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At least 199 records · Page 11Linked to original sources
PubMed1985

Kinetic rates of tryptic digestion of bovine cardiac myofibrils. An improved measurement of cross-bridge dissociation.

The rates of tryptic digestion of the 50/20-kDa junction in myosin in cardiac myofibrils were determined under various solvent conditions. This cleavage reaction is slow in the rigor solvent and proceeds at a fast rate in the presence of MgATP. When the reaction solvent contains 50% ethylene glycol, the digestion of myosin in the presence of MgATP occurs at the same rate as in myofibrils relaxed by Mg adenyl-5'-yl imidodiphosphate (AMP-PNP). It is shown that with the help of two reference rates of digestion, for attached and dissociated myosin heads, the initial cleavage rates of myosin in the presence of nucleotides accurately measure the dissociation of cross-bridges from actin in myofibrils. Under physiological salt conditions and at 24 degrees C, MgADP, MgPPi, and MgAMP-PNP cause only small cross-bridge detachment (less than or equal to 15%) in cardiac myofibrils. The dissociation of myosin from actin is greatly increased by lowering the solvent temperature to 4 degrees C. Lowering the salt concentration of the solvent from 0.1 to 0.01 M NaCl has the most pronounced effect on the rates of myosin digestion in the presence of MgATP. In the low salt medium a substantial fraction of myosin heads (at least 30%) appears to be attached to actin in the presence of 5 mM MgATP.

Actomyosin↗
PubMed1984

Comparison of M-line and other myofibril components during reversible phorbol ester treatment.

The events occurring during phorbol ester mediated destruction of myofibrils in differentiated muscle cells were followed at the fluorescence and electron microscope levels using antibodies which bind troponin-T, a newly discovered 185 000 dalton M-line protein called myomesin and muscle type creatine kinase. The following series of events is proposed. Within one day of phorbol ester treatment, Z-bands and thin filaments, including troponin-T, are absent from many myofibrils resulting in the rapid loss of longitudinal and lateral alignment. A-bands become randomly oriented and clustered into ever smaller compartments within the rounding, myosac-like, multinucleated cells until after 3 days of treatment they too disappear. The M-line proteins are always present in existing A-bands. These results suggest that the Z-band and associated structures are responsible for the maintenance of alignment and the lateral register of myofibrils, whereas the M-line is responsible for the structural integrity of the A-band. When phorbol ester is removed, the cells revert to a myotube morphology and within 2 to 3 days are filled with myofibrils. A comparison of the appearance of troponin-T and the 185 000 dalton myomesin in the recovery period to their appearance during normal myofibrillogenesis reveals that these proteins are more temporally co-ordinated during myofibrillogenesis than in the phorbol ester experimental system.

Animals↗
PubMed1983

Troponin subunit stoichiometry and content in rabbit skeletal muscle and myofibrils.

The quantity and molar ratio of the three troponin subunits to actin were determined in rabbit psoas muscle, muscle homogenates (800 X g pellet), and purified myofibrils. Proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The quantities of the separated proteins were determined directly from the gel slices by amino acid analysis after correction for losses and background. The molar ratio of actin, troponin T, troponin I, and troponin C was found to be 6.99:1:05:1:04:0.92 in purified myofibrils and was not significantly different (p greater than 0.05) from those obtained from 800 X g pellets of muscle homogenates or intact muscle tissue. Isolated troponin purified by several different procedures also had a 1:1:1 subunit ratio although the variability was much greater than that found in myofibrils. The troponin content of rabbit psoas muscle and myofibrils was 91 +/- 16 and 770 +/- 110 pmol/mg, respectively.

Actins↗
PubMedDate not supplied

A morphometric study of myocardial mitochondria and myofibrils in turkey poults during development of furazolidone-induced cardiomyopathy.

Furazolidone (FZ) at a dose of 700 ppm was fed to turkey poults 2-5 weeks posthatching. At 3, 4, and 5 weeks of age, six poults each were sacrificed from control and FZ-fed groups. Samples of tissue from the outer free walls of the ventricles and the interventricular septum were removed and processed for electron microscopy. Volumetric density of mitochondria and myofibrils was determined by the point-counting technique. Significant reductions (p less than or equal to .05) in the volumetric density of mitochondria were observed at 5 weeks of age in the ventricular free walls of round-heart poults, and at 4 and 5 weeks of age in the interventricular septum. Significant reductions (p less than or equal to .05) in the volumetric density of myofibrils were noted at 4 weeks of age in the left ventricular free wall of FZ-fed normal poults and in the interventricular septum of round-heart poults. At 5 weeks of age, a significant increase (p less than or equal to .05) in myofibrils was observed in the left ventricular free wall of FZ-fed normal poults. Data confirms that the volumetric densities of mitochondria and myofibrils are significantly altered during development of FZ-induced hypertrophy.

Animals↗
PubMed1995

Phosphofructokinase binding to myofibrils in fish muscle: influences of ionic strength and metabolite levels on enzyme complex formation.

The interaction of 6-phosphofructo-1-kinase (PFK) with myofibrils was assessed using the purified proteins from rainbow trout white skeletal muscle. More than 70% of PFK activity was bound at pH values between 6 and 7 but higher pHs dissociated the complex. Increasing salt concentrations also reduced PFK binding, with greater sensitivity to salt at pH 7.0 vs pH 6.6. Substrates and allosteric effectors also reduced enzyme binding to myofibrils; 50% of enzyme was released at 0.23, 0.24, 1.3, and 2.0 mM for fructose 6-phosphate, Mg.ATP, ATP and AMP, respectively. However, the addition of a protein crowding agent, poly(ethylene)glycol, greatly enhanced PFK binding to myofibrils, particularly at high pH (8.0) or high [KCl]. The studies suggest that reversible binding of PFK to myofibrils may be an important factor in the control of anaerobic glycolysis in vivo, especially under the cellular conditions of burst swimming exercise.

Actins↗
PubMed1993

Scanning electron microscopy of muscle myofibrils after high pressure freezing and freeze-substitution-staining.

A novel approach to study the three dimensional ultrastructure of organelles and cells by means of scanning electron microscopy is described. Muscle myofibrils have been used in the development of the techniques since their structure is well characterized using conventional electron microscopic methods. Myofibrils in rigor buffer (with no cryo-protectants or pressure sealants) were frozen at high pressure (2300 bar) within specially designed chambers. The frozen specimens were then freeze-substituted-stained with methanol containing tungsten and iron salts and finally critical point dried. These methods allowed scanning electron microscopic observations of the organization of individual filaments within whole myofibrils over several sarcomeres. Images obtained showed excellent structural preservation with three dimensional information which is not available with other electron microscopic techniques. Success in these approaches was ascribed to (a) rapid and uniform freezing at high pressure without ice segregation patterns, (b) uniform electro-conductivity of the specimen closely attached to the polished carbon piston/carrier, and (c) good electron emission (secondary and back-scattered) from the metal incorporated into the myofibril structure without additional coating.

Animals↗
PubMed1975

Microscopic observations on the interaction of heavy meromyosin-S-1 and actin in myofibrils.

The binding of the proteolytic myosin fragment, HMM-S-1, to the non-overlapping part of actin filaments in intact myofibrils can be demonstrated under the phase contrast microscope as a contrast reversal of striation. The same effect can be seen on ghost myofibrils (after myosin extraction) where the whole length of the I-filaments is bare. HMM-S-1-loaded ghost myofibrils contracted upon addition of ATP in agrement with the recent report of Oplatka et al. (1974a, b) but under the same conditions ghost myofibrils not treated with HMM-S-1) also contracted. If the ghosts were prepared under conditions more favourable to myosin extraction, contraction became nil or negligible even when we loaded then ghosts with S-1. Thus we attribute the effect described by Oplatka's group to a small numer of residual myosin filaments in the ghosts.

Actins↗
PubMed1995

Morphological characters and disturbance of myofibril assembly in avian myoblasts fused with HVJ (Sendai virus).

We have been studying myogenic differentiation, especially the mechanism of myotube formation, using quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus, QM-RSV cells. Myogenic differentiation of QM-RSV cells can be controlled by the incubation temperature. When cultured at 35.5 degrees C, a permissive temperature for the virus, the cells proliferate but do not differentiate, whereas, when incubated at 41.0 degrees C, a non-permissive temperature, they proceed through myogenic differentiation and then form myotubes. Presumptive QM-RSV cells cultured at 35.5 degrees C were fused with hemagglutinating virus of Japan (HVJ, Sendai virus) and the morphological characteristics of the fused myoblasts were compared with the myotubes formed spontaneously at 41.0 degrees C. The artificially fused myoblasts were tubular, like the spontaneously formed myotubes, and their tubular form was based on their microtubules. However, assembly of myofibrils was seen in spontaneously formed myotubes cultured at 41.0 degrees C, but not in artificially fused myoblasts cultured at 35.5 degrees C. Instead of myofibrils, round or rod-shaped structures were seen in artificially fused myoblasts. These structures were stained with rhodamine-labeled phalloidin but not anti-myosin antibody and were formed at 35.5 degrees C. On shift up to 41.0 degrees C, these structures disappeared, and myofibrils were formed, suggesting that the actin-positive structures in artificially fused myoblasts at 35.5 degrees C were disassembled forms of myofibrils.

Actins↗
PubMed1975

Removal of Z-lines and alpha-actinin from isolated myofibrils by a calcium-activated neutral protease.

A calcium-activated factor (CaAF) has been isolated and partially purified from the post-myofibrillar supernatant fraction of rabbit skeletal muscle. The 200-fold purified CaAF hydrolyzed denatured casein, [3-H]acetyl hemoglobin, and N-ethyl[3-H]maleimide-labeled alpha-actinin. The proteolytic activity has a pH optimum at 6.9 and is dependent on the presence of Ca2+ (optimum concentration, 10 mM). Digestion of isolated myofibrils with CaAF results in removal of Z-lines and in a parallel loss of a 90, 000-dalton protein that has a mobility identical with that of alpha-actinin as determined by polyacrylamide gel electrophoresis. A protein with the properties of alpha-actinin (identical electrophoretic mobility, and ability to accelerate the Mg2+-activated ATPase of reconstituted actomyosin) was isolated from the supernatant of CaAF-treated myofibrils. The release of alpha-actinin from myofibrils by the calcium-activated neutral protease occurs in the absence of detectable change in the electrophoretic profiles of the other myofibrillar proteins, or in the ethylene glycol bis(beta-aminoethyl ether)-N, N' tetraacetic acid (EGTA) sensitivity of Mg2+-activated ATPase. In contrast to the specific removal of Z-lines and of alpha-actinin by CaAF, trypsin treatment of myofibrils results in extensive degradation of myosin heavy chains and of the inhibitory component of troponin (TN-I), and in loss of EGTA sensitivity of myofibrillar ATPase. The degradation of TN-I and loss of EGTA sensitivity occur before the Z-line disappearance.

Animals↗
PubMed1993

Effect of troponin T-treatment on the composition of rabbit skeletal myofibrils.

The effect of several conditions of troponin T-treatment was examined on the SDS-gel electrophoretic patterns of rabbit skeletal myofibrils. More troponin C and I were removed from the myofibrils after troponin T-treatment at acidic pH (5.6-6.2) than after treatment at neutral pH (6.8-7.4). Incubation of the myofibrils (0.4 mg/ml) in the presence of 0.043 mg/ml troponin T at a pH 6.2 for 60 min at 25 degrees C resulted in the removal of most troponin C and I from the myofibrils. Troponin T1 (N-terminal 158 residue fragment of troponin T of 259 residues) showed much lower troponin C.I removing activity than troponin T.

Animals↗
PubMed1995

Pathogenesis of heart myofibril lesion in experimental vitamin D-induced cardionecrosis.

Multifocal cardionecrosis has been produced in rats by treatment with 3 x 100,000 iu vitamin D3 (calciol). The effects of hypervitaminosis D on rat heart myofibril structure and total protease activity was investigated. Proteolytic enzyme activity of heart muscle homogenate was determined in two independent ways, and was approximately two times higher in the necrotic heart homogenate than in the control rat heart. Electron microscopic examinations showed structural derangements. Myofibrils isolated from necrotic heart exhibited significant changes of ultrastructure in the region of Z-line and I-band. Myofibril enzyme activity (Mg(2+)-ATPase) measurements demonstrated functional deficits as well. Under different conditions of myofibril isolation, it was shown that both ultrastructural and enzymatic lesions appear to be mediated by calcium-activated proteolytic enzymes operating in situ. Our results indicate that the increased proteolytic activity caused by vitamin D treatment leads to the in situ damage of proteins of the heart contractile system.

Animals↗
PubMed1993

Active tension generation in isolated skeletal myofibrils.

Single or double myofibrils isolated from rabbit psoas muscle were suspended between a fine needle and an optical force transducer. By using a photodiode array, the length of every sarcomere along the specimen could be measured. Relaxed specimens exhibited uniform sarcomere lengths and their passive length-tension curve was comparable to that of larger specimens. Most specimens could be activated and relaxed four to five times before active force levels began to decline; some specimens lasted for 10-15 activation cycles. Active tension (20-22 degrees C) was reproducible from contraction to contraction. The contractile response was dependent on initial sarcomere length. If initially activated at sarcomere lengths of > or = 2.7 microns, one group of sarcomeres usually shortened to sarcomere lengths of 1.8-2.0 microns, while the remaining sarcomeres were stretched to longer lengths. Myofibrils that were carefully activated at shorter initial sarcomere lengths usually contracted homogeneously. Both homogeneous and inhomogeneous contractions produced high levels of active tension. Calcium sensitivity was found to be comparable to that in larger preparations; myofibrils immersed in pCa 6.0 solution generated 30% of maximal tension, while pCa 5.5-4.5 resulted in full activation. Active tension at full overlap of thick and thin filaments ranged from 0.34 to 0.94 N mm-2 (mean of 0.59 N mm-2 +/- 0.13 SD. n = 65). Even allowing for a maximum of 20% nonmyofibrillar space in skinned or intact muscle fibres, the mean tension generated by isolated myofibrils per cross-sectional area is higher than by fibre preparations.

Animals↗
PubMed2004

About the T-system in the myofibril-free sarcoplasm of the frog muscle fibre.

Previous investigations of the T-system in skeletal muscle fibres described the inter-myofibrillar relationships between T-tubules and the sarcoplasmic reticulum. They disregarded the arrangement of the T-system in the myofibril-free sarcoplasm in the area of muscle fibre nuclei. In the present investigation, the T-system was filled by means of lanthanum incubation and the myofibril-free sarcoplasm was ultrastructural examined by means of thin (< or = 100 nm) as well as thick sections (> 300 nm-1 microm) with the electron microscope. The investigation of thick sections revealed that T-tubules meander through this myofibril-free sarcoplasm and tangle up at the poles of muscle fibre nuclei and in the area of fundamental nuclei of the motor end plate. They are, far from myofibrils, in proximity to these nuclei, the Golgi apparatus and mitochondria. On basis of this proximity and their openings at the muscle fibre surface, a contribution at the drainage of metabolic products and at the local calcium control is discussed.

Animals↗
PubMed2000

Dynamic distribution of an antigen involved in the differentiation of avian myoblasts: II. Possible association of beta1 integrin with myofibril organization.

Previous studies have shown that a monoclonal antibody, H-145, inhibits myotube formation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) [Hyodo and Kim, 1994: Exp. Cell Res. 212:120-131]. The antigen recognized by H-145 (H-145 antigen), which is a glycoprotein with a molecular mass of about 116 kDa, is related to a step immediately before myoblast fusion. To determine the functional significance of H-145 antigen, we examined its dynamic state during myogenic differentiation of QM-RSV cells. H-145 antigen showed a unique and discrete distribution. In immature myotubes immediately after myoblast fusion, many ring-like structures of H-145 antigen appeared on the ventral surface of the cells, encircling the actin dots detected simultaneously by immunofluorescence and interference reflection microscopy. The core of the ring-like structures was filled with the termini of actin bundles, mainly formed by alpha-actin. Other cytoskeletal-associated proteins, such as vinculin and alpha-actinin, were also associated with these structures. The ring-like structures of H-145 antigen were observed only during a restricted period when myoblasts fused actively, suggesting their relationships to myotube formation and an early stage of myofibril formation. With maturation of the myotubes, most of the H-145 antigen became redistributed in linear arrays on the apical cell surface and was probably associated with the termini of actin bundles to organize myofibrils, suggesting that the antigen was also related to maturation of myotubes. Experiments using monoclonal antibodies against chick beta1 integrin showed that H-145 antigen is beta1 integrin or a very closely related derivation. Thus H-145 antigen (beta1 integrin) is possibly involved in both myoblast fusion and the myofibril organization in myotubes.

Actins↗
PubMed2001

Genetics of the Drosophila flight muscle myofibril: a window into the biology of complex systems.

This essay reviews the long tradition of experimental genetics of the Drosophila indirect flight muscles (IFM). It discusses how genetics can operate in tandem with multidisciplinary approaches to provide a description, in molecular terms, of the functional properties of the muscle myofibril. In particular, studies at the interface of genetics and proteomics address protein function at the cellular scale and offer an outstanding platform with which to elucidate how the myofibril works. Two generalizations can be enunciated from the studies reviewed. First, the study of mutant IFM proteomes provides insight into how proteins are functionally organized in the myofibril. Second, IFM mutants can give rise to structural and contractile defects that are unrelated, a reflection of the dual function that myofibrillar proteins play as fundamental components of the sarcomeric framework and biochemical "parts" of the contractile "engine".

Animals↗
PubMed1993

Contractile protein dynamics of myofibrils in paired adult rat cardiomyocytes.

The purpose of this study was to determine how quickly contractile proteins are incorporated into the myofibrils of freshly isolated cardiomyocytes and to determine whether there are regions of the cells that are more dynamic than others in their ability to incorporate the proteins. Paired cardiomyocytes joined at intercalated discs and single cells were isolated from adult rats, and microinjected 3 hours later with fluorescently labeled actin, alpha-actinin, myosin light chains and vinculin. The cells were fixed and permeabilized at various period, 5 seconds and longer, after microinjection. Actin became incorporated throughout the I-Bands in as short a time as 5 seconds. The free edges of the cells, which were formerly intercalated discs, exhibited concentrations of actin greater than that incorporated in the I-Bands. This extra concentration of actin was not detected, however, at intact intercalated discs connecting paired cells. Alpha-actinin was incorporated immediately into Z-Bands and intercalated discs. Vinculin, also, was localized at the Z-Bands and at intercalated discs, but in contrast to alpha-actinin, there was a higher concentration of vinculin in the region of the intact intercalated discs. Both alpha-actinin and vinculin were concentrated at the free ends of the cells that were formerly parts of intercalated discs. Myosin light chains were observed to incorporate into the A-Bands in periods as short as 5 seconds. These results suggest that the myofibrils of adult cardiomyocytes may be capable of rapid isoform transitions along the length of the myofibrils. The rapid accumulation of fluorescent actin, alpha-actinin, and vinculin in membrane sites that were previously parts of intercalated discs, may reflect the response to locomotory activity that is initiated in these areas as cells spread in culture. A similar response after an injury in the intact heart could allow repair to occur.

Actins↗
PubMed1994

Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils.

The morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin-binding protein (ABP) filamin is associated with stress fiber-like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z-disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z-disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two-dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non-striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14-somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms.

Animals↗
PubMedDate not supplied

Ca-activated neutral protease and its inhibitors: in vitro effect on intact myofibrils.

SDS gel electrophoresis of troponin and myosin extracted from chicken myofibrils pretreated with Ca-activated neutral protease showed a pattern similar to that observed in the muscles of patients with Duchenne muscular dystrophy, that is, a decrease in troponin-I and troponin-C with relative preservation of troponin-T and degradation of the heavy chain of myosin. Also, alpha-actinin and troponin-C were released from myofibrils. Two inhibitors of Ca-activated neutral protease, leupeptin and E-64, inhibited the degradative action of Ca-activated neutral protease on intact myofibrils in vitro.

Agmatine↗