X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction.

To clarify the extensibility of thin actin and thick myosin filaments in muscle, we examined the spacings of actin and myosin filament-based reflections in x-ray diffraction patterns at high resolution during isometric contraction of frog skeletal muscles and steady lengthening of the active muscles using synchrotron radiation as an intense x-ray source and a storage phosphor plate as a high sensitivity, high resolution area detector. Spacing of the actin meridional reflection at approximately 1/2.7 nm-1, which corresponds to the axial rise per actin subunit in the thin filament, increased about 0.25% during isometric contraction of muscles at full overlap length of thick and thin filaments. The changes in muscles stretched to approximately half overlap of the filaments, when they were scaled linearly up to the full isometric tension, gave an increase of approximately 0.3%. Conversely, the spacing decreased by approximately 0.1% upon activation of muscles at nonoverlap length. Slow stretching of a contracting muscle increased tension and increased this spacing over the isometric contraction value. Scaled up to a 100% tension increase, this corresponds to a approximately 0.26% additional change, consistent with that of the initial isometric contraction. Taken together, the extensibility of the actin filament amounts to 3-4 nm of elongation when a muscle switches from relaxation to maximum isometric contraction. Axial spacings of the layer-line reflections at approximately 1/5.1 nm-1 and approximately 1/5.9 nm-1 corresponding to the pitches of the right- and left-handed genetic helices of the actin filament, showed similar changes to that of the meridional reflection during isometric contraction of muscles at full overlap. The spacing changes of these reflections, which also depend on the mechanical load on the muscle, indicate that elongation is accompanied by slight changes of the actin helical structure possibly because of the axial force exerted by the actomyosin cross-bridges. Additional small spacing changes of the myosin meridional reflections during length changes applied to contracting muscles represented an increase of approximately 0.26% (scaled up to a 100% tension increase) in the myosin periodicity, suggesting that such spacing changes correspond to a tension-related extension of the myosin filaments. Elongation of the myosin filament backbone amounts to approximately 2.1 nm per half sarcomere. The results indicate that a large part (approximately 70%) of the sarcomere compliance of an active muscle is caused by the extensibility of the actin and myosin filaments; 42% of the compliance resides in the actin filaments, and 27% of it is in the myosin filaments.     

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.     

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)     

Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles

Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 Å) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles.We also show that any increase of intensity on the 59 Å and 51 Å layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.     

New X-ray diffraction observations on vertebrate muscle: organisation of C-protein (MyBP-C) and troponin and evidence for unknown structures in the vertebrate A-band

Previous low-angle X-ray diffraction studies of various vertebrate skeletal muscles have shown the presence of two rich layer-line patterns, one from the myosin heads and based on a 429 A axial repeat, and one from actin filaments and based on a repeat of about 360-370 A. In addition, meridional intensities have been seen from C-protein (MyBP-C; at about 440 A and its higher orders) and troponin (at about 385 A and its orders). Using preparations of intact, relaxed, bony fish fin muscles and the ID-02 low-angle X-ray camera at the ESRF with a 10 m camera length we have now seen numerous, hitherto unreported, sampled, X-ray layer-lines many of which do not fit onto the previously observed repeats and which require interpretation. The new reflections all fall on the normal ("vertical") hexagonal lattice row-lines in the highly sampled, almost "crystalline", low-angle diffraction X-ray patterns from bony fish muscle, indicating that they all arise from the muscle A-band. However, they do not fall on a single axial repeat. In direct confirmation of our previous analysis, some of these new reflections are explained by the interaction in resting muscle between the N-terminal ends of myosin-bound C-protein molecules with adjacent actin filaments, possibly through the Pro-Ala-rich region. Other newly observed reflections lie on a much longer repeat, but they are most easily interpreted in terms of the arrangement of troponin on the actin filaments. If this is so, then the implication is that the actin filaments and their troponin complexes are systematically arranged in the fish muscle A-band lattice relative to the myosin head positions, and that these newly observed X-ray reflections, when fully analysed, will report on the shape and distribution of troponin molecules in the resting muscle A-band. The less certain contributions of titin and nebulin to these new reflections have also been tested and are described. Many of the new reflections do not appear to come from these known structures. There must be structural features of the A-band that have not yet been described.     

Changes in the pitch of the myosin helix preceding cross-brdige attachment in oscillatory contractions of frog sartorius muscle

When glycerinated fibres in presence of 5·10^?7M Ca²⁺ were oscillated at a frequency of 10 Hz, the X-ray intensities at 429 Å and at the small radial position of the 191 Å layer-line change in an inverse pattern. This rearrangement of the pitch of the myosin helix precedes by at least 10 msec the intensity increase on the 59 Å X-ray layer-line, which provides a measure for cross-bridge attachment to the actin. The perfect match of the new pitch of the myosin helix with the pitch of the actin helix may ensure optimal cross-bridge attachment.     

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.     

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.     

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.     

Time-resolved x-ray diffraction study of the troponin-associated reflexions from the frog muscle.

The vertebrate skeletal muscle gives rise to a series of x-ray reflexions indexed as orders (n) of 77 nm, the even orders being meridional whereas the odd orders being near-meridional. The diffraction intensities associated with these reflexions originate from the axial period of 39 nm attributable to the repeat of troponin-tropomyosin on the thin filament. In the present study, the x-ray intensities of the furthest inner reflexions, A2 (n = 2) reflexion at an axial spacing of 1/39 nm-1 and A4 (n = 4) reflexion at 1/19 nm, of this series were measured with a time resolved manner. Upon activation of the frog striated muscle, the two reflexions underwent biphasic time courses of the intensity changes. With A2 reflexion, a rapid intensity increase by 16%, being completed by the time when tension rises to 5%, was followed by a slow intensity decrease down to 50%, which was associated with the tension rise. In both phases, lateral widths remained unchanged. A4 reflexion also behaves in the same way, although the first phase (the intensity increase) was not clear due to unsatisfactory statistics. We interpret phase one as being caused by conformational change of the troponin-tropomyosin complex upon binding of Ca2+ to troponin, whereas phase two being due to direct contribution of the mass of the myosin heads bound to the thin filament, although possible contribution of conformational changes of the regulatory proteins to phase two is not excluded. The results indicated that the calcium activation of the thin filament leads the onset of the actomyosin interaction.     

Evidence for Unique Structural Change of Thin Filaments upon Calcium Activation of Insect Flight Muscle

Upon activation of living or skinned vertebrate skeletal muscle fibers, the sixth X-ray layer-line reflection from actin (6th ALL) is known to intensify, without a shift of its peak position along the layer line. Since myosin attachment to actin is expected to shift the peak towards the meridian, this intensification is considered to reflect the structural change of individual actin monomers in the thin filament. Here, we show that the 6th ALL of skinned insect flight muscles (IFMs) is rather weakened upon isometric calcium activation, and its peak shifts away from the meridian. This suggests that the actin monomers in the two types of muscles change their structures in substantially different manners. The changes that occurred in the 6th ALL of IFM were not diminished by lowering the temperature from 20 to 5 °C, while active force was greatly reduced. The inclusion of 100 μM blebbistatin (a myosin inhibitor) did not affect the changes either. This suggests that calcium binding to troponin C, rather than myosin binding to actin, causes the structural change of IFM actin.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Evidence for structurally different attached states of myosin cross-bridges on actin during contraction of fish muscle.

Using data from fast time-resolved x-ray diffraction experiments on the synchrotrons at Daresbury and (Deutsches Elektronen Synchrotron [DESY]), it is shown that during contraction of fish muscle there are at least two distinct configurations of myosin cross-bridges on actin, that they appear to have different tension producing properties and that they probably differ in the axial tilt of the cross-bridges on actin. Evidence is presented for newly observed myosin-based layer lines in patterns from active fish muscle, together with intensity changes of the actin layer lines. On the equator, the 110 reflection changes much faster (time for 50% change t1/2 = 21 +/- 4 ms after activation) than the 100 reflection (t1/2 = 35 +/- 8 ms) and tension (t1/2 = 41 +/- 3 ms) during the rising phase of tetanic contractions. These and higher order reflections have been used to show the time course of mass attachment at actin during this rising phase. Mass arrival (t1/2 = 25 ms) precedes tension by approximately 15 ms. Analysis has been carried out to evaluate the effects of changes in sarcomere length during the tetanus. It is shown that any such effects are very small. Difference "equatorial" electron density maps between active muscle at a time when mass arrival at actin is just complete, but the tension is still rising, and at a later time well into the tension plateau, show that the structural difference between the lower and higher force states corresponds to mass movement consistent with axial swinging of heads from a nonstereospecific actin attached state (low force) to a more stereospecific (high force) state.     

Arrangement of myosin heads in relaxed thick filaments from frog skeletal muscle

The distribution of myosin heads on the surface of frog skeletal muscle thick filaments has been determined by computer processing of electron micrographs of isolated filaments stained with tannic acid and uranyl acetate. The heads are arranged in three strands but not in a strictly helical manner and so the structure has cylindrical symmetry. This accounts for the "forbidden" meridional reflections seen in diffraction patterns. Each layer-line therefore represents the sum of terms of Bessel orders 0, +/- 3, +/- 6, +/- 9 and so on. These terms interact so that, unlike a helical object without terms from overlapping Bessel orders, as the azimuth is changed, the amplitude on a layer-line at a particular radius varies substantially and its phase does not alter linearly. Consequently, a three-dimensional reconstruction cannot be produced from a single view. We have therefore used tilt series of three individual filaments to decompose the data on layer-lines 0 to 6 into terms of Bessel orders up to +/- 9 using a least-squares procedure. These data had a least-squares residual of 0.32 and enabled a three-dimensional reconstruction to be obtained at a nominal resolution of 6 nm. This showed, at a radius of about 10 nm, three strands of projecting morphological units with three units spaced along each strand every 42.9 nm axially. We have identified these units with pairs of myosin heads. Successive units along a strand are perturbed axially, azimuthally and radially from the positions expected if the structure was perfectly helical. This may simply be a consequence of steric restrictions in packing the heads on the thick filament surface, but could also reflect an underlying non-helical arrangement of myosin tails, which would be consistent with the thick filament shaft being constructed from three subfilaments in which the tails were arranged regularly. There was also material at a radius of about 6 nm spaced 42.9 nm axially, which we tentatively identified with accessory proteins. The filament shaft had a pronounced pattern of axial staining.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-activation?

Low-angle X-ray diffraction patterns from relaxed fruitfly (Drosophila) flight muscle recorded on the BioCat beamline at the Argonne Advanced Photon Source (APS) show many features similar to such patterns from the "classic" insect flight muscle in Lethocerus, the giant water bug, but there is a characteristically different pattern of sampling of the myosin filament layer-lines, which indicates the presence of a superlattice of myosin filaments in the Drosophila A-band. We show from analysis of the structure factor for this lattice that the sampling pattern is exactly as expected if adjacent four-stranded myosin filaments, of repeat 116 nm, are axially shifted in the hexagonal A-band lattice by one-third of the 14.5 nm axial spacing between crowns of myosin heads. In addition, electron micrographs of Drosophila and other flies (e.g. the house fly (Musca) and the flesh fly (Sarcophaga)) combined with image processing confirm that the same A-band superlattice occurs in all of these flies; it may be a general property of the Diptera. The different A-band organisation in flies compared with Lethocerus, which operates at a much lower wing beat frequency (approximately 30 Hz) and requires a warm-up period, may be a way of optimising the myosin and actin filament geometry needed both for stretch activation at the higher wing beat frequencies (50 Hz to 1000 Hz) of flies and their need for a rapid escape response.     

Helical reconstruction of frozen-hydrated scallop myosin filaments.

Native myosin filaments from scallop striated muscle that have been rapidly frozen in relaxing solutions appear to be well preserved in vitreous ice. Electron micrographs of samples at -177 degrees C were recorded with an electron dose of 10 e/A2 at 1.5 microns defocus. After filament images were straightened by spline-fitting, several transforms showed well-defined layer-lines arising from the helical structure of the filament. A set of 17 near-meridional layer-lines has been collected and corrected for background and for phase and amplitude contrast functions. Preliminary helical reconstructions from this still incomplete data set reveal aspects of structure that were not apparent from earlier analysis of negatively stained filaments from scallop muscle. Individual pear-shaped myosin heads now appear to be well resolved from each other and from the filament backbone. The two heads of each myosin molecule appear to be splayed apart axially. The reconstructions also reveal that the filament backbone has a polygonal shape in cross-section, and that it appears to contain seven peripherally located subfilaments.     

X-ray diffraction patterns from mammalian heart muscle

We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart.     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)