A structural origin of latency relaxation in frog skeletal muscle

A time-resolved x-ray diffraction study at a time resolution of 0.53 ms was made to investigate the structural origin of latency relaxation (LR) in frog skeletal muscle. Intensity and spacing measurements were made on meridional reflections from the Ca-binding protein troponin and the thick filament and on layer lines from the thin filament. At 16 degrees C, the intensity and spacing of all reflections started to change at 4 ms, simultaneously with the LR. At 0 degrees C, the intensity of the troponin reflection and the layer lines from the thin filament and the spacing of the 14.3-nm myosin meridional reflection, but not the spacing of other myosin meridional reflections, began to change at approximately 15 ms, when the LR also started. Intensity of myosin-based reflections started to change later. When the muscle was stretched to non-overlap length, the intensity and spacing changes of the myosin reflections disappeared. The simultaneous spacing change of the 14.3-nm myosin meridional reflection with the LR suggests that detachment of myosin heads that are bound to actin in the resting muscle is the cause of the LR.     

Time-resolved X-ray diffraction studies of frog skeletal muscle isometrically twitched by two successive stimuli using synchrotron radiation

In order to clarify the delay between muscular structural changes and mechanical responses, the intensity changes of the equatorial and myosin layer-line reflections were studied by a time-resolved X-ray diffraction technique using synchrotron radiation. The muscle was stimulated at 12-13 degrees C by two successive stimuli at an interval (80-100 ms) during which the second twitch started while tension was still being exerted by the muscle. At the first twitch, the intensity changes of the 1.0 and 1.1 equatorial reflections reached 65 and 200% of the resting values, and further changes to 55 and 220% were seen at the second twitch, respectively. Although the second twitch decreased not only the time to peak tension but also that to the maximum intensity changes of the equatorial reflections (in both cases, about 15 ms), the delay (about 20 ms) between the intensity changes and the development of tension at the first twitch were still observed at the second twitch. On the other hand, the intensities of the 42.9 nm off-meridional and the 21.5 nm meridional myosin reflections decreased at the first twitch to the levels found when a muscle was isometrically tetanized, and no further decrease in their intensities was observed at the second twitch. These results indicate that a certain period of time is necessary for myosin heads to contribute to tension development after their arrival in the vicinity of the thin filaments during contraction.     

Time-resolved synchrotron X-ray diffraction studies of a single frog skeletal muscle fiber. Time courses of intensity changes of the equatorial reflections and intracellular Ca2+ transients.

Time-resolved X-ray equatorial diffraction studies on a single frog skeletal muscle fiber were performed with a 10 ms time resolution using synchrotron radiation in order to compare the time courses of the molecular changes of contractile proteins and the intracellular Ca2+ transient during an isometric twitch contraction at 2.7 degrees C. Measurements of the Ca2+ transient using aequorin as an intracellular Ca2+ indicator were conducted separately just before and after the X-ray experiments under very similar experimental conditions. The results, which showed a similar time course of tension to that observed in the X-ray experiment, were compared with the aequorin light signal, tension and the intensity changes of the 1,0 and 1,1 equatorial reflections. No appreciable change in both reflection spacings indicated that the effect of internal shortening of the muscle was minimized during contraction. The intensity change of the equatorial reflections generally occurred after the aequorin light signal. In the rising phase, the time course of increase in the 1,1 intensity paralleled that of the rise of the light signal and the intensity peak occurred 20-30 ms after the peak of the light signal. The decrease in the 1,0 intensity showed a time course similar to that of tension and the intensity minimum roughly coincided with the tension peak, coming at 80-90 ms and about 60 ms after the peaks of the light signal and the 1,1 intensity change, respectively. In the relaxation phase, the 1,1 intensity seemed to fall rapidly just before the tension peak and then returned to the original level in parallel with the decay of tension. The 1,0 intensity returned more slowly than the tension relaxation. Thus, the change of the 1,1 intensity was faster than that of the 1,0 intensity in both the rising and relaxation phases. When the measured aequorin light signal was corrected for the kinetic delay of the aequorin reaction with a first-order rate constant of either 50 or 17 s-1, the peak of the corrected light signal preceded that of the measured one by approx. 30 ms. Thus, the peak of the Ca2+ transient appeared earlier than the peaks of the 1,1 and 1,0 intensity changes by 50-60 and 110-120 ms, respectively. The time lag between the extent of structural change and the Ca2+ transient is discussed in relation to the double-headed attachment of a cross-bridge to actin.     

X-ray diffraction studies on muscle regulation

Using the intensity of the outer part of the second actin layer line as an indicator of thin filament conformation in vertebrate muscle we were able to identify the four different states of rest, and the three states induced by the presence of Ca²⁺ ions, rigor bridge attachment and actively cycling bridges, respectively. These findings are in qualitative agreement with a number of biochemical studies by Eisenberg and Greene and others, indicating that activation of the thin filament depends both on Ca²⁺ ions and crossbridge binding. Yet quantitatively, the biochemical data and our structural data are contradictory. Whereas the biochemical studies suggest a strong coupling between structural changes of the thin filament and the ATPase activity, the structural studies indicate that this is not necessarily the case.Troponin molecules also change their conformation upon activation depending on both Ca²⁺ ions and crossbridge binding as demonstrated by the early part of the time course of the thin filament meridional reflections in contracting frog muscle.Low ionic strength which has been shown by Brenner and collaborators to increase weakly binding crossbridges in relaxed rabbit psoas muscle does not influence the intensity of the second actin layer line in this muscle. Yet in contracting frog muscle the increase of the second actin layer line increases very rapidly in one step, suggesting that weak binding bridges which are attached to actin prior to force production may indeed influence the thin filament conformation. It therefore appears that weakly bound bridges in the low ionic strength state do not have the same effect on the thin filament conformation as weakly bound bridges in an actively contracting muscle.Arthropod muscles like the thin filament regulated lobster muscles differ from vertebrate muscle in not showing an increase of the second layer line during contraction, which may have to do with differences in crossbridge attachment. The myosin-regulated molluscan muscle ABRM shows a large increase on the second actin layer line upon phasic contraction and a much smaller increase in catch or rigor, indicating that actively cycling bridges influence the thin filament conformation differently than catch or rigor bridges.Several pieces of evidence which we have briefly outlined in this paper suggest that the thin filament conformational changes we have observed do not arise solely from tropomyosin movements and that conformational changes of actin domains should be considered.     

An X-Ray Diffraction Study on Early Structural Changes in Skeletal Muscle Contraction

Structural changes in frog skeletal muscle were studied using x-ray diffraction with a time resolution of 0.53–1.02 ms after a single electrical stimulus at 8°C. Tension began to drop at 6 ms (latency relaxation), reached a minimum at 8 ms, and then twitch tension developed. The intensity of the meridional reflection at 1/38.5 nm⁻¹, from troponin molecules on the thin filament, began to increase at 4–5 ms and reached a maximum at ~12 ms. The meridional reflections based on the myosin 43-nm repeat began to decrease when the tension began to develop. The peak position of the third-order myosin meridional reflection began to shift toward the higher angle at ~5 ms, reached a maximum shift (0.02%) at 10 ms, and then moved toward the lower angle. The intensity of the second actin layer line at 1/18 nm⁻¹ in the axial direction, which was measured at 12°C, began to rise at 5 ms, whereas the latency relaxation started at 3.5 ms. These results suggest that 1), the Ca²⁺-induced structural changes in the thin filament and a structural change in the thick filament have already taken place during latency relaxation; and 2), the Ca²⁺ regulation of the thin filament is highly cooperative.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

X-ray diffraction indicates that active cross-bridges bind to actin target zones in insect flight muscle.

We report the first time-resolved study of the two-dimensional x-ray diffraction pattern during active contraction in insect flight muscle (IFM). Activation of demembranated Lethocerus IFM was triggered by 1.5-2.5% step stretches (risetime 10 ms; held for 1.5 s) giving delayed active tension that peaked at 100-200 ms. Bundles of 8-12 fibers were stretch-activated on SRS synchrotron x-ray beamline 16.1, and time-resolved changes in diffraction were monitored with a SRS 2-D multiwire detector. As active tension rose, the 14.5- and 7.2-nm meridionals fell, the first row line dropped at the 38.7 nm layer line while gaining a new peak at 19.3 nm, and three outer peaks on the 38.7-nm layer line rose. The first row line changes suggest restricted binding of active myosin heads to the helically preferred region in each actin target zone, where, in rigor, two-headed lead bridges bind, midway between troponin bulges that repeat every 38.7 nm. Halving this troponin repeat by binding of single active heads explains the intensity rise at 19.3 nm being coupled to a loss at 38.7 nm. The meridional changes signal movement of at least 30% of all myosin heads away from their axially ordered positions on the myosin helix. The 38.7- and 19.3-nm layer line changes signal stereoselective attachment of 7-23% of the myosin heads to the actin helix, although with too little ordering at 6-nm resolution to affect the 5.9-nm actin layer line. We conclude that stretch-activated tension of IFM is produced by cross-bridges that bind to rigor's lead-bridge target zones, comprising < or = 1/3 of the 75-80% that attach in rigor.     

Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by x-ray diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.     

Effects of pressure on equatorial x-ray fiber diffraction from skeletal muscle fibers.

When skeletal muscle fibers are subjected to a hydrostatic pressure of 10 MPa (100 atmospheres), reversible changes in tension occur. Passive tension from relaxed muscle is unaffected, rigor tension rises, and active tension falls. The effects of pressure on muscle structure are unknown: therefore a pressure-resistant cell for x-ray diffraction has been built, and this paper reports the first study of the low-angle equatorial patterns of pressurized relaxed, rigor, and active muscle fibers, with direct comparisons from the same chemically skinned rabbit psoas muscle fibers at 0.1 and 10 MPa. Relaxed and rigor fibers show little change in the intensity of the equatorial reflections when pressurized to 10 MPa, but there is a small, reversible expansion of the lattice of 0.7 and 0.4%, respectively. This shows that the order and stability of the myofilament lattice is undisturbed by this pressure. The rise in rigor tension under pressure is thus probably due to axial shortening of one or more components of the sarcomere. Initial results from active fibers at 0.1 MPa show that when phosphate is added the lattice spacing and equatorial intensities change toward their relaxed values. This indicates cross-bridge detachment, as expected from the reduction in tension that phosphate induces. 10 MPa in the presence of phosphate at 11 degrees C causes tension to fall by a further 12%, but not change is detected in the relative intensity of the reflections, only a small increase in lattice spacing. Thus pressure appears to increase the proportion of attached cross-bridges in a low-force state.     

Structure and periodicities of cross-bridges in relaxation, in rigor, and during contractions initiated by photolysis of caged Ca2+.

Ultra-rapid freezing and electron microscopy were used to directly observe structural details of frog muscle fibers in rigor, in relaxation, and during force development initiated by laser photolysis of DM-nitrophen (a caged Ca2+). Longitudinal sections from relaxed fibers show helical tracks of the myosin heads on the surface of the thick filaments. Fibers frozen at approximately 13, approximately 34, and approximately 220 ms after activation from the relaxed state by photorelease of Ca2+ all show surprisingly similar cross-bridge dispositions. In sections along the 1,1 lattice plane of activated fibers, individual cross-bridge densities have a wide range of shapes and angles, perpendicular to the fiber axis or pointing toward or away from the Z line. This highly variable distribution is established very early during development of contraction. Cross-bridge density across the interfilament space is more uniform than in rigor, wherein the cross-bridges are more dense near the thin filaments. Optical diffraction (OD) patterns and computed power density spectra of the electron micrographs were used to analyze periodicities of structures within the overlap regions of the sarcomeres. Most aspects of these patterns are consistent with time resolved x-ray diffraction data from the corresponding states of intact muscle, but some features are different, presumably reflecting different origins of contrast between the two methods and possible alterations in the structure of the electron microscopy samples during processing. In relaxed fibers, OD patterns show strong meridional spots and layer lines up to the sixth order of the 43-nm myosin repeat, indicating preservation and resolution of periodic structures smaller than 10 nm. In rigor, layer lines at 18, 24, and 36 nm indicate cross-bridge attachment along the thin filament helix. After activation by photorelease of Ca2+, the 14.3-nm meridional spot is present, but the second-order meridional spot (22 nm) disappears. The myosin 43-nm layer line becomes less intense, and higher orders of 43-nm layer lines disappear. A 36-nm layer line is apparent by 13 ms and becomes progressively stronger while moving laterally away from the meridian of the pattern at later times, indicating cross-bridges labeling the actin helix at decreasing radius.     

Two attached non-rigor crossbridge forms in insect flight muscle

We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.     

Time-resolved x-ray study of effect of sinusoidal length change on tetanized frog muscle.

Time-resolved x-ray diffraction studies were done on frog skeletal muscles with synchrotron radiation by applying sinusoidal length changes of frequency 10 Hz and amplitude approximately 1% to isometrically contracting muscles at approximately 17 degrees C. Distinct periodic intensity changes were observed in the 14.3-nm myosin meridional reflection and the equatorial 1,0 and 1,1 reflections. Response of the 14.3-nm reflection to the sinusoidal length change was nonlinear, as evidenced by a large second harmonic in its oscillatory intensity change, whereas the response of the equatorial 1,1 reflection was closely linear, as evidenced by almost sinusoidal intensity change. Intensity change of the 1,0 reflection was nearly antiphase to that of the 1,1 reflection. Integral widths of the 14.3-nm meridional reflection measured along the meridian and of the equatorial 1,1 reflection remained almost constant during tension development, while that of the 1,0 reflection tended to decrease. The widths of the 14.3-nm meridional reflection perpendicular to the meridian and of the equatorial 1,0 reflection appeared to undergo oscillatory changes in response to the sinusoidal length changes.     

Effect of muscle storage and stimulation on the intensity of Z-reflection of its equatorial x-ray pattern

The maintenance of frog sartorius muscle in Ringer solution over a long period of time (24 h or more) increases Z-reflection intensity of equatorial X-ray pattern relative to that from fresh (1-2 h) dissecting muscle at sarcomere length more than 2.3 m. Long stimulation deteriorates the equatorial X-ray pattern from muscle. Then with the course of time Z-reflection intensity increases several times, Z and reflections merge, and rigor equatorial X-ray pattern is registered.     

Caldesmon inhibits formation of strongly bound myosin cross-bridges and activates an ability of weakly bound cross-bridges to transform actin monomers to the off-conformation

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.     

The Fraction of Myosin Motors That Participate in Isometric Contraction of Rabbit Muscle Fibers at Near-Physiological Temperature

The duty ratio, or the part of the working cycle in which a myosin molecule is strongly attached to actin, determines motor processivity and is required to evaluate the force generated by each molecule. In muscle, it is equal to the fraction of myosin heads that are strongly, or stereospecifically, bound to the thin filaments. Estimates of this fraction during isometric contraction based on stiffness measurements or the intensities of the equatorial or meridional x-ray reflections vary significantly. Here, we determined this value using the intensity of the first actin layer line, A1, in the low-angle x-ray diffraction patterns of permeable fibers from rabbit skeletal muscle. We calibrated the A1 intensity by considering that the intensity in the relaxed and rigor states corresponds to 0% and 100% of myosin heads bound to actin, respectively. The fibers maximally activated with Ca²⁺ at 4°C were heated to 31–34°C with a Joule temperature jump (T-jump). Rigor and relaxed-state measurements were obtained on the same fibers. The intensity of the inner part of A1 during isometric contraction compared with that in rigor corresponds to 41–43% stereospecifically bound myosin heads at near-physiological temperature, or an average force produced by a head of ∼6.3 pN.     

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.     

Structural changes of cross-bridges on transition from isometric to shortening state in frog skeletal muscle

Structural changes in the myosin cross-bridges were studied by small-angle x-ray diffraction at a time resolution of 0.53 ms. A frog sartorius muscle, which was electrically stimulated to induce isometric contraction, was released by approximately 1% in 1 ms, and then its length was decreased to allow steady shortening with tension of approximately 30% of the isometric level. Intensity of all reflections reached a constant level in 5-8 ms. Intensity of the 7.2-nm meridional reflection and the (1,0) sampling spot of the 14.5-nm layer line increased after the initial release but returned to the isometric level during steady shortening. The 21.5-nm meridional reflection showed fast and slow components of intensity increase. The intensity of the 10.3-nm layer line, which arises from myosin heads attached to actin, decreased to a steady level in 2 ms, whereas other reflections took longer, 5-20 ms. The results show that myosin heads adapt quickly to an altered level of tension, and that there is a distinct structural state just after a quick release.     

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)     

Study of the mechanics and small-angle equatorial x-ray pattern of the frog skeletal muscle during transition and rigor at different temperatures

Mechanical characteristics and low-angle equatorial X-ray patterns from frog sartorius muscle passing into iodoacetate rigor under isometric conditions at temperatures 2 degrees-25 degrees C were studied. It is ascertained that during the rigor tension development at all the temperatures Z-reflection intensity increases and those of the (10), (11), (20), (21) and (30) reflections decrease. The last three reflections disappear then still in the phase of the rigor tension development. It is found that the sarcomere lengths remain not always invariable, especially at high temperatures, when the muscle passes into rigor, and can both decrease and increase in the sample place which is investigated by means of X-ray diffraction method. It is shown that the decrease of the I10/I11 relation in some experiments at high temperatures is only due to the sarcomere length decrease. The merging time of the Z and (11) reflections depends both on the temperature and on the sarcomere length change. Thus essential changes correlated with the rigor tension development, and resulted in the Z-reflection intensity increase take place in tetragonal lattice of Z-band and in the I-band region located near Z-band. In A-band the hexagonal lattice order change for the worse is marked only. It is proposed that the mechanism of the rigor tension development differs from that of tension development in ordinary contraction of the skeletal muscle.     

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 degrees 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.