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 subfragment-1 attachment to actin. Expected effect on equatorial reflections

The characteristic equatorial X-ray pattern from a relaxed vertebrate skeletal muscle changes when the muscle is activated. In particular, there is a simultaneous decrease in the intensity of the first reflection (I10) and increase in the intensity of the second (I11). This observed change is almost reciprocal. When compared with the predictions of computer modeling, it produces a strong argument that the intensity change is due to a redistribution of myosin heads (myosin subfragment-1 or S-1), which results from the formation and configuration changes of actin-myosin links. Computer modeling shows that different actin-S-1 configurations will give different numerical values for I10 and I11, assuming the same number of attachments. For a given configuration, the intensity changes are a nonlinear function of attachment number, so that direct scaling of force to reflection intensity may be difficult. Data from active muscle are consistent with the notion that in different states of active muscle, i.e. shortening or isometric, there are different average configurations of actin-myosin attachment and different numbers of actin-myosin links.     

X-ray diffraction observations of chemically skinned frog skeletal muscle processed by an improved method.

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.     

Structural changes during activation of frog muscle studied by time-resolved X-ray diffraction

The pattern given by contracting frog muscle can be followed with high time resolution using synchrotron radiation as a high-intensity X-ray source. We have studied the behaviour of the second actin layer-line (axial spacing of approximately 179 A) at an off-meridional spacing of approximately 0.023 A-1, a region of the diagram that is sensitive to the position of tropomyosin in the thin filaments. In confirmation of earlier work, we find that there is a substantial increase in the intensity of this part of the pattern during contraction. We find that the reflection reaches half its final intensity about 17 milliseconds after the stimulus at 6 degrees C. The changes in the equatorial reflections, which arise from movement of crossbridges towards the thin filaments, occur with a delay of about 12 to 17 milliseconds relative to this change in the actin pattern. In over-stretched muscle, where thick and thin filaments no longer overlap, the changes in the actin second layer-line still take place upon stimulation with a time course and intensity similar to that observed at full overlap. This indicates that tropomyosin movement, in response to calcium binding to troponin, is the first structural step in muscular contraction, and is the prerequisite for myosin binding. A change in intensity similar to that found in contracting muscle is seen in rigor, where tropomyosin is probably locked in the active position. During relaxation the earlier stages in the decrease in intensity of the second actin layer-line take place significantly sooner after the last stimulus than tension decay. In over-stretched muscles the intensity decay is appreciably faster than in the same muscles at rest length, where attached crossbridges may interfere with the return of tropomyosin to its resting position.     

Changes associated with glycolytic-enzyme binding in the equatorial X-ray-diffraction pattern of glycerinated rabbit psoas muscle.

The binding of fructose biphosphate aldolase to the thin filaments of glycerinated rabbit psoas muscle produces a significant change in its low-angle X-ray-diffraction pattern. The intensity of the (11) reflection relative to that of the (10) reflection increases by 26 +/- 3% (mean +/- S.E.M.), which is consistent with the increase in the mass of the thin filaments produced by enzyme binding. A similar effect is found with a mixture of aldolase and glyceraldehyde 3-phosphate dehydrogenase. The significance of the change in intensity is considered with reference to the interpretation of the equatorial patterns obtained from muscles in different physiological states. The magnitude of the increase in the relative intensity of the (11) reflection is lower than that observed between relaxed and contracting muscle and does not bring into question the interpretation linking changes in these patterns to cross-bridge movement. However, the effect due to enzyme binding may be important when making detailed interpretations of these changes. It may also be related to an unusual pattern sometimes observed in cardiac muscle.     

Time-Resolved Structural Studies on Insect Flight Muscle after Photolysis of Caged-ATP

The time course of structural changes occurring on ATP-induced relaxation of glycerinated insect flight muscle from the rigor state has been investigated using synchrotron radiation as a source of high intensity x rays and photolysis of caged-ATP to produce a rapid rise in ATP concentration. Temporal resolutions of 1 ms for the strongest equatorial reflections and 5 ms for the 14.5 nm meridional reflection are attainable from single events (i.e., without averaging over several cycles). The equatorial intensity changes completely, the meridional intensity partially, towards their respective relaxed values on a much faster time scale than relaxation of tension. The results suggest that actively cycling bridges present shortly after ATP-release are either too few in number to be detected in the equatorial diffraction pattern or that their structure is different from that of rigor bridges.     

Ultrastructure of a molluscan smooth muscle during phasic contractions and in the relaxed state

Tension and X-ray diffraction patterns are not always correlated in the smooth anterior retractor muscle (ABRM) of Mytilus edulis. The muscle produces equatorial intensity profiles of X-ray diffraction patterns corresponding to either a relaxed or a contracted structure. During phasic contractions, comprising a contracted as well a a relaxed phase, the diffracted intensity on the equator at 0.003 A^?1 changes within the first 10s after onset of stimulation. The tension reaches a maximum after about the same time. The time dependence of this intensity change during phasic contraction has been measured. It shows that the tension decays within 10s, but the relaxed structure needs 30–40 s to reestablish. There is no difference between the observed intensities from the tonic and phasic contracted states. Inactivated muscles with minimum tension, normally termed relaxed, can have either a “contracted” or a relaxed structure.     

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.     

Relation between the intensity of low-angle equatorial reflections of x-ray diffraction patterns of frog skeletal muscle and sarcomere length

Dependence of the intensities of low-angle equatorial reflections from frog live resting sartorius muscle on sarcomere length between 1.95 micron and 3.1 micron were studied in stretch and shortening regimes. It is found that intensities of the (10), (20), (30) and Z-reflections increase at sarcomere length increase from about 2 micron, reach maximum value at sarcomere length between 2.3 micron and 2.7 micron, and then fall at further increase of the sarcomere length. The (11) and (21) intensities decrease at sarcomere length increase. A conclusion is drawn that tetragonal lattice of the thin filaments near Z-line gives essential contribution to Z-reflection together with Z-line. It is proposed that hexagonal lattice of A-band and tetragonal lattice of the thin filaments distort each other at sarcomere length less than 2.3 micron and have the most order at sarcomere length between 2.3 micron and 2.7 micron. At further increase of the sarcomere length the packing of both lattices deteriorates apparently due to other factors than in the case of the short sarcomere length.     

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.     

Changes in the X-ray diffraction pattern from rigor muscles by application of external length changes

The effect of external force on the X-ray pattern from frog muscles in rigor was studied by a time-resolved diffraction technique. When sinusoidal length changes (1.5–3% of the muscle length, 5Hz) were applied to the muscle, the 14.3 nm intensity decreased during the releasing phase and increased during the stretching phase. The intensity ratio of the equatorial 1,0 and 1,1 reflections did not change, nor were there any appreciable intensity changes in the 5.9 nm and 5.1 nm reflections during the length change. Experiments were also done with the relaxed muscles and no change was seen in any reflection, indicating that the rigor linkages are needed to produce the 14.3 nm intensity change. Thus the distinct effect of the length change was detected only on the 14.3 nm reflection. These results suggest no large conformational changes are induced in both the distal part of the myosin head attached to actin and the actin filament during the oscillation. It is therefore most probable that the proximal portion of myosin heads including S-2 contributes to the intensity change in response to the length change (see, also ref.21). When the muscle was stretched beyond the filament overlap, the 14.3 nm intensity change was suppressed to less than 50% of that of the slack length. It was also found that the tension change delayed the intensity change during the length oscillation. However, this delay of the tension change as observed in the muscle at the slack length was lacking in the overstretched muscle, indicating that the 14.3 nm intensity change may arise partly from a portion other than the crossbridges.     

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.     

Preliminary X-ray crystallographic data on phospholipase C from Bacillus cereus.

Low-angle X-ray diffraction shows that, despite the well-defined regular axially projected structure, there is no long-range lateral order in the packing of molecules in native (undried) or dried elastoidin spicules from the fin rays of the spurhound Squalus acanthias. The equatorial intensity distribution of the X-ray diffraction pattern from native elastoidin indicates a molecular diameter of 1.1 nm and a packing fraction for the structure projected on to a plane perpendicular to the spicule (fibril) axis of 0.31 (the value for tendon is much higher at around 0.6). Density measurements support this interpretation. When the spicule dries the packing fraction increases to 0.43 but there is still no long-range order in the structure. The X-ray diffraction patterns provide no convincing evidence for any microfibrils or subfibrils in elastoidin. Gel electrophoresis shows that the three chains in the elastoidin molecule are identical. The low packing fraction for collagen molecules in elastoidin explains the difference in appearance between electron micrographs of negatively stained elastoidin and tendon collagen. In elastoidin, but not in tendon collagen, an appreciable proportion of the stain is able to penetrate between the collagen molecules.     

Knockdown of fast skeletal myosin-binding protein C in zebrafish results in a severe skeletal myopathy

Myosin-binding protein C (MyBPC) in the muscle sarcomere interacts with several contractile and structural proteins. Mutations in the cardiac isoform (MyBPC-3) in humans, or animal knockout, are associated with cardiomyopathy. Function of the fast skeletal isoform (MyBPC-2) in living muscles is less understood. This question was addressed using zebrafish models, combining gene expression data with functional analysis of contractility and small-angle x-ray diffraction measurements of filament structure. Fast skeletal MyBPC-2B, the major isoform, was knocked down by >50% using morpholino antisense nucleotides. These morphants exhibited a skeletal myopathy with elevated apoptosis and up-regulation of factors associated with muscle protein degradation. Morphant muscles had shorter sarcomeres with a broader length distribution, shorter actin filaments, and a wider interfilament spacing compared with controls, suggesting that fast skeletal MyBPC has a role in sarcomere assembly. Active force was reduced more than expected from the decrease in muscle size, suggesting that MyBPC-2 is required for optimal force generation at the cross-bridge level. The maximal shortening velocity was significantly increased in the MyBPC-2 morphants, but when related to the sarcomere length, the difference was smaller, reflecting that the decrease in MyBPC-2B content and the resulting myopathy were accompanied by only a minor influence on filament shortening kinetics. In the controls, equatorial patterns from small-angle x-ray scattering revealed that comparatively few cross-bridges are attached (as evaluated by the intensity ratio of the 11 and 10 equatorial reflections) during active contraction. X-ray scattering data from relaxed and contracting morphants were not significantly different from those in controls. However, the increase in the 11:10 intensity ratio in rigor was lower compared with that in controls, possibly reflecting effects of MyBPC on the cross-bridge interactions. In conclusion, lack of MyBPC-2 results in a severe skeletal myopathy with structural changes and muscle weakness.     

Characterization of a non-indexible equatorial x-ray reflection from frog sartorius muscle.

X-ray equatorial reflections from frog sartorius muscle were studied using a position sensitive detector. A weak reflection appeared between the 10 and 11 peaks which did not index on the hexagonal filament lattice. This reflection, first reported by Elliott et al. (1967), was further characterized. The spacing of the reflection varied in direct proportion to that of the 10 peaks for sarcomere lengths between 2·0 μm and 3·0 μm. Its intensity appeared relatively insensitive to length changes. Optical diffraction patterns from electron micrographs of oblique sections through muscle gave ratios for the spacings of the myosin filaments and the Z-disc lattice that correlated very closely with the X-ray results. It is suggested that the Z-disc structure is the major source of this nonindexible reflection.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cross-bridge attachment and stiffness during isotonic shortening of intact single muscle fibers.

Equatorial x-ray diffraction pattern intensities (I10 and I11), fiber stiffness and sarcomere length were measured in single, intact muscle fibers under isometric conditions and during constant velocity (ramp) shortening. At the velocity of unloaded shortening (Vmax) the I10 change accompanying activation was reduced to 50.8% of its isometric value, I11 reduced to 60.7%. If the roughly linear relation between numbers of attached bridges and equatorial signals in the isometric state also applies during shortening, this would predict 51-61% attachment. Stiffness (measured using 4 kHz sinusoidal length oscillations), another putative measure of bridge attachment, was 30% of its isometric value at Vmax. When small step length changes were applied to the preparation (such as used for construction of T1 curves), no equatorial intensity changes could be detected with our present time resolution (5 ms). Therefore, unlike the isometric situation, stiffness and equatorial signals obtained during ramp shortening are not in agreement. This may be a result of a changed crossbridge spatial orientation during shortening, a different average stiffness per attached crossbridge, or a higher proportion of single headed crossbridges during shortening.     

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.     

Time-resolved X-ray diffraction study on poly[(R)-3-hydroxybutyrate] films during two-step-drawing: generation mechanism of planar zigzag structure

Uniaxially oriented films with high tensile strength were processed from ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] (P(3HB)) by a method combining hot-drawing near the melting point of the polymer and two-step-drawing at room temperature. In a two-step-drawn and subsequently annealed film, P(3HB) molecular chains fall into two states: 2/1 helix (alpha-form) and planar zigzag (beta-form) conformations. The mechanism for generating the beta-form during two-step-drawing was investigated by time-resolved synchrotron wide- and small-angle X-ray scattering measurements (WAXD and SAXS), together with the measurement of stress-strain curves. It was found that the improvement of mechanical properties is due to not only the orientation of molecular chains but also the generation of the beta-form during the drawing. The crystal and molecular structures of the alpha-form remained unchanged until the yield point of the stress-strain curve. At the yield point, the long period obtained from SAXS doubled and a new reflection indicative of the beta-form was observed on the equatorial line in WAXD. The intensity of the reflection from the beta-form increased with an increase in the two-step-drawing ratio at room temperature. The SAXS pattern changed from a two-point reflection along the meridian to a cross pattern with streaking on the equatorial line, demonstrating the close alignment of shish-kebab structures. The reflection intensity, crystal orientation and crystal size of the alpha-form decreased during two-step-drawing. Based on these results, the beta-form is mainly introduced from the orientation of free molecular chains in the amorphous regions between alpha-form lamellar crystals, but the structural transformation of molecular chains also occurs from the alpha-form to the beta-form at the deformed lamellar crystals.     

X-ray evidence for radial cross-bridge movement and for the sliding filament model in actively contracting skeletal muscle

Equatorial X-ray diffraction patterns have been studied from muscles at rest, during contraction and in rigor. It is confirmed that the relative intensity ($\text{I}\text{1,0}\text{I}\text{1,1}$) of the two main equatorial reflections depends both on the sarcomere length and on the state of the muscle; in any one state the ratio $\text{I}\text{1,0}\text{I}\text{1,1}$ increases as the sarcomere length of the muscle increases, while at any fixed sarcomere length the ratio is smaller for contracting muscle than for resting muscle and smaller still for rigor muscles. The change of $\text{I}\text{1,0}\text{I}\text{1,1}$ with change of state at constant sarcomere length is interpreted as being due to radial movement of cross-bridges: the average movement during contraction being about 40% of that in rigor.Over the whole range of sarcomere length studied (between 1.8 and 2.7 μm) there was no evidence for any change in lattice spacing when a muscle contracts isometrically.Muscles were studied generating tension after they had shortened actively against a load. The lattice spacings and intensity ratio $\text{I}\text{1,0}\text{I}\text{1,1}$ both changed during active shortening in a way entirely consistent with the sliding filament theory of contraction.