the binding of ATP to acto-myosin subfragment 1 was investigated using the same method. The inhibition constants of caged ATP and ADP were found to be 0.35 mM and 50 microM, respectively. This inhibitory effect of caged ATP on ATP binding accounts for the lower rate of ATP binding to fibers, deduced from caged ATP [(0.5-1) x 10(6) M-1 s-1], than that reported for acto-S1 (3.5 x 10(6) M-1 s-1) [Goldman, Y. E., Hibberd, M. G., & Trentham, D. R. (1984) J. Physiol. (London) 354, 577].

Adenosine Diphosphate↗
PubMed1982

Binding of calcium and magnesium to myosin in skeletal muscle myofibrils.

Binding profiles for divalent cation to myosin have been obtained in myofibrils where myosin is assembled in arrays typical of the in vivo organization. Protection by Ca2+ and Mg2+ ions of the regions of myosin susceptible to chymotryptic attack provided the means to monitor metal ion binding. The effect of various concentrations of divalent cations on the chymotryptic digestion patterns was assessed by densitometry of Coomassie Blue stained gels obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by the rate of myofibrillar solubilization. The results indicate the presence of two classes of binding sites differing in affinity by 4 orders of magnitude. The fractional saturation of the high-affinity site associated with the 5,5'-dithiobis(2-nitrobenzoic acid)-dissociable light chains of myosin regulated the production of subfragment 1 of myosin. From the digestion profiles as a function of metal ion concentration, binding constants for Mg2+ and Ca2+ were obtained. The value for Mg2+ was 5.7 x 10(6) M-1, which is about 1 order of magnitude higher than the most recently determined values for free myosin in solution; the value for Ca2+ was 6.3 x 10(6) M-1. Binding to the low-affinity site regulated the production of the heavy meromyosin fragment and yielded association constants for Ca2+ and Mg2+ of 0.9 x 10(3) and 0.7 x 10(3) M-1, respectively.

Animals↗
PubMed1998

Assembly of force-expressed troponin-I isoforms in myofibrils of cultured cardiac and fast skeletal muscle cells as studied by epitope tagging.

The isoform-specific assembly of cardiac and skeletal muscle troponin-I (CTnI and FTnI, respectively) on to myofibrils (MFs) was investigated. Epitope tagging was used to monitor the intracellular localization of exogenously introduced constructs to myofibrillar structures in cultured chicken cardiac and fast skeletal (breast) muscle cells. Exogenous CTnI and FTnI were incorporated into endogenous MFs of cardiac and breast muscle cells with high affinity, respectively. In the case of CTnI and FTnI with breast and cardiac muscle cells respectively, CTnI was not incorporated into breast MFs but FTnI was assembled on to cardiac MFs. To determine which portion of TnI is responsible for incorporation into these MFs, we constructed chimeric TnIs with the head and tail of CTnI replaced by those of FTnI. The behaviour of these chimeras depends on the tail of TnIs. These results suggest that the tail regions of TnIs bind to cardiac and breast MFs, and that this affinity of TnI tails is responsible for the assembly of FTnI on to cardiac MFs.

Amino Acid Sequence↗
PubMed2004

Not just a plasma membrane protein: in cardiac muscle cells alpha-II spectrin also shows a close association with myofibrils.

Spectrin and its associated proteins are essential for the integrity of muscle cells and there is increasing evidence for their involvement in signalling pathways as well as having a structural function in mediating stress. Spectrin is a multigene family and it is essential to determine which isoforms are present and their location in the cell. In heart muscle, we have found that one spectrin isoform, alphaII-spectrin, is strongly represented and, using immunofluorescence, we show that it lies within the contractile fibres near the Z-disc as well as on the cardiomyocyte plasma membrane. Electron microscopy of immunogold-labelled cryosections reveals statistically significant clustering of gold particles near the Z-disc, within and close to the edge of myofibrils. betaII-spectrin and ankyrin-R and G are both known to occupy this region. We suggest that alphaIIbetaII spectrin tetramers with ankyrin organise and/or stabilise cardiac muscle cell membrane components relative to the contractile apparatus.

Animals↗
PubMed1990

Increased association of ribosomes with myofibrils during the skeletal-muscle hypertrophy induced either by the beta-adrenoceptor agonist clenbuterol or by tenotomy.

Ribosome distribution in skeletal-muscle myofibres was investigated by immunohistochemistry and microdensitometry by using anti-(60 S ribosomal subunit) antibodies. Administration of the beta-adrenoceptor agonist clenbuterol caused an increase in the staining of the myofibrillar region with this antibody relative to that found in the subsarcolemmal cytoplasm. A similar effect was observed during hypertrophy of the plantaris muscle following severance of the tendon to the gastrocnemius. The results suggest that increased association of ribosomes with the myofibrils occurs during muscle hypertrophy.

Animals↗
PubMed2003

Damped elastic recoil of the titin spring in myofibrils of human myocardium.

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor. Application of a slack-test protocol revealed that the passive shortening velocity (Vp) of nonactivated cardiomyofibrils depends on: (i) initial sarcomere length, (ii) release-step amplitude, and (iii) temperature. Selective digestion of titin, with low doses of trypsin, decelerated myofibrillar passive recoil and eventually stopped it. Selective extraction of actin filaments with a Ca2+-independent gelsolin fragment greatly reduced the dependency of Vp on release-step size and temperature. These results are explained by the presence of viscous forces opposing myofibrillar passive recoil that are caused mainly by weak actin-titin interactions. Thus, Vp is determined by two distinct factors: titin elastic recoil and internal viscous drag forces. The recoil could be modeled as that of a damped entropic spring consisting of independent worm-like chains. The functional importance of myofibrillar elastic recoil was addressed by comparing instantaneous Vp to unloaded shortening velocity, which was measured in demembranated, fully Ca2+-activated, human cardiac fibers. Titin-driven passive recoil was much faster than active unloaded shortening velocity in early phases of isotonic contraction. Damped myofibrillar elastic recoil could help accelerate active contraction speed of human myocardium during early systolic shortening.

Actins↗
PubMed1962

A study of myofibril sarcomere structure during contraction.

In the present investigation of cross-striated muscle fibers of axolotl, we succeeded in observing in one field of vision of the electron microscope all the stages of myofibril contraction. This allowed us to avoid errors in establishing the sequence of individual contraction stages. Our studies reveal a new contraction stage which appears at the shortening of the sarcomere below 74 per cent of the "resting length" but prior to the formation of typical "maximally shortened" sarcomeres, characterized by strong "contraction bands." At this stage, in the center of the sarcomere, at either side of the M line, a "secondary anisotropic" band arises which widens with further sarcomere contraction. At either side of this band, at the place of the former ("primary") anisotropic band, a "secondary isotropic" band is formed. A scheme of successive stages of contraction of the sarcomere is presented. The mechanisms of contraction for the first stage (from 100 to 79 per cent of the "resting length") and for the last stage (from 74 to 58 per cent of the "resting length") seem to be different. While the sliding of myofilaments with respect to one another can be assumed for the first stage, it is the spiralization of these structures which is the most likely explanation for the last stage. (An Abstract in German also appears at the end of this article.)

Actin Cytoskeleton↗
PubMed1962

The elongation of myofibrils from the indirect flight muscle of Drosophila.

Myofibrils which lengthen by several per cent in the presence of ATP and magnesium ions were prepared by teasing indirect flight muscle of Drosophila in solutions containing ethylenediaminetetraacetate. A study was made of the hydrogen ion, magnesium ion, ATP, and potassium chloride concentrations with which this effect could be observed. The lack of elongation with pyrophosphate and several nucleoside triphosphates suggests that the lengthening is ATP specific. A relaxing factor system comparable to that described for rabbit muscle was not demonstrable, as elongated fibrils did not shorten with calcium ions, carnosine, or digitonin.

Animals↗
PubMed1995

Involvement of transglutaminase in myofibril assembly of chick embryonic myoblasts in culture.

Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross-striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross-striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100-insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle.

Animals↗
PubMed1977

Polarity of actin filaments at the initial stage of myofibril assembly in myogenic cells in vitro.

The polarity of thin filaments in relation to thick filaments in developing muscle cells in vitro was investigated. The majority of thin filaments exhibited the right polarity and spatial position similar to that seen in mature myofibrils. It appears that the interaction between thick and thin filaments exists in the initial phases of myofibrillogenesis. Cortical microfilaments are found to have their polarities arranged randomly.

Actins↗
PubMed1981

Fluorescence microscope study of the binding of added C protein to skeletal muscle myofibrils.

The binding of extra C protein to rabbit skeletal muscle myofibrils has been investigated by fluorescence microscopy with fluorescein-labeled C protein or unmodified C protein plus fluorescein-labeled anti-C protein. Added C protein binds strongly to the I bands, which is consistent with its binding to F actin in solution (Moos, C., C. M. Mason, J. M. Besterman, I. M. Feng, and J. H. Dubin. 1978. J. Mol. Biol. 124:571-586). Of particular interest, the binding to the I band is calcium regulated: it requires a free calcium ion concentration comparable to that which activates the myofibrillar ATPase. This increases the likelihood that C protein-actin interaction might be physiologically significant. When I band binding is suppressed, binding in the A band becomes evident. It appears to occur particularly near the M line, and possibly at the edges of the A band as well, suggesting that those parts of the thick filaments that lack C protein in vivo may nevertheless be capable of binding added C protein.

Animals↗
PubMed1953

The relationship between cytochondria and myofibrils in pigeon skeletal muscle.

In pigeon breast muscle the mitochondria are the principal site of oxidative metabolism, whereas the myofibrils are incapable of oxidizing intermediates of the Krebs cycle. The mitochondria contain the oxidative enzymes, and the sarcosomes are associated with a factor which accelerates the mitochondrial oxidative rate. The maintenance of myofibrillar contractility and structure is closely correlated with preservation of mitochondrial oxidative capacity and structure. By use of fluoride and dinitrophenol the connection between mitochondrial metabolism and myofibrillar behavior is shown to occur through the process of oxidative phosphorylation.

Animals↗
PubMed1999

Differential epitope tagging of actin in transformed Drosophila produces distinct effects on myofibril assembly and function of the indirect flight muscle.

We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments.

Actins↗
PubMed2005

Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Amino Acid Sequence↗
PubMed2000

Effect of Arg145Gly mutation in human cardiac troponin I on the ATPase activity of cardiac myofibrils.

In order to determine the functional consequences of the Arg145Gly mutation in troponin I found in familial hypertrophic cardiomyopathy, human cardiac troponin I and its mutant were expressed in Escherichia coli and purified, and then their effects on the ATPase activity of porcine cardiac myofibrillar preparations from which both troponins C and I had been depleted were examined. Both the wild-type and mutant troponin Is suppressed the ATPase activity of the troponin C.I-depleted myofibrils, but the maximum inhibition caused by mutant troponin I was weaker than that by wild-type troponin I. In the Ca(2)(+)-activation profile of the myofibrillar ATPase activity after reconstitution with both troponins I and C, the Ca(2)(+)-sensitivity with mutant troponin I was higher than that with wild-type troponin I, whereas the maximum level of the ATPase activity with mutant troponin I was lower than that with wild-type troponin I. These findings strongly suggest that the Arg145Gly mutation in human cardiac troponin I modulates the Ca(2)(+)-regulation of contraction by impairing the interaction of troponin I with both actin-tropomyosin and troponin C.

Adenosine Triphosphatases↗
PubMed1988

Identification of troponin-I of crayfish myofibrils.

Crayfish (Procambarus clarkii) myofibrils contain two basic proteins of molecular weights of 25,000 and 23,000. Both of the two proteins inhibit actomyosin ATPase as the vertebrate troponin-I does. These results differ from the previous one that troponin-I of crayfish (Astacus leptodactylus) showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).

Adenosine Triphosphatases↗