Equilibrium of the actin-tropomyosin interaction

The actin-tropornyosin interaction was studied by means of light-scattering. The experimental data were analysed on the basis of the model of co-operative binding of large ligands to a one-dimensional lattice with overlapping binding sites. The affinity of tropomyosin for actin filaments was dependent on the magnesium concentration. A fivefold increase of the magnesium concentration (from 0·5 mm to 2·5 mm) enhanced the equilibrium constant twofold (from 700 to 1600 m^?1) for the isolated binding of tropomyosin molecules to actin filaments. At low magnesium concentrations (0·5 mm), tropomyosin molecules were bound to isolated binding sites on an actin filament about 600 times more weakly than to contiguous binding sites. At increased magnesium concentrations (2·5 mm), the tendency of tropomyosin to bind contiguously increased twofold. Due to the co-operative nature of the actin-tropomyosin interaction, a small change in the magnesium concentration may cause a great change of the structural organisation of the complex. A small enhancement of the magnesium concentration (from 1 mm to 1·5 mm) caused bare filaments to be covered almost completely with tropomyosin. The length of tropomyosin clusters and the number of gaps on actin filaments depended strongly on the magnesium concentration. From the values of the experimentally determined equilibrium constants, it was concluded that the end-to-end interaction of tropomyosin was not strong enough to bring about all-or-none behaviour, where actin filaments of physiological length (~1000 nm) are either completely covered with or completely free of tropomyosin.     

Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils

Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.     

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.     

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)     

Transverse elasticity of myofibrils of rabbit skeletal muscle studied by atomic force microscopy

Surface structure of myofibrils of rabbit skeletal muscle and their transverse elasticity were studied by atomic force microscopy. Images of myofibrils had a periodic structure characteristic of sarcomeres of skeletal muscle fibers. The transverse elasticity distribution in the sarcomere was determined based on force-distance curves measured at various loci of single myofibrils. The Z-line in rigor myofibrils was the most rigid in all the loci of myofibrils studied under various physiological conditions. The overall transverse elasticity of myofibrils decreased in the order in rigor solution > +AMPPNP solution > relaxing solution. The "apparent" transverse Young's modulus of myofibrils estimated at the overlap region between thin and thick filaments was 84.0 +/- 18.1, 37.5 +/- 14.0, and 11.5 +/- 3.5 kPa in rigor, +AMPPNP, and relaxing solution respectively.     

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.     

Effect of calcium ion on the interaction of aldolase with rabbit muscle myofibrils

A partition equilibrium study has shown calcium ion to be a noncompetitive inhibitor of aldolase adsorption by rabbit muscle myofibrils. This inhibition is interpreted quantitatively in terms of a 10-fold decrease in the intrinsic association constant for the aldolase-myofibril interaction upon Ca2+ binding to either or both of the low-affinity troponin sites associated with regulation of muscle contraction.     

Measurement of the fraction of myosin heads bound to actin in rabbit skeletal myofibrils in rigor

Tryptic digestion of rabbit skeletal myofibrils at physiological ionic strength and pH results in cleavage of the myosin heavy chain at one site giving two bands (M_r = 200,000 and 26,000) on sodium dodecyl sulfate/polyacrylamide gels. Following addition of sodium pyrophosphate (to 1 mm) to dissociate the myosin heads from actin, tryptic proteolysis results in production of three bands, 160K², 51K and 26K, with a 74K band appearing as a precursor of the 51K and 26K species. Under these conditions, there is insignificant cleavage of heavy chain to the heavy and light meromyosins. Trypsin-digested myofibrils yield the same amount of rod as native myofibrils when digested with papain. These results indicate that actin blocks tryptic cleavage of the myosin heavy chain at a site 74K from the N terminus. From measurements of the amount of 51K species formed by digestion of rigor fibers at various sarcomere lengths, we estimate that at least 95% of the myosin heads are bound to actin at 100% overlap of thick and thin filaments. Hence all myosin molecules can bind to actin, and consequently both heads of a myosin molecule can interact simultaneously with actin filaments under rigor conditions.     

Myonase is localized in skeletal muscle myofibrils

A novel chymotrypsin-like proteinase termed myonase was previously purified from MDX-mouse skeletal muscle [Hori et al. (1998) J. Biochem. 123, 650-658]. Western blots and immunohistochemical analyses showed that myonase was present within myocytes of both MDX-mouse and control mouse, and subcellular fractionation showed that it was associated with myofibrils. No significant difference was observed on Western blots between the amounts of myonase in myofibrils of MDX-mouse and control mouse, but the amount of myonase recoverable as a pure protein was 5-10-fold more when MDX-mouse was the source of the skeletal muscle. Myofibrils also possessed an endogenous inhibitor of myonase, whose inhibitory activity at physiological pH (pH 7.4) depended on salt concentration, stronger inhibition being observed at a low salt concentration. Inhibition at alkaline pH (pH 9) was weak and independent of salt concentration. Myonase in myofibrils was partially released at neutral pH by a high salt concentration (>0.6 M NaCl). However, even at 4 M NaCl, more than 80% of myonase remained within the myofibrils. Under alkaline conditions, release of myonase from myofibril was more extensive. At pH 12, myonase was almost completely present in the soluble fraction. Release of myonase under these conditions coincided with the solubilization of other myofibrillar proteins.     

The content of troponin, tropomyosin, actin, and myosin in rabbit skeletal muscle myofibrils

A stacking sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to resolve and quantify all the major myofibrillar protein components (actin, myosin, tropomyosin, and troponin C, T, and I). Quantification was achieved by densitometry of the fast green-stained gels calibrated with the use of purified proteins. The approximate molar ratios of these proteins in rabbit muscle are: actin: myosin: tropomyosin: troponin T: troponin I: troponin C = 7:1:1:1:1:1. On the basis of these results and available structural information one obtains an estimate of 254 myosin molecules per thick filament.     

Postmortem changes in the actin-myosin interaction of rabbit skeletal muscle

Postmortem changes in the actin-myosin interaction were studied by determining the amount of thick and thin filaments dissociated by ATP. The amount of separated filaments was very small in myofibrils prepared from muscles in rigor, while it increased markedly during post-rigor storage of muscles. Electron microscopically, separated thick and thin filaments prepared from stored muscles were similar to freshly prepared ones and no signs of proteolytic degradation of either type of filament could be observed. A protein which was released from myofibrils (probably from Z discs) on Ca2+-treatment seemed to be most closely related to the post-rigor dissociation of thick filaments from thin filaments.     

Mechanical characterization of skeletal muscle myofibrils.

A new instrument, based on a technique described previously, is presented for studying mechanics of micron-scale preparations of two to three myofibrils or single myofibrils from muscle. Forces in the nanonewton to micronewton range are measurable with 0.5-ms time resolution. Programmed quick (200-microseconds) steps or ramp length changes are applied to contracting myofibrils to test their mechanical properties. Individual striations can be monitored during force production and shortening. The active isometric force, force-velocity relationship, and force transients after rapid length steps were obtained from bundles of two to three myofibrils from rabbit psoas muscle. Contrary to some earlier reports on myofibrillar mechanics, these properties are generally similar to expectations from studies on intact and skinned muscle fibers. Our experiments provide strong evidence that the mechanical properties of a fiber result from a simple summation of the myofibrillar force and shortening of independently contracting sarcomeres.     

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 °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 MgATP, 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.     

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.     

Creatine kinase binds more firmly to the M-band of rabbit skeletal muscle myofibrils in the presence of its substrates

Creatine kinase (CK) (E.C. 2.7.3.2) buffers cellular ATP concentration during fluctuating ATP turnover. Muscle cytosolic CK isoform interacts with various subcellular structures where it is functionally coupled with relevant ATPases. However, how this interaction affects its activity is not known. We have therefore studied the interaction of CK with myofibrils and the role of different conformational states of CK molecule induced by ATP, phosphocreatine, ADP and the ATP-creatine pair. Purified rabbit psoas myofibrils with CK specific activity of 0.4 ± 0.02 IU/mg were used. The exchange rates between the myofibrillar M-band and its surroundings were measured with fluorofore conjugated CK (IAF) by the Fluorescence Lost in Photobleaching (FLIP) method within a very narrow pH range 7.1–7.15. For CK-IAF without docked substrates, the time derivative of the initial loss of the fluorescent signal within the M-band equalled −3.26 at the fifth second and the decrease reached 82% by the 67th second. For CK-IAF with added substrates, the derivatives fell into the range of −0.95 to −1.30, with respective decreases from 16 to 46% at the 67th second. The results show that the substrates slowed down the exchange rate. This indicates that the strength of the bond between CK and the M-band of myofibrils increased.     

Sarcomere dynamics in skeletal muscle myofibrils during isometric contractions

The main goal of this study was to evaluate the dynamics of sarcomeres during isometric activation of skeletal muscle myofibrils. Rabbit psoas myofibrils (n=14) were attached between a pair of cantilevers for force measurements at one side and a rigid glass needle at the other side, and their images were used for measurements of individual sarcomere lengths (SL) during contractions. Myofibrils were set at average SL between 2.13 and 3.06 μm, and were activated and held isometric for 20–35 s during which SL and force were continuously measured. SL dispersion increased from the rest state to activation, but it remained mostly constant during the activation period. Even with the length non-uniformity developed during myofibril activation, most sarcomeres stabilized their length changes during the isometric contraction. As a result, sarcomeres contracted at different degrees of filament overlap while producing similar forces. When the myofibrils were separated in two groups that produced force at averaged short (≤2.5 μm) or long (≥2.5 μm) SL, the initial non-uniformity was greater in long lengths, but changes observed in sarcomeres during the activation period were similar, suggesting that sarcomere stability is not length-dependent.     

Nonlinear force-length relationship in the ADP-induced contraction of skeletal myofibrils

The regulatory mechanism of sarcomeric activity has not been fully clarified yet because of its complex and cooperative nature, which involves both Ca²⁺ and cross-bridge binding to the thin filament. To reveal the mechanism of regulation mediated by the cross-bridges, separately from the effect of Ca²⁺, we investigated the force-sarcomere length (SL) relationship in rabbit skeletal myofibrils (a single myofibril or a thin bundle) at SL > 2.2 μm in the absence of Ca²⁺ at various levels of activation by exogenous MgADP (4-20 mM) in the presence of 1 mM MgATP. The individual SLs were measured by phase-contrast microscopy to confirm the homogeneity of the striation pattern of sarcomeres during activation. We found that at partial activation with 4-8 mM MgADP, the developed force nonlinearly depended on the length of overlap between the thick and the thin filaments; that is, contrary to the maximal activation, the maximal active force was generated at shorter overlap. Besides, the active force became larger, whereas this nonlinearity tended to weaken, with either an increase in [MgADP] or the lateral osmotic compression of the myofilament lattice induced by the addition of a macromolecular compound, dextran T-500. The model analysis, which takes into account the [MgADP]-and the lattice-spacing-dependent probability of cross-bridge formation, was successfully applied to account for the force-SL relationship observed at partial activation. These results strongly suggest that the cross-bridge works as a cooperative activator, the function of which is highly sensitive to as little as ≤1 nm changes in the lattice spacing.     

Compositional studies of myofibrils from rabbit striated muscle

The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thick-filament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as alpha-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Z-line proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack M-and Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.