Rigor Fibers Research Articles

Changes in the filament lattice of skeletal muscle fibres induced by changes in osmotic pressure, sarcomere length and the number of crossbridges

Equatorial small-angle X-ray diffraction patterns offer the possibility of obtaining detailed knowledge about interfilament distance and mass distribution of the myofilament lattice of skeletal muscle. These patterns of skinned frog-muscle fibres have been studied as a function of changes in the incubation solution using the Synchrotron Radiation Source in Daresbury (England). The results of these X-ray diffraction measurements and the results of measurements of the elastic properties of single muscle fibres, with microsecond time resolution, enabled an insight to be gained into the mechanism of muscle contraction. Interfilament distance decreased with increasing osmotic pressure in the absence of free calcium, whereas the mass distribution within the filament lattice did not change. Similar changes in the interfilament distance could be obtained either by decreasing the ionic strength in the absence of free calcium or by increasing the concentration of free calcium of the incubation solution. In both cases, however, mass distribution within the filament lattice also changed, which indicates that under these conditions the number of operating actomyosin complexes in the muscle fibre (crossbridges) tends to increase. The changes in interfilament distance and mass distribution that occur which depend on the free calcium concentration can be explained by a changed number of force-generating crossbridges. In the case of changes induced by ionic strength two effects interfere. Only part of the change in lattice spacing can be explained by an increased number of weakly attached crossbridges; the remaining part is caused by a process similar to that in the case of osmotic pressure. The effect of weakly attached crossbridges in the absence of free calcium under normal ionic strength conditions is small. Therefore, the large decrement in interfilament distance due to an increased sarcomere length, in the absence of free calcium, is not caused by weakly attached crossbridges. Changes in sacromere length, in the presence of free calcium, resulted in changes in the number of force-generating crossbridges. However, the observed effect on interfilament distance cannot be explained solely by the variation of the number of force-generating crossbridges. The results of X-ray diffraction patterns and stiffness measurements of fibres in rigor solutions [absence of ATP (adenosine triphosphate) and calcium] and fibres in calcium solutions indicated that the (force-generating) crossbridges in the latter differ from crossbridges in rigor fibres.

Characterization of radial force and radial stiffness in Ca(2+)‐activated skinned fibres of the rabbit psoas muscle.

1. When chemically skinned muscle fibres are activated by Ca2+ at an ionic strength of 170 mM, the spacing between the filaments has been shown to decrease with increasing force, suggesting that the cross-bridges can generate force not only in the axial but also in the radial direction. In the present study, radial force and radial stiffness of activated single skinned rabbit psoas fibres were studied by X-ray diffraction. The responses of the lattice spacing to changes in osmotic pressure by application of dextran T500, which is equivalent to force applied in the radial direction, was examined. The radial force generated by the attached cross-bridges was calculated, with the approximation that a negligible fraction of cross-bridges was attached in the relaxed muscle at the same ionic strength of 170 mM. 2. The active radial force was found to be a slightly non-linear function of lattice spacing, reaching zero at 34 nm. The radial force was compressive at lattice spacing greater than 34 nm and expansive at less than 34 nm. 3. The active axial force, on the other hand, was found to be much less affected by the application of dextran T500. Active axial force increased by 4% to a plateau at 4% dextran T500 and then decreased by 10% at 8% dextran T500. 4. While not under osmotic pressure, the radial force of the activated fibre was determined to be 400 pN (single thick filament)-1. This is of the same order of magnitude as the axial force. The radial stiffness was also comparable to the axial stiffness at 7 pN (thick filament)-1 (0.1 nm)-1. 5. The radial elasticity of the fully activated fibre differs significantly from that of the fibre in rigor. The radial stiffness exhibited by fibres in rigor was approximately five times higher, at 30 pN (thick filament)-1 (0.1 nm)-1 and the point where the radial force reached zero was 38 nm. 6. In the activated state, the point at which radial force reaches zero is independent of the level of Ca2+ activation, i.e. independent of the number of cross-bridges attached to actin in the force-generating state. We suggest that the zero-force point is equivalent to the equilibrium point of a spring and is an intrinsic property of the radial elasticity of the cross-bridge. 7. It is concluded that activated and rigor cross-bridges exhibit a spring-like property in the radial direction.(ABSTRACT TRUNCATED AT 400 WORDS)

Orientation of spin-labeled light chain-2 exchanged onto myosin cross-bridges in glycerinated muscle fibers

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the angular distribution of a spin label attached to rabbit skeletal muscle myosin light chain 2. A cysteine reactive spin label, 3-(5-fluoro-2,4-dinitroanilino)-2,2,5,5- tetramethyl-1-pyrrolidinyloxy (FDNA-SL) was bound to purified LC2. The labeled LC2 was exchanged into glycerinated muscle fibers and into myosin and its subfragments. Analysis of the spectra of labeled fibers in rigor showed that the probe was oriented with respect to the fiber axis, but that it was also undergoing restricted rotations. The motion of the probe could be modeled assuming rapid rotational diffusion (rotational correlation time faster than 5 ns) within a "cone" whose full width was 70 degrees. Very different spectra of rigor fibers were obtained with the fiber oriented parallel and perpendicular to the magnetic field, showing that the centroid of each cone had the same orientation for all myosin heads, making an angle of approximately 74 degrees to the fiber axis. Binding of light chains or labeled myosin subfragment-1 to ion exchange heads immobilized the probes, showing that most of the motion of the probe arose from protein mobility and not from mobility of the probe relative to the protein. Relaxed labeled fibers produced EPR spectra with a highly disordered angular distribution, consistent with myosin heads being detached from the thin filament and undergoing large angular motions. Addition of pyrophosphate, ADP, or an ATP analogue (AMPPNP), in low ionic strength buffer where these ligands do not dissociate cross-bridges from actin, failed to perturb the rigor spectrum. Applying static strains as high as 0.16 N/mm2 to the labeled rigor fibers also failed to change the orientation of the spin label. Labeled light chain was exchanged into myosin subfragment-1 (S1) and the labeled S1 was diffused into fibers. EPR spectra of these fibers had a component similar to that seen in the spectra of fibers into which labeled LC2 had been exchanged directly. However, the fraction of disordered probes was greater than seen in fibers. In summary, the above data indicate that the region of the myosin head proximal to the thick filament is ordered in rigor, and disordered in relaxation.

Conformations of crossbridges in contracting skeletal muscle

Conformations of crossbridges during isometric contraction were observed and compared with those in rigor state. Rabbit skeletal muscle fibers were chemically skinned, rapidly frozen during isometric contraction or in the rigor state, then freeze-substituted. Longitudinal and cross sections were observed. Crossbridges in contracting fibers are arranged on the thin filament, retaining a periodicity of approximately 14.3 nm with some variability, while crossbridges in rigor fibers tend to attach to actin target zones which have a periodicity of approximately 36 nm. Rigor crossbridges are wide near the thin filament; crossbridges in contracting fibers are thinner and have more uniform width from the proximal region to the distal region. The majority of the mass of the crossbridges during contraction is not as close to the thin filament as in rigor fibers. Shapes of crossbridges during contraction are variable, especially when viewed along the filament axis.

Dynamic X-ray diffraction measurements following photolytic relaxation and activation of skinned rabbit psoas fibres

1) The ATP binding and crossbridge dissociation in muscle fibres is as fast as in solution, has a Q10 ca. 2-3, and is not measurably strain sensitive. 2) The final ADP release from the AM.ADP state achieved by adding ADP to rigor fibres must be greater than or equal to 69 sec-1 at 10 degrees C, and the combination of this rate and the ADP rebinding rate at 1 mM ADP limits the ATP induced crossbridge dissociation rate at greater than 2 mM ATP, but these kinetics were not strain sensitive. The strain sensitive steps must occur earlier on the attached pathway. 3) On activation, the equatorial changes thought to reflect crossbridge attachment are faster than tension production. The 10 intensity may change slightly ahead of the 11. This rate was not very temperature sensitive unlike the tension producing step in the mechanism. 4) The re-equilibration of equatorial intensity levels was much faster on activation from the rigor state than from the relaxed state. We conclude that crossbridges do not necessarily move far from the thin filaments when they detach in a fully activated thin filament system. 5) The 14.3 nm meridional intensity increases greater than 200% on fibre activation at 24 degrees C. The structural reorganisation of the heads responsible for this increase is associated with the tension generating step in the ATPase mechanism rather than the initial binding of bridges.

Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers

We have measured the microsecond rotational motions of myosin heads in contracting rabbit psoas muscle fibers by detecting the transient phosphorescence anisotropy of eosin-5-maleimide attached specifically to the myosin head. Experiments were performed on small bundles (10-20 fibers) of glycerinated rabbit psoas muscle fibers at 4 degrees C. The isometric tension and physiological ATPase activity of activated fibers were unaffected by labeling 60-80% of the heads. Following excitation of the probes by a 10-ns laser pulse polarized parallel to the fiber axis, the time-resolved emission anisotropy of muscle fibers in rigor (no ATP) showed no decay from 1 microsecond to 1 ms (r infinity = 0.095), indicating that all heads are rigidly attached to actin on this time scale. In relaxation (5 mM MgATP but no Ca2+), the anisotropy decayed substantially over the microsecond time range, from an initial anisotropy (r0) of 0.066 to a final anisotropy (r infinity) of 0.034, indicating large-amplitude rotational motions with correlation times of about 10 and 150 microseconds and an overall angular range of 40-50 degrees. In isometric contraction (MgATP plus saturating Ca2+), the amplitude of the anisotropy decay (and thus the amplitude of the microsecond motion) is slightly less than in relaxation, and the rotational correlation times are about twice as long, indicating slower motions than those observed in relaxation. While the residual anisotropy (at 1 ms) in contraction is much closer to that in relaxation than in rigor, the initial anisotropy (at 1 microsecond) is approximately equidistant between those of rigor and relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers, e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 ° relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spinlabeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 ° and 66 °, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5′-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.

Orientation of the backbone structure of myosin filaments in relaxed and rigor muscles of the housefly: Evidence for non-equivalent crossbridge positions at the surface of thick filaments

The orientation of the backbone structure of myosin filaments of relaxed and rigor fibers of the flight muscles of the housefly, Musca domestica, relative to the actin filaments has been investigated. In relaxed muscles 23% of the myosin filaments have gaps in the wall of their shaft located opposite the surrounding actin filaments, while in 77% the subfilament pairs of the wall are thus located. These are the expected values if the backbone orientation is random. In rigor muscles 40% of the thick filaments have their gaps opposite the actins and 60%, the subfilament pairs are opposite the actins. This increase in the percentage of filaments with gaps opposite the actins therefore results from binding of the crossbridges in rigor with change in rotational orientation of the backbone. The findings are related to a model of Beinbrech et al. (1988) in which two populations of crossbridges have been postulated: one originating at the surface of the thick filaments, the other coming from within the gap between the subfilament pairs.

Myosin crossbridge orientation in demembranated muscle fibres studied by birefringence and X-ray diffraction measurements

Muscle contraction is generally thought to involve changes in the orientation of myosin crossbridges during their ATP-driven cyclical interaction with actin. We have investigated crossbridge orientation in equilibrium states of the crossbridge cycle in demembranated fibres of frog and rabbit muscle, using a novel combination of techniques: birefringence and X-ray diffraction. Muscle birefringence is sensitive to both crossbridge orientation and the transverse spacing of the contractile filament lattice. The latter was determined from the equatorial X-ray diffraction pattern, allowing accurate characterization of the orientation component of birefringence changes. We found that this component decreased when relaxed muscle fibres were put into rigor at rest length, and when either the ionic strength or temperature of relaxed fibres was lowered. In each case the birefringence decrease was accompanied by an increase in the intensity of the (1,1) equatorial X-ray reflection relative to that of the (1,0) reflection. When fibres that had been stretched largely to eliminate overlap between actin- and myosin-containing filaments were put into rigor, there was no change in the orientation component of the birefringence. When isolated myosin subfragment-1 was bound to these rigor fibres, the orientation component of the birefringence increased. The birefringence changes at rest length are likely to be due to changes in the orientation of myosin crossbridges, and in particular of the globular head region of the myosin molecules. In relaxed fibres from rabbit muscle, at 100 mm ionic strength, 15 °C, the long axis of the heads appears to be relatively well aligned with the filament axis. When fibres are put into rigor, or the temperature or ionic strength is lowered, the degree of alignment decreases and there is a transfer of crossbridge mass towards the actin-containing filaments.

Myosin cross-bridge orientation in rigor and in the presence of nucleotide studied by electron spin resonance

The tilt series electron spin resonance (ESR) spectrum from muscle fibers decorated with spin labeled myosin subfragment 1 (S1) was measured from fibers in rigor and in the presence of MgADP. ESR spectra were measured at low amplitude modulation of the static magnetic field to insure that a minimum of spectral lineshape distortion occurs. Ten tilt series ESR data sets were fitted simultaneously by the model-independent methodology described in the accompanying paper (Burghardt, T. P., and A. R. French, 1989. Biophys. J. 56:525-534). By this method the average and standard error in the mean of order parameters for the probe angular distribution were calculated for the two states of the fiber investigated. The average order parameters were used to reconstruct the probe angular distribution in two dimensions, one angular dimension corresponding to a polar angle measured relative to the fiber axis, and the other a torsional angular degree of freedom of the probe. We find that the probe angular distributions for the rigor and MgADP states of the fiber differ such that the rigor distribution is broader and shifted relative to the distribution in the presence of MgADP. The shape of the rigor distribution suggests the presence of two probe orientations, one similar to that in the presence of MgADP, and another at a different orientation. The shape of the distribution in the presence of MgADP suggests that the binding of the nucleotide to the rigor cross-bridge shifts the spin population into a more homogeneous one by causing a cross-bridge rotation.

Effect of adenosine triphosphate analogues on skeletal muscle fibers in rigor

It is commonly believed, for both vertebrate striated and insect flight muscle, that when the ATP analogue adenyl-5'-yl imidodiphosphate (AMPPNP) is added to the muscle fiber in rigor, it causes the fiber to lengthen by 0.15%. This has been interpretated (Marston S.B., C.D. Roger, and R.T. Tregear. 1976. J. Mol. Biol. 104:263-267) as suggesting (a) that in rigor the crossbridge is fixed to, i.e., almost never detaches from the actin filament; (b), that the crossbridge remains fixed to the actin filament after AMPPNP addition; and (c) that the ability of AMPPNP to cause apparent lengthening of a muscle fiber is due to its ability to cause a conformational change in the myosin crossbridge that has an axial component of approximately 1.6 nm/half-sarcomere. The present study, done only on chemically-skinned rabbit psoas fibers, confirms that AMPPNP can cause muscle fibers to lengthen by 0.15% but only for a narrow set of experimental conditions. When experimental conditions are varied over a wider range, it becomes apparent that the extent of lengthening of a rigor muscle fiber upon AMPPNP addition depends almost entirely on the strain present in the rigor fiber before AMPPNP addition. Addition of AMPPNP to an unstrained rigor fiber (one supporting zero tension), induces zero length change while addition of AMPPNP to very highly strained rigor fibers induces length changes greater than 0.15%. The data thus do not support the hypotheses that the crossbridges remain fixed to the actin filament after AMPPNP addition and that the size of the apparent length change induced by AMPPNP is related to the size of the axial component of a conformational change. Instead, the data support the idea that the ability of AMPPNP to cause lengthening of a rigor muscle fiber is related to its ability to accelerate the rate at which strained crossbridges detach from actin and reattach in positions in lesser strain. The data do not rule out a conformational change upon AMPPNP binding, they simply make clear that any attempt to measure a force response conceivably due to a conformational change, would be more than obscured by the force changes due to crossbridges detaching and reattaching in positions of lesser strain.

Differential behavior of two cysteine residues on the myosin head in muscle fibers

We have previously shown that the orientation of (iodoacetamido)tetramethylrhodamine labels on SH1 thiol of S-1 moieties changes when MgADP is added to the fibers in rigor [Borejdo, J., Assulin, O., Ando, T., & Putnam, S. (1982) J. Mol. Biol. 158, 391-414. Burghardt, T.P., Ando, T., & Borejdo, J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7515-7519]. Here we report the results of experiments in which the SH2 of S-1 was labeled with maleimidorhodamine. The specificity of modification of thiols was checked by measuring the stoichiometry of attached dye, by determining the extent of the decrease in EDTA (K+)- and Ca2+-ATPase activities, and by the localization of the dyes on peptides containing SH1 and/or SH2. Labeled S-1 was diffused into single glycerinated fibers of rabbit psoas muscle, and the orientation of chromophores was measured by fluorescence detected dichroism. The dye attached to SH1 was oriented at 65 degrees with respect to the fiber axis in rigor and at 51 degrees in the presence of MgADP, regardless of whether SH2 was modified or not. The dye on SH2 was oriented near 42 degrees both in the presence and in the absence of ADP, regardless of whether SH1 was modified or not. Our results show that rhodamine oriented differently when attached to SH2 compared with when attached to SH1 and that in the former placement it was not sensitive to MgADP. We think this indicates that the SH2-containing region has a mobility different from that of the SH1-containing region, i.e., that this is evidence for internal flexibility of S-1.

Elastic properties of relaxed, activated, and rigor muscle fibers measured with microsecond resolution

Tension responses due to small and rapid length changes (completed within 40 microseconds) were obtained from skinned single-fiber segments (4- to 7-mm length) of the iliofibularis muscle of the frog incubated in relaxing, rigor, and activating solution. The fibers were skinned by freeze-drying. The first 500 microseconds of the responses for all three conditions could be described with a linear model, in which the fiber is regarded as a rod composed of infinitesimally small identical segments, containing an undamped elastic element, two damped elastic elements and a mass in series. An additional damped elastic element was needed to describe tension responses of activated fibers up to the first 5 ms. Consequently phase 1 and phase 2 of activated fibers can be described with four apparent elastic constants and three time constants. The results indicate that fully activated fibers and fibers in rigor have similar elastic properties within the first 500 microseconds of tension responses. This points either to an equal number of attached cross-bridges in rigor and activated fibers or to a different number of attached cross-bridges in rigor and activated fibers and nonlinear characteristics in rigor cross-bridges. Mass-shift measurements obtained from equatorial x-ray diffraction patterns support the latter possibility.

Relaxation of muscle fibers with adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and by laser photolysis of caged ATP[gamma S]: evidence for Ca2+-dependent affinity of rapidly detaching zero-force cross-bridges.

The relationship between the mechanical and biochemical states of the muscle cross-bridge cycle and the control of contraction were investigated by using the nucleotide analogs adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and caged ATP[gamma S] [the O-1(2-nitrophenyl)ethyl P3-ester of ATP[gamma S]]. ATP[gamma S] interacts with actomyosin in a manner similar to ATP but is hydrolyzed (by a factor of 500) more slowly. Generation of ATP[gamma S] by photolysis of caged ATP[gamma S] within a permeabilized fiber in rigor in the absence of Ca2+ relaxed tension and stiffness as occurs with ATP. The transient rise in tension prior to final relaxation observed with photolysis of caged ATP was absent with caged ATP[gamma S]. This result suggests that following detachment of a cross-bridge, ATP is normally hydrolyzed before force generation. In the presence of Ca2+, photolysis of caged ATP[gamma S] within rigor fibers caused tension to relax fully but significant stiffness remained. Stiffness also developed without concomitant tension when Ca2+ concentration was raised from less than 1 nM to 30 microM in the presence of ATP[gamma S]. The amplitude of the tension response to ramp stretches in the presence of Ca2+ and ATP[gamma S] increased with ramp stretch velocity, suggesting that the cross-bridges have detachment rate constants extending into the 10(3) s-1 range. The results provide evidence that the Ca2+-regulatory system can directly control attachment of cross-bridges into states before the power stroke.

Passive interaction between sliding filaments in the osmotically compressed skinned muscle fibers of the frog

Shortening and lengthening velocities, instantaneous stiffness, and tension transients after stretch were measured in compressed muscle fibers from the frog in the presence or absence of polyvinylpyrrolidone (PVP K30) or Dextran T70. Both shortening and lengthening velocities clearly decreased with the concentration of polymer. In the presence of polymer, "passive" stiffness was observed in relaxing solution depending on fiber diameter, and stiffness increased further by activation. This increase by activation above "passive" stiffness was nearly constant in the wide range of polymer concentrations. These active and "passive" stiffnesses were found to be dependent on sarcomere length. The stiffness of a compressed rigor fiber was indicated to be composed of constant rigor stiffness and a variable "passive" one. The tension transient after stretch in a compressed active or rigor fiber was also indicated to be composed of two kinds of transients. The above results suggest that (a) there exist two kinds of interactions in parallel in a compressed active or rigor fiber: one active or rigor and another "passive" between sliding filaments, and (b) the decrease in shortening velocity in a compressed fiber may be brought about by this "passive" interaction.

Probe studies of the MgADP state of muscle cross-bridges: microscopic and wavelength-dependent fluorescence polarization from 1,5-IAEDANS-labeled myosin subfragment 1 decorating muscle fibers.

The microscopic and wavelength-dependent fluorescence polarization signals from the 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (1,5-IAEDANS) labeled subfragment 1 of myosin (S-1) decorating muscle fibers in rigor and in the presence of MgADP are measured. Using microscopic fluorescence polarization, we select a small uniform volume (approximately 0.1 micron 3) from the muscle fiber and detect a high degree of angular order. From these data we show that the probe angular distribution from fibers in rigor is quantitatively different from that present when MgADP is bound to S-1. Using wavelength-dependent fluorescence polarization, we vary the wavelength of the excitation light and thereby change the direction that the probe absorption dipole makes with a reference frame fixed in S-1. From these data we show that the binding of MgADP to S-1 causes an angular reorientation of S-1 relative to the actin filament. The difference between the angular distribution of probes for the rigor vs. MgADP states cannot be accounted for by the addition of random probes. The microscopic fluorescence polarization experiments suggest that the earlier attempts to distinguish a rigor from a MgADP probe angular distribution by using the 1,5-IAEDANS probe failed due to the lower resolution of the optical technique employed. The wavelength-dependent fluorescence polarization experiments indicate that the probe dipole orientation on S-1, at the typically used excitation and emission wavelengths, is not ideal for detecting the orientation change in the rigor to MgADP angular transition.

Lattice swelling with the selective digestion of elastic components in single-skinned fibers of frog muscle

Changes in the 1.0 lattice spacing during trypsin (0.25 micrograms/ml) treatment in mechanically skinned single fibers of frog muscle was examined by an x-ray diffraction method at various sarcomere lengths. The resting tension of a relaxed fiber was decreased by trypsin treatment but the stiffness of a rigor fiber was not, suggesting that elastic components were selectively digested. With progression of the digestion, the lattice spacing increased remarkably at longer sarcomere lengths and finally became independent of the sarcomere length. The increase in the lattice spacing was proportional to the decrease in the resting tension. These results support our previous suggestion (Higuchi, H., and Y. Umazume, 1986, Biophys. J., 50:385-389) that the lattice spacing decreases at long lengths due to compressive force exerted by a lateral elastic component that connects thick filaments to an axial elastic component. Consequently, it is unlikely that the decrease in the lattice spacing is determined by a decrease in the repulsive force between thick and thin filaments with stretching a fiber.

Force generation by muscle fibers in rigor: a laser temperature-jump study.

A clear prediction of the helix-coil model for force generation in muscle is that force should be produced when the equilibrium (helix-coil) of a rigor (or activated) fiber is perturbed by a temperature jump near the melting temperature of the light meromyosin/heavy meromyosin hinge. An infrared, iodine-photodissociation laser was used to heat the fibers by approximately equal to 5 degrees C in under 1 mus. Under ionic conditions where rigor bridges are predominantly associated with the thick filament backbone, an abrupt drop in tension typical of normal thermoelastic expansion was seen. A similar response was observed below 41 degrees C for thick filament-released rigor bridges. Above this temperature, a rubber-like thermoelastic response was obtained typical of a helix-coil transition. At temperatures near 50 degrees C, the amount of force generated by a rigor fiber was large and comparable to that seen for an activated fiber at 5 degrees C. The relaxation spectra of force generation obtained for both systems (rigor and activated) show a step change followed by a biexponential kinetic process. The reciprocal relaxation times and amplitudes for these individual processes in activated and rigor fibers differ only by factors of 2-4. Force generation in the rigor muscle appears to arise from melting in the subfragment 2 hinge region of the myosin molecule since binding of subfragment 2 to the thick filament backbone inhibits force production. No significant force generation was observed following temperature jumps of relaxed fibers.

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range. An effective actin concentration in the millimolar range suggests that the strength of actin binding to cross-bridges in fibers may be several orders of magnitude weaker than the strength of ATP binding. Previously, it has been assumed that these two quantities were equal, as this gives the minimum energy loss when ATP dissociates the cross-bridge from actin (Morales, 1980, J. Supramol. Struct., 3:105:1975; Eisenberg, E.,Hill, T. L. and Y. Chen, 1980, Biophys. J., 29:195).

Stiffness of carbodiimide-crosslinked glycerinated muscle fibres in rigor and relaxing solutions at high salt concentrations.

In this article, we have applied a crosslinking technique with a water-soluble carbodiimide to single glycerol-extracted muscle fibres from the rabbit. We have measured the stiffness of the fibres in a relaxing solution at high salt concentration. These fibres were crosslinked to varying extents in the rigor state. The relaxing solution caused uncrosslinked crossbridge heads (S1) to detach. High salt concentrations were used because the fibres were not activated by the crosslinked crossbridges under these conditions, although they were at physiological ionic strength. We found a linear correlation between the extent of S1 crosslinking to thin filaments and the stiffness and that the stiffness in the relaxing solution of muscle fibres with all the S1 heads crosslinked to thin filaments was the same as the rigor stiffness of the fibres before crosslinking. We conclude that the sarcomere compliance is mostly a property of the crossbridges (with more than 65% of the crossbridge compliance in the S1 portions and less than 35% in the S2 portion) and little of other sarcomere structures. In an earlier paper [Kimura & Tawada, Biophys. J. 45, 603-10 (1984)], we demonstrated that the S2 portion of the crossbridge was stiff. It then follows that the crossbridge compliance, and thus the sarcomere compliance, is a property of the S1 heads. Assuming that the S1 portion of the crossbridges in rigor strained muscle fibres is bent, we calculated the Young's modulus of the S1 portion and found that it is about 10(2) MN m-2. Because this order of magnitude is reasonable in terms of globular protein elasticity, bending is likely to be the nature of the S1 compliance in rigor muscle fibres.