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PubMed1993

The binding of fluorescent phallotoxins to actin in myofibrils.

Fluorescence microscope observation of myofibrils incubated with rhodamine-phalloidin and coumarine-phallacidin showed an initial appearance of fluorescence bands at the Z-lines and near the middle of the sarcomeres indicating preferential binding of dye to actin subunits located at both actin filament ends. After long incubation times (1-3 h) however, a final pattern is reached which consists of fluorescent Z-lines in the center of uniformly labelled actin bands, with greater fluorescence in the Z-lines than in the uniform region outside the Z-lines. Increasing the temperature or the ionic strength increased the rate of change to the final pattern. These data indicate: (1) that the ends of the actin filament are kinetically more accessible to phallotoxins; (2) at long times when equilibrium binding presumably occurs, the concentration of actin subunits in the Z-band is greater than in the rest of the sarcomere.

Actins↗

PubMed1982

Myofibril and sarcoplasmic reticulum changes with exercise and growth.

The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), spring (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p less than or equal to 0.05). In the 51 day E group ATPase activity (0.378 +/- 0.009 mumol Pi X mg-1 X min-1) was greater than at 10 and 21 days (0.307 +/- 0.006 and 0.323 +/- 0.008 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). No change occurred in the S group from 10 to 21 and 51 days (p greater than or equal to 0.05). Both the 21 and 51 day S (0.318 +/- 0.011 and 0.399 +/- 0.010 mumol Pi X mg-1 X min-1) and E (0.323 +/- 0.008 and 0.378 +/- 0.009 mumol Pi X mg-1 X min-1) groups had higher activity compared to the C group (0.193 +/- 0.029 and 0.172 +/- 0.031 mumol Pi X mg-1 X min-1) (p less than or equal to 0.05). Maturation (10--51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca2+ binding (p less than or equal to 0.05) while Ca2+ uptake ability did not change (p greater than or equal to 0.05). SR yield, Ca2+ binding and uptake were not altered with S training (p greater than or equal to 0.05). The E training resulted in greater Ca2+ uptake at 51 days compared to C and S (p less than or equal to 0.05), with no change in Ca2+ binding (p greater than or equal to 0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.

Adenosine Triphosphatases↗

PubMed1992

Proteolytic activity of isolated lamb calpains on myofibrils under the conditions of pH, Ca2+ concentration and temperature existing in postmortem muscle.

The two calcium-activated neutral proteinases (calpains I and II) and their specific inhibitor were isolated by ion exchange chromatography in DEAE-Sephacel from lamb skeletal muscle (longissimus dorsi). Their proteolytic activities were then determined using myofibrils as substrate. The Ca2+ requirements were different for each form of the enzyme: calpain I needed only 50 mumol Ca2+ for half-maximal activity, while the other isoenzyme, calpain II, needed 1,000 mumol Ca2+ for reaching 50% of its maximum activity. Both calpains showed a relevant activity in the pH range 5.5-6.5 (over 40% of maximum activity found at pH 7.5). With regard to the effect of temperature, both isoenzymes retained about 25% of their activity at 25 degrees C with a temperature reduction down to 4 degrees C. It is concluded that calpain I is an active protease under conditions similar to that prevalent in lamb meat during postmortem storage.

Animals↗

PubMed1990

Morphometric comparison of mitochondria and myofibrils of cardiomyocytes between hypertrophic and dilated cardiomyopathies.

We performed an ultrastructural, morphometric comparison of mitochondria and myofibrils of cardiomyocytes using endomyocardial biopsy specimens in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Biopsies came from the right ventricular side of the interventricular septum in nine patients with HCM, nine with DCM, and nine controls with arrhythmia and/or ST depression. Morphometric analysis was carried out using electron microscopic photographs and an image analyser. Mitochondria were significantly greater in number and smaller in size in HCM than in the control group. In DCM, the size of mitochondria was also significantly smaller than in the control group, although their number was similar to that of the control group. No statistically significant difference was found regarding the size of mitochondria between HCM and DCM. The percentages of both mitochondrial and myofibrillar areas in cytoplasm were smaller in the DCM than the HCM and control groups, though no difference was seen between the latter two. The ratio of mitochondrial area to myofibrillar area was almost the same in each group. These results suggest increased mitochondrial function to match hypertrophic cardiomyocytes in HCM, and decreased mitochondrial function and cardiomyocytic contractility in DCM.

Adolescent↗

PubMed1991

Vertebrate muscle Z-line structure: an electron microscopic study of negatively-stained myofibrils.

Structural features of the Z-lines of rabbit psoas muscle myofibrils have been studied in the electron microscope with a negative staining technique. The results obtained suggest the presence of about 20 nm periodicity in the structural organization of the Z-line region: a band pattern of five bands of extra density spaced about 20 nm apart was revealed in the Z-region and the Z-filaments connecting actin filaments from neighbouring sarcomeres often appeared to be positioned at intervals of 17-20 nm. An electron microscopic investigation of the interaction in vitro of two major Z-line proteins, alpha-actinin and F-actin, indicated that the positions of alpha-actinin bridges between actin filaments are defined by relative azimuthal positions of actin subunits. A possible arrangement of actin-linking macromolecular bridges in the Z-region is considered. It is supposed that the arrangement of the Z-filaments is related to the helical symmetry of actin-containing filaments. Also, the banded appearance of the Z-region is interpreted as arising from the arrangement of crossbridges connecting thin filaments of the same sarcomeres.

Actinin↗

PubMed2003

The mode of myofibril remodelling in human skeletal muscle affected by DOMS induced by eccentric contractions.

Myofibrillar Z-disc streaming and loss of the desmin cytoskeleton are considered the morphological hallmarks of eccentric contraction-induced injury. The latter is contradicted by recent studies where a focal increase of desmin was observed in biopsies taken from human muscles with DOMS. In order to determine the effects of eccentric contraction-induced alterations of the myofibrillar Z-disc, we examined the distribution of alpha-actinin, the Z-disc portion of titin and the nebulin NB2 region in relation to actin and desmin in DOMS biopsies. In biopsies taken 2-3 days and 7-8 days after exercise, we observed a significantly higher number of fibres showing focal areas lacking staining for alpha-actinin, titin and nebulin than in biopsies taken from control or 1 h after exercise. None of these proteins were part of Z-disc streamings but instead they were found in distinct patterns in areas characterised by altered staining for desmin and actin. These were preferentially seen in regions with increased numbers of sarcomeres in parallel myofibrils. We propose that these staining patterns represent different stages of sarcomere formation. These findings therefore support our previous suggestion that muscle fibres subjected to eccentric contractions adapt to unaccustomed activity by the addition of new sarcomeres.

Actins↗

PubMed2006

No direct effect of creatine phosphate on the cross-bridge cycle in cardiac myofibrils.

Creatine phosphate (CP) and creatine kinase (CK) are involved in the rapid resynthesis of ATP and thereby serve to stabilize ATP concentration and to maintain free ADP low inside cardiac muscle cells during contraction. Recently, it has been suggested from experiments in permeabilized multicellular preparations that CP/CK also regulate the kinetics of the actomyosin interaction (cross-bridge cycle) and may explain contractile dysfunction, for instance, during ischemia. However, the reported effects of CP/CK may be confounded by diffusion limitations in multicellular preparations in which inorganic phosphate (P(i)) and ADP may significantly accumulate during contraction. To test this hypothesis, we measured force production and the rates of force development (k (ACT) and k (TR)) in isolated cardiac myofibrils, in which rapid concentration changes of Ca(2+), CP/CK, and P(i) were imposed using a rapid perfusion change system. The results showed that CP/CK did not influence maximum force-generating capacity, whereas P(i) markedly reduced force and increased the rates of force development. No effects of CP/CK on the rates of force development were observed, consistent with the notion that CP/CK do not exert a direct effect on the actomyosin interaction.

Actomyosin↗

PubMed1983

Equilibrium partition studies of the interaction between aldolase and myofibrils.

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 M-1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10-12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 M-1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25-30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca2+ and fructose-1,6-bisphosphate that then prevail.

Animals↗

PubMed1985

Effect of thermodynamic nonideality on the subcellular distribution of enzymes: adsorption of aldolase to muscle myofibrils.

An expression is derived whereby allowance may be made for the effects of thermodynamic nonideality on the biphasic interaction of a macromolecular solute with an immobilized reactant. This quantitative description, written in terms of activity coefficients expressed as virial coefficients on the basis of excluded volume, also takes into account the space-filling effect of an inert macromolecule present in the reaction mixture. Advantage is then taken of the theory to consider the effect of bovine serum albumin on the interaction of aldolase with bovine cardiac muscle myofibrils in I 0.158 imidazole-chloride buffer, pH 6.8. Partition equilibrium studies are used to establish that inclusion of a moderate concentration (14 mg/ml) of serum albumin in reaction mixtures leads to a 35-40% increase in the apparent binding constant written in terms of reactant molarities, and that the enhancement is attributable entirely to nonideality inasmuch as the same thermodynamic binding constant pertains. This investigation of thermodynamic nonideality arising from the space-filling effects of inert macromolecules on enzyme partition reinforces the possibility that some enzymes may be distributed between soluble and adsorbed states in the highly concentrated macromolecular environment of the cell cytoplasm.

Adsorption↗

PubMed1976

Effects of ionic strength on calcium binding to rabbit skeletal myofibrils, thin filaments and myosin.

Calcium binding by rabbit skeletal myosin, thin filaments and myofibrils was measured in solutions with and without 2 mM MgATP and with ionic strengths adjusted with KCl to 0.05, 0.10 and 0.14 M. Free Mg2+ was held constant at 1 mM, pH at 7.0 and temperature at 25 degrees C. In the presence of MgATP, the relation between free Ca2+ and myofibrillar bound calcium shifted to the left as ionic strength was decreased from 0.14 to 0.05 M. In the absence of MgAPT, myofibrillar calcium binding was enhanced over a wide range of free Ca2+ concentration, but calcium binding was no longer a function of ionic strength. Similarly, calcium binding by thin filaments and myosin was unaffected by changes in ionic strength from 0.05 to 0.14 M. In view of evidence that cross-bridge connections between thick and thin filaments increase as ionic strength decreases, our results suggest that these connections enhance myofibrillar calcium binding. These results thus confirm previous data of Bremel and Weber (Bremel, R. D. and Weber, A. (1972) Nature New Biol. 238, 97-101) who first showed that nucleotide-free cross-bridge connections enhance thin filament calcium binding.

Animals↗

PubMed1978

Purification and characterization of a myosin-cleaving protease from rat heart myofibrils.

A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin.

Animals↗

PubMed1981

Purification of cathepsin D from rabbit skeletal muscle and its action towards myofibrils.

Cathepsin D (EC 3.4.23.4) was purified from rabbit skeletal muscle using acetone-dried muscle powder as starting material. After the acetone-dried powder was extracted with 0.2 mM ATP, the extract was fractionated with acetone an subjected to DEAE-Sephadex A-50 and Sephadex G-100 column chromatography. Rechromatography on a Sephadex G-100 column resulted in a purified preparation. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed one major band of 42,000 daltons and some bands of contaminants. Since gel filtration also indicated a value of 42,000 daltons for the enzyme, it was concluded that muscle cathepsin D has no subunit structure. The enzyme acted optimally towards myofibrils around pH3, resulting in the degradation of the myosin heavy chain and production of a 30,000-dalton component.

Animals↗

PubMed1986

Age changes of myofibrils of human minor pectoral muscle.

Micro- and ultramicromeasuring studies have been made on the age changes of surgically resected minor pectoral muscles in 200 Japanese females of various ages (26-80 years). With advancement of age, muscle fibers of type I decreased in number and increased in size, and the age-related increase in size is not considered to be due to an increase in number of myofibrils but seems to be due to an increase in their volume resulting from an increase in the number of myofilaments.

Adult↗

PubMed1991

Chemomechanical coupling in actomyosin system: an approach by in vitro movement assay and kinetic analysis of ATP hydrolysis by shortening myofibrils.

On the basis of our recent studies of the sliding distance of actin filaments during one ATP cycle on the surface of myosin-coated glass surface and ATP hydrolysis by rapidly shortening myofibrils, the molecular mechanism of chemomechanical coupling is considered. We conclude that the myosin head can repeat many active cyclic interactions with actins to drive the actin filaments over a long distance during one ATP cycle, and that the distance is variable depending on the load.

Actomyosin↗

PubMed1985

Association of chicken pectoralis muscle phosphorylase with the Z-line and the M-line of myofibrils: comparison with 'amorphin', the amorphous component of the Z-line.

By immunofluorescence technique, glycogen phosphorylase, one of the soluble glycolytic enzymes, was shown to be localized in the Z-line of chicken pectoralis muscle myofibrils, in addition to the M-line, as previously reported by Heizmann, C.W. and Eppenberger, H.M. (Heizmann, C.W. and Eppenberger, H.M. (1978) J. Biol. Chem. 253, 270-277). After extraction of thick filaments by a solution containing pyrophosphate and high salt (Hasselbach-Schneider solution), or after extraction of thin and thick filaments by a solution containing 0.6 M KI, phosphorylase still remained in the Z-line. Amorphin (Mr 85 000) was reported by Chowrashi, P.K. and Pepe, F.A. (Chowrashi, P.K. and Pepe, F.A. (1982) J. Cell Biol. 94, 565-573) as a new Z-line amorphous component. The amino acid composition of amorphin reported by them was very similar to that of phosphorylase b reported by Heizmann and Eppenberger. The partially purified 85 kDa protein, according to Chowrashi and Pepe, showed cross-reactivity to anti-phosphorylase serum and phosphorylase activity. Although amorphin was reported to be eluted from DEAE-column chromatography around the gradient of 0.5 M KCl, little protein was eluted around 0.5 M KCl in our experiments, and the 85 kDa protein which we identified as phosphorylase b was eluted around 0.2 M KCl. Hence, it should be said that the major 85 kDa protein extracted according to Chowrashi and Pepe was phosphorylase b.

Animals↗

PubMed2000

The effect of inorganic phosphate on force generation in single myofibrils from rabbit skeletal muscle.

In striated muscle, force generation and phosphate (P(i)) release are closely related. Alterations in the [P(i)] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P(i)] in single or very thin bundles (1-3 microM in diameter; 5 degrees C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P(i)] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P(i)]. Steady-state isometric force decreases linearly with an increase in log[P(i)] in the range 500 microM to 10 mM (slope -0.4/decade). Between 5 and 200 microM P(i), the slope of the relation is smaller ( approximately -0.07/decade). The rate constant of force development (k(TR)) increases with an increase in [P(i)] over the same concentration range. After rapid jumps in [P(i)], the kinetics of both the force decrease with an increase in [P(i)] (k(Pi(+))) and the force increase with a decrease in [P(i)] (k(Pi(-))) were measured. As observed in skinned fibers with caged P(i), k(Pi(+)) is about three to four times higher than k(TR), strongly dependent on final [P(i)], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P(i)] is slower: k(Pi(-)) is indistinguishable from k(TR) measured at the same [P(i)] and has the same calcium sensitivity.

Adenosine Diphosphate↗

PubMed1986

Saturation transfer electron paramagnetic resonance study of the mobility of myosin heads in myofibrils under conditions of partial dissociation.

The rotational motion of rigidly spin-labeled myosin heads of glycerinated myofibrils as reflected in saturation-transfer EPR spectra behaves to a first approximation as though the heads consist of two populations with different rotational motions. An immobilized fraction has a correlation time (tau 2) of approximately 0.5 ms, comparable to that of spin-labeled subfragment-1 (S1) bound to thin filaments, while a mobile fraction has a tau 2 of 10 microseconds, comparable to that of the heads of purified myosin filaments. The effects of nonhydrolyzable ATP analogues, potassium pyrophosphate (PPi), or adenylyl imidodiphosphate, Ca2+, temperature, or ionic strength on the spectra can be analyzed in terms of the fraction of myosin heads immobilized by attachment to thin filaments, without requiring changes in the motion of either attached or detached heads.

Adenylyl Imidodiphosphate↗

PubMed1984

ATPase activity and force production in myofibrils and twitch characteristics in intact muscle from neonatal, adult, and senescent rat myocardium.

To determine whether age-associated alterations in the cardiac muscle twitch could be related to altered myofibrillar ATPase activity or to an altered force-pCa relationship, these variables were measured in rat cardiac preparations across a broad age range. Between 2 and 6 months, maximum ATPase activity in detergent treated myofibrils decreased approximately two fold (0.166 +/- 0.01 v. 0.078 +/- 0.02 microM Pi/min X mg protein, P less than 0.001), but did not change with further aging (12 or 24 months). The Ca2+-dependent force in thin 'Triton skinned' papillary muscles was not age-related. ATPase activity and force exhibited identical Ca2+ sensitivity from the submicromolar to micromolar range: for ATPase activity pCa for 50% activity averaged 6.1 and Hill coefficients averaged 4.5; pCa for 50% force development was 6.1 and Hill coefficients of the force-pCa relation averaged 4.5; no age differences in these parameters were observed. In the intact muscles prior to skinning, neither twitch force nor the maximum rate of force production were age-related; however, indices of the time course of contraction, time to peak force, half relaxation time, and their sum, increased progressively, changing by approximately 30% from 2 to 24 months (P less than 0.001). Since the decline in ATPase activity occurred over the maturational period only, and did not change with further ageing, while the twitch duration changed progressively with age, it is concluded that the twitch prolongation of the senescent myocardium cannot be directly related to the age-related decline in myofibrillar ATPase activity.

Adenosine Triphosphatases↗