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At least 127 records · Page 7Linked to original sources
PubMed2004

Does phosphate release limit the ATPases of soleus myofibrils? Evidence that (A)M. ADP.Pi states predominate on the cross-bridge cycle.

The ATPases (+/-Ca2+) of myofibrils from rabbit soleus (a slow muscle) and psoas (a fast muscle) have different Ea: -Ca2+, 78 and 60 kJ/mol and +Ca2+, 155 and 71 kJ/mol, respectively. At physiological temperatures, the two types of myofibrillar ATPase are very similar and yet the mechanical properties of the muscles are different (Candau et al. (2003) Biophys J 85: 3132-3141). Muscle contraction relies on specific interactions of the different chemical states on the myosin head ATPase pathway with the thin filament. An explanation for the Ea data is that different states populate the pathways of the two types of myofibril because the rate limiting steps are different. Here, we put this to the test by a comparison of the transient kinetics of the initial steps of the ATPases of the two types of myofibril at 4 degrees C. We used two methods: rapid flow quench ('cold ATP chase': titration of active sites, ATP binding kinetics, k(cat); 'Pi burst': ATP cleavage kinetics) and fluorescence stopped-flow (MDCC-phosphate binding protein for free Pi; myofibrillar tryptophan fluorescence for myosin head-thin filament detachment and ATP cleavage kinetics). We find that, as with psoas myofibrils, the most populated state on the cross-bridge cycle of soleus myofibrils, whether relaxed or activated, is (A)M.ADP.Pi. We propose a reaction pathway that includes several (A)M.ADP.Pi sub-states that are either 'weak' or 'strong', depending on the mechanical condition.

Adenosine Diphosphate↗
PubMed1996

The effect of a thiadiazinone derived Ca2+ sensitizer on the responsiveness of Mg(2+)-ATPase to Ca2+ in myofibrils isolated from stunned and nonstunned porcine and human myocardium.

Previously, we showed, in an in situ porcine model, that the thiadiazinone derivative [+]EMD 60263, a putative Ca2+ sensitizer with minimal phosphodiesterase III inhibitory properties, increased contractility more profoundly in stunned than in nonstunned myocardium. The aim of the present investigation was to study the mechanism of action by determining the in vitro effects of [+]EMD 60263 on the Ca2+ responsiveness of the Mg(2+)-dependent ATPases of myofibrils and sarcoplasmic reticulum membrane vesicles, isolated from normal ventricle of swine and hypertrophic septum of cardiomyopathic patients. Contamination of the myofibrils with sarcoplasmic reticulum membranes was excluded by testing the effect of the sarcoplasmic reticulum Ca(2+)-pumping ATPase inhibitor thapsigargin. The plasma concentrations at which [+]EMD 60263 exerted its inotropic effect in the in situ porcine model were found to be submicromolar. [+]EMD 60263 stimulated concentration-dependently (1-10 microM) the submaximally activated Mg(2+)-ATPases (at pCa 6.1) of pig heart myofibrils. [+]EMD 60263 (10 microM) shifted the pCa50 of porcine myofibrillar Ca(2+)-stimulated, Mg(2+)-dependent ATPase from 6.00 +/- 0.05 to 6.67 +/- 0.05, whereas the [-]enantiomer EMD 60264 had no significant effect. Although the effect was much less at 1 and 3 microM, [+]EMD 60263 (10 microM) also stimulated maximal myofibrillar Mg(2+)-ATPase activity. The Hill coefficient, reflecting the steepness of the fitted pCa/Mg(2+)-ATPase curve at half-maximal activation, was not affected by [+]EMD 60263 (10 microM). [+]EMD 60263 (10 microM) had no effect on sarcoplasmic reticulum Ca(2+)-stimulated, Mg(2+)-dependent ATPase from swine heart. The thiadiazinone derivative [+]EMD 57033 (10 microM), but not its [-]enantiomer EMD 57439, had similar, although less potent, effects on pig heart myofibrillar Mg(2+)-ATPase activity as compared to [+]EMD 60263. [+]EMD 60263 (3 microM) produced a significantly larger leftward shift of the pCa2+/Mg(2+)-ATPase activity curve of myofibrils isolated from the stunned compared to the adjacent nonstunned myocardium (Delta pCa50s caused by the presence of [+]EMD 60263 amounted to +0.57 +/- 0.04 and +0.42 +/- 0.05, respectively) in the in situ porcine model. The effects of [+]EMD 60263 on myofibrillar Mg(2+)-ATPase of hypertrophic human heart were identical to those observed with porcine heart myofibrils. The results indicate that the positive inotropic action of [+]EMD 60263 observed in the in situ porcine model of stunned myocardium may be primarily due to myofilament sensitization to Ca2+, and that this compound may have a similar action on diseased human myocardium.

Animals↗
PubMed1988

Attenuation of ultrasound in suspensions of bovine muscle myofibrils and myosin.

The attenuation of 1.5-7 MHz ultrasound was measured over the pH range 3-7 in 100 mM KCl suspensions of bovine M. semitendinosus myofibrils, precipitated myosin and the residue of myofibrils after partial extraction of myosin. In all fractions attenuation showed a similar dependence on pH over the range 3-7, with a broad, substantial maximum in the region of pH 4.5-pH 5.5 and similar mass attenuation coefficients (per g protein). At pH 7 and 7 MHz these were 3.49 +/- 0.20 cm2 g-1 in the myofibrils, 3.26 +/- 0.31 cm2 g-1 in the myofibrilar residue and 2.83 +/- 0.68 cm2 g-1 in the precipitated myosin. Measurements at 5.3 MHz of precipitated myosin over a wider pH range revealed an attenuation titration curve similar to that previously observed in homogenates of muscle and muscle myofibrils, with substantial peaks at about pH 5 and 11.5, and a shoulder perhaps indicating a small underlying peak at about pH 8-9. Myosin dissolved in 800 mM KCl gave attenuation levels that were typically 50% lower than precipitated myosin e.g. at pH 7 and 7 MHz: 2.83 +/- 0.68 cm2 g-1 in the precipitated form, 1.29 +/- 0.10 cm2 g-1 in solution. These results indicated that: (a) attenuation by myosin filaments contributed substantially to the total attenuation in suspensions of myofibrils and (b) the peak in the myofibrilar attenuation is caused, or substantially contributed to, by a process taking place in the myosin component.

Animals↗
PubMed2005

Concentration-dependent suppressive effect of shrimp head protein hydrolysate on dehydration-induced denaturation of lizardfish myofibrils.

To utilize fishery waste products as functional food material, the shrimp head protein hydrolysate (SHPH) was produced from three species of shrimp wastes, Northern pink shrimp, Endeavour shrimp and black tiger shrimp, by enzymatic hydrolysis. The SHPH was used as a natural food preservative by adding to lizardfish myofibrils at concentrations ranging from 2.5% to 10%. Their effects on the state of water and the denaturation of myofibrils during dehydration were evaluated. The amount of monolayer and multilayer water in myofibrils containing SHPH were higher than those without SHPH (control). DSC analyses revealed that the amount of unfrozen water increased significantly after addition of SHPH. The Ca-ATPase inactivation rate of myofibrils containing SHPH decreased during dehydration while 5-7.5% concentrations of SHPH exhibited optimum effect regardless of the species. The results implicated that SHPH can be used as an alternative food preservative for suppressive the dehydration-induced denaturation of myofibrils.

Animals↗
PubMed1980

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.

Actins↗
PubMed1998

ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.

Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (kF), and a deceleration to a slow phase (kS). Evidence is given that kF is the ATPase rate of shortening myofibrils. V0 is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V0 and kF are 2.4 microm half-sarcomere(-1) s(-1) and 4.6 s(-1), and their Km values are 33 and 200 microM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar kF is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.

Adenosine Triphosphatases↗
PubMed1984

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges.

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Selective deletion of the NH2-terminal variable region of cardiac troponin T in ischemia reperfusion by myofibril-associated mu-calpain cleavage.

The structure of the NH2-terminal region of troponin T (TnT) is hypervariable among the muscle type-specific isoforms and is also regulated by alternative RNA splicing. This region does not contain binding sites for other thin filament proteins, but alteration of its structure affects the Ca2+ regulation of muscle contraction. Here we report a truncated cardiac TnT produced during myocardial ischemia reperfusion. Amino acid sequencing and protein fragment reconstruction determined that it is generated by a posttranslational modification selectively removing the NH2-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle fibers promoted production of the NH2-terminal truncated cardiac TnT (cTnT-ND), indicating a myofibril-associated proteolytic activity. Mu-calpain is a myofibril-associated protease and is known to degrade TnT. Supporting a role of mu-calpain in producing cTnT-ND in myocardial ischemia reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. Mu-calpain treatment of the cardiac myofibril and troponin complex specifically reproduced cTnT-ND. In contrast, mu-calpain treatment of isolated cardiac TnT resulted in nonspecific degradation, suggesting that this structural modification is relevant to physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils overexpressing fast skeletal muscle TnT produced similar NH2-terminal truncations of the endogenous and exogenous TnT, despite different amino acid sequences at the cleavage site. With the functional consequences of removing the NH2-terminal variable region of TnT, the mu-calpain-mediated proteolytic modification of TnT may act as an acute mechanism to adjust muscle contractility under stress conditions.

Amino Acid Sequence↗
PubMed2003

Solid phase microextraction-gas chromatography for quantifying headspace hexanal above freeze-dried chicken myofibrils.

A method using solid phase microextraction (SPME) combined with gas chromatography/mass spectrometry (GC/MS) was developed and used to determine the oxidation of freeze-dried chicken myofibrils spiked with methyl linoleate. Freeze-dried chicken myofibrils were found to act as a significant reservoir for hexanal. Recovery of hexanal emissions from the headspace above spiked myofibrils was 95% using a 5 min sampling time, with a total analysis time of approximately 12 min/sample. The SPME-GC/MS working linear response was from 0.01 to 10 mg hexanal/L (r( 2) = 0.995). Freeze-dried chicken myofibrils with added methyl linoleate (0.6 mmol/g of protein) were stored at 50 degrees C at water activities of 0.30 and 0.75 for 0, 12, 27, and 50 h. Lipid oxidation was determined using SPME-GC/MS to measure headspace hexanal concentration, the thiobarbituric acid reactive substances assay (TBARS) to quantify malonaldehyde, and a conjugated diene assay. Lipid oxidation was influenced by storage time and water activity. A strong correlation (r = 0.938) existed between SPME-GC/MS and TBARS. The use of SPME-GC/MS was a sensitive and rapid method for detecting hexanal as an indicator of lipid oxidation in chicken myofibrils.

Aldehydes↗
PubMed2000

Early structural changes in myosin rod upon heating of carp myofibrils.

Upon heating carp myofibrils at 40 degrees C, the amount of myosin that is soluble and monomeric dropped very quickly, roughly 5 times faster than the ATPase inactivation. This rapid decrease of solubility was well explained by a rapid denaturation of the rod portion as measured by chymotryptic digestibility. Chymotryptic digestion of heated myofibrils in a low-salt medium with EDTA generated a reduced amount of rod and subfragment-1 (S-1). The decrease of S-1 produced from the heated myofibrils was consistent with the ATPase inactivation. The decrease of rod produced from the heated myofibrils was explained by the increased susceptibility of the heavy meromyosin (HMM)/light meromyosin (LMM) junction to chymotryptic. It was, therefore, concluded that the fastest event occurring in the myosin molecule upon heating of myofibrils is the irreversible exposure of the HMM/LMM junction.

Animals↗
PubMed2001

Overexpression of miniparamyosin causes muscle dysfunction and age-dependant myofibril degeneration in the indirect flight muscles of Drosophila melanogaster.

Miniparamyosin (mPM) is a protein of invertebrate muscle thick filaments. Its similarity to paramyosin (PM) suggests that it regulates thick filament and myofibril assembly. To determine its role in muscle structure and function we overexpressed mPM in muscles of Drosophila melanogaster. Surprisingly, myofibrils accumulating excess mPM assemble nearly normally, with thick filament electron density and sarcomere length unaffected. Myofibrils in some indirect flight muscle groups are misaligned and young flies exhibit a moderate level of flight impairment. This phenotype is exacerbated with age. Transgenic flies undergo progressive myofibril deterioration that increases flight muscle dysfunction. Our observations indicate that the correct stoichiometry of mPM is important for maintenance of myofibril integrity and for the proper function of the flight musculature.

Aging↗
PubMedDate not supplied

Random walk analysis of restricted metabolite diffusion in skeletal myofibril systems.

The purpose of this work was the development of a basal mathematical model for the diffusion of low-molecular metabolites in a skeletal muscle cell. A three-dimension diffusion of low-molecular particles was simulated by a Monte-Carlo method (random walks of diffusing molecules). The model takes into account the following structural elements: (i) a regular lattice of actin and myosin filaments inside a myofibril; (ii) the membranes of sarcoplasmic reticulum and mitochondria surrounding the myofibrils; (iii) a set of myofibrils inside a skeletal muscle cell. We simulated diffusion of particles in the bulk of intracellular water phase and their reflections from the rigid surfaces of intracellular structures. The model allowed to calculate the apparent coefficients of particle diffusion in the axial and radial directions, Dparallel(app) and Dperpendicular(app), respectively. In accordance with experimental data from literature, the coefficient Dparallel(app) was independent of time. The coefficient of radial diffusion Dperpendicular(app) decreased with time to steady state values similar to that determined by the NMR diffusion spectroscopy methods. The interactions of diffusing particles with thin and thick filaments of myofibrils could explain the decrease in the Dperpendicular(app) value by a factor of 20%. The collisions of particles with myofilaments began to reveal themselves as a gradual decrease in the Dperpendicular(app) value at early stages of diffusion (t1/2 approximately equal to 0.05 microsec). The contribution of particle reflections from the membranes of sarcoplasmic reticulum and mitochondria to the retardation of the radial diffusion was about of 20-30%, depending on porosity of a membranous shield around the myofibril. For conventional sizes of a membranous shield (diameter 2 microm), the interactions of particles with the shield caused a decrease in the Dperpendicular(app) value with a half-time t1/2 approximately equal to 0.5 msec. This time is essentially lower by a factor about of 100 than that found in published NMR measurements. When we considered diffusion of particles inside a cell compartment confined to impermeable membranous shield, the reflection of particles from this shield led the drastic decrease in the radial diffusion coefficient (Dperpendicular(app) --> porportional to when t --> porportional to). This pattern of the Dperpendicular(app)(t) time-course might be expected in the NMR measurements on skeletal muscle tissue where a sarcolemma represents an impermeable shield for ATP and PCr molecules.

Models, Theoretical↗
PubMed1980

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

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

3',5'-Cyclic-AMP Phosphodiesterases↗
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↗
PubMed1989

Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions.

The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.

Adenosine Diphosphate↗
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↗
PubMed1982

Direct extraction of G-actin from the myosin-removed myofibrils under the conditions of low ionic strength.

Muscle actin is, in most cases, prepared from an acetone-dried powder of the myosin-removed myofibrils under low-salt conditions in the presence of ATP. In this paper, it is shown that G-actin can be directly extracted from the myosin-removed myofibrils without acetone treatment. The extraction conditions are the same as those used for the extraction of G-actin from the dried powder: extraction of the myosin-removed myofibrils for 1 h with 2 mM Tris-HCl, pH 8.0, in the presence of 0.5 mM ATP. However, the crude G-actin directly extracted from the myosin-removed myofibrils loses its polymerizability after prolonged extraction. Measurements of inorganic phosphate and thin layer chromatography of the adenine nucleotides of the crude G-actin solution show that free ATP added to the extraction buffer is sequentially hydrolyzed to ADP and AMP, and then finally converted to IMP. The instability of the G-(ADP)-actin, depolymerized from the ends of actin filaments, explains the loss in polymerizability of G-actin during the extraction. Residual ATPase, adenylate kinase, and deaminase contained in the myofibrils may account for the decomposition of ATP.

Actins↗