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At least 19 recordsLinked to original sources
PubMed1976

Modulation of Ca2+ control of dog and rabbit cardiac myofibrils by Mg2+. Comparison with rabbit skeletal myofibrils.

Increases in free Mg2+ from 0.04 to 10.0 mM with constant pH 7.0 TO 0.10 M ionic strength, and 2 mM MgATP2- caused a rightward shift of the free Ca-relative ATPase relation for both cardiac skeletal myofibrils. The specific activity of cardiac myofibrillar ATPase over a wide range of free Ca2+ was, however, depressed in 0.04 vs. 1.0 mM Mg2+, whereas a similar decrease in free Mg2+ slightly enhanced skeletal myofibrillar ATPase. Lowering free Mg2+ from 1.0 to 0.04 mM caused similar increases in cardiac and skeletal myofibrillar bound calcium, which were largely attributable to increased calcium binding to myofibrillar myosin. Raising free Mg2+ from 1.0 to 10.0 mM caused only a slight decrease of skeletal myofibrillar bound calcium, and this change was attributable to myofibrillar myosin. The same increase in free Mg2+ caused cardiac myofibrils to bind increased amounts of calcium and this change was not attributable to myofibrillar myosin. By subtracting calcium bound to myofibrillar myosin, we were able to estimate calcium binding by myofibrillar troponin. The transition between basal and maximal ATPase in 1.0 and 10 mM Mg2+ was found to be assocciated with binding of an additional 2 mol/mol of either skeletal or cardiac myofibrillar troponin.

Adenosine Triphosphatases↗
PubMed1976

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.

Adenosine Diphosphate↗
PubMed1979

Studies on the post-mortem fragmentation of myofibrils.

1. There was a close relationship between the fragmentation of myofibrils and the tension developed during post-mortem contraction of muscle. The extent of fragmentation was at its maximum when the sarcomeres attained a length of 2.0 to 2.2 micron. 2. The rate of fragmentation of myofibrils depended upon the calcium ion concentration within a range of 10(-5) to 2 x 10(-2) M, with a minimum at pH 6.5. The fragmentation of myofibrils free from muscle fibers was not affected by 10 mM iodoacetate, an irreversible inhibitor of calcium-activated factor (CAF). 3. Incubation of myofibrils with 10 mM CaCl2 caused the release of about 12% of the total myofibrillar proteins after homogenization. The protein solution contained little alpha-actinin, and considerable amounts of 54,000- and 76,000-dalton components which seem to originate from the Z-line. SDS-polyacrylamide gels of troponin prepared from the incubated myofibrils did not change with time of incubation. These findings are in contrast with the proteolytic degradation of Z-lines by CAF treatment, in which alpha-actinin and 87,000 dalton component are released. 4. These data directly demonstrate that the in vitro fragmentation of post-mortem muscle (i.e. duirng its conversion into myofibrils upon mechanical homogenization) is different from that induced by CAF. The possible role of calcium ions during in vitro fragmentation of myofibrils is discussed.

Animals↗
PubMed1979

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.

Actinin↗
PubMed1976

Mechanism of myofibril growth and proliferation in fish muscle.

The mechanisms of myofibril growth proliferation were investigated in the red and white muscles of fish. In both types of muscle the ratio of lattice filament spacings between the Z disk and M line was found to be greater than that required for perfect transformation of a square into a hexagonal lattice. This mismatch was considered to result in the thin filaments being pulled obliquely instead of at right angles to the Z disk. The angle of pull of the thin filaments was measured in longitudinal sections. The splitting process was found to decrease the degree of pull. Splitting was also observed in transverse sections of the peripheral myofibrils. In both red and white fibres these myofibrils were found to commence splitting when they reached a size of approximately 1-2 mum diameter. Evidence from ultrastructural and autoradiographical studies suggested that growth of the myofibrils within the fibres is centrifugal. The outermost myofibrils appear to be the ones which are being built up and which split. The data indicated that in fish muscle a considerable number of filaments may be added to the daughter regions whilst splitting of the myofibril is still continuing.

Animals↗
PubMed1978

Cardiac myofibril disorientation and Z band abnormalities in idiopathic cardiomyopathy. An electron microscope study.

Morphological alterations in idiopathic cardiomyopathy were examined and their clinicopathological significance was discussed. Six cases of congestive type and five of hypertrophic type chosen from 22 patients according to our strict criteria were examined by light and electron microscopy. The diameter values of cardiac muscle fibers were broadly distributed with multiple peaks in congestive cases and with 4 peaks in the hypertrophic cases in contrast to the 2 peaks in controls. The chief electron microscopic alterations consisted of myofibril disorientation and Z band abnormalities. The congestive cases were characterized by decreased and disdarrayed myofibrils (loose myofibril disorientation), wheras the hypertrophic cases by abundant myofibrils characteristically entangled with each other (tight myofibril disorientation). The Z band showed 3 types of abnormalities. Type 1 was characterized by widening, bifurcation and overriding of Z bands while the regular sarcomere was retained. The changes were more conspicuous in the hypertrophic cases than in the congestive cases. Type 2 was represented by clumping of Z band substance, and Type 3 by an interwoven form of electron dense fibers. Types 2 and 3 were seen in some of the hypertrophic and congestive cases. The tight myofibril disorientation and Type 1 of Z band abnormalities were considered important cytopathological clues for the diagnosis of the hypertrophic type of idiopathic cardiomyopathy.

Adult↗
PubMed1977

Connectin, an elastic protein of muscle. Its abundance in cardiac myofibrils.

Pure myofibrils were isolated from bovine heart by sucrose layer ultracentrifugation. Cardiac myofibrils thus prepared contained more protein as insoluble stroma than skeletal muscle. The insoluble stroma largely consisted of connectin, an elastic protein of muscle. The connectin content in cardiac myofibrils was about 18% of the total myofibrillar protein and was three times that in skeletal myofibrils. In view of the role of connectin as an elastic component of muscle, the abundance of connectin in cardiac myofibrils may be responsible for keeping myofibrils short at rest. This would account for the more effective tension generation in cardiac muscle on passive stretching due to blood inflow (Stirling's law).

Amino Acids↗
PubMed1979

Differential response of myofibrils and 10-nm filaments to a cocarcinogen.

Multinucleated myotubes containing large numbers of striated myofibrils and large numbers of longitudinally-oriented 10-nm filaments were treated with the cocarcinogen phorbol-12-myristate-13-acetate (PMA) for 24, 48 or 72 hours. The inhibitory effects of PMA on the accumulation of myofibrils was evident within 24 hours, and by 72 hours virtually all striated myofibrils had disappeared. In contrast, the density of the 10-nm filaments was greatly enhanced in these myofibril-depleted myotubes. These effects were not due to a generalized cytotoxicity, for PMA stimulated the replication of the presumptive myoblasts and fibroblasts present in these cultures. 24 hours after removing the PMA, these myotubes assembled a new set of striated myofibrils and the density of 10-nm filaments diminished proportionately.

Animals↗
PubMed1977

Localization of creatine kinase isoenzymes in myofibrils. I. Chicken skeletal muscle.

Purified, repeatedly washed, skeletal muscle myofibrils contain approx. 0.2 U of creatine kinase (CK) activity (equivalent to 2.5 micrograms CK) per milligram dry weight; this firmly bound CK activity is estimated to represent 3-5% of the total cellular CK. It had been shown previously that the myofibrillar CK, which can be quantitatively extracted at low ionic strength and purified to homogeneity, is very similar, if not identical, to the bulk MM-CK. It is shown that the two protein preparations also have the same peptide pattern after cyanogen bromide fractionation and very similar specific activities, confirming their identity. The earlier demonstration that the bound CK is specifically located at the M-lines of isolated myofibrils has been confirmed by immunofluorescence. Antibodies directed against purified MM- and BB-CK were used in the indirect fluorescent antibody technique to study the specificity of myofibril binding sites for different forms of CK. With myofibrils from adult muscle, which has only MM-CK, as well as from early developmental stages in which BB-CK is the predominant isoenzyme, M-type CK was localized exclusively at the M-line, while greater or lesser amounts of B-type CK were found at the Z-line. The data provide strong evidence that the MM-CK at the M-lines in skeletal myofibrils is not adventitiously bound but is rather an integral element in the M-line structure. The amount of CK bound is reasonably consistent with the earlier proposal that the CK molecules might be the transverse M-bridges and appears to be sufficient to regenerate all of the ATP hydrolyzed during muscle contraction.

Animals↗
PubMed1979

Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase.

Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl-labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line.

Animals↗
PubMed1978

Preparation of a myosin-extracted "ghost" myofibril Sephadex conjugate column and its application to the separation of myosin subfragment-1 giving and not giving the initial burst of phosphate.

A "ghost" myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP can be prepared in a few days by conjugating "ghost" myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments. It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-1, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the "ghost" myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem, 80, 1371-1380), which used S-1 treated with CMB, was applied to the "ghost" myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A separation method based essentially on the principle employed by Taniguichi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.

Actins↗
PubMed1975

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Actomyosin↗
PubMed1976

Dry ice fixation of myofibrils for scanning electron microscopy.

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.

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↗
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↗
PubMed1978

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gröschel-Stewart.

Animals↗
PubMed1979

Desmin and vimentin coexist at the periphery of the myofibril Z disc.

Two-dimensional gel electrophoresis has revealed that vimentin, the predominant subunit of intermediate filaments in cells of mesenchymal origin, is a component of isolated skeletal myofibrils. It thus coexists in mature muscle fibers with desmin, the major subunit of muscle intermediate filaments. Antisera to desmin and vimentin, shown to be specific for their respective antigens by two-dimensional immunoautoradiography, have been used in immunofluorescence to demonstrate that vimentin has the same distribution as desmin in skeletal muscle. Both desmin and vimentin surround each myofibril Z disc and form honeycomb-like networks within each Z plane of the muscle fiber. This distribution is complementary to that of alpha-actinin within a given Z plane. Desmin and vimentin may thus be involved in maintaining the lateral registration of sarcomeres by transversely linking adjacent myofibrils at their Z discs. This linkage would support and integrate the fiber as a whole, and provide a molecular basis for the cross-striated appearance of skeletal muscle.

Animals↗
PubMed1978

Myofibril content of histochemical fibre types in rat skeletal muscle.

A modification of the histochemical method for myosin ATPase was used to determine the myofibril complement, mean myofibril size and myofibrillar packing of defined muscle fibre types in rat skeletal muscle. Fast muscle fibres (Types IIA and IIB) were found to have smaller myofibrils and a lower packing density than slower (Type I) fibres. These findings were discussed with respect to their relevance in estimations of muscle strength from histological and histochemical preparations of muscle cross-sections.

Animals↗