X-ray study of myosin heads in contracting frog skeletal muscle

Using synchrotron radiation, the behaviour of the diffuse X-ray scatter was investigated in the relaxed and active phases of auxotonic and isometric contractions. Muscles were stimulated tetanically for 0.75 of a second, leaving intervals of three minutes between successive contractions. In isometric contractions the scatter is very asymmetric, which means that the myosin heads have a strongly preferred orientation. During tension rise the scatter expands in the meridional direction and contracts in the equatorial direction, the maximal local intensity change being about 20%. The shape change indicates that on average the myosin heads become oriented more perpendicularly to the fibre axis. The distribution of orientations at peak tension is quite different from that we found previously in X-ray scattering data from rigor muscles. In auxotonic contractions where muscles shorten against an increasing tension the scatter is practically circularly symmetrical. This suggests that during shortening the myosin heads go evenly through a wide range of orientations. It is concluded that the results from both the auxotonic and isometric experiments provide strong support for the rotating myosin head model. In isometric contractions the transition between the relaxed phase and peak tension is accompanied by an overall increase in scattering intensity of about 10%: this corresponds to a relative increase in the fraction of disordered myosin heads by almost 30%.     

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.     

Structural characteristics and aggregation of myosin heads in skeletal muscles

Native conformational modifications of rabbit skeletal muscle myosin and its subfragment-1 (S-1) within the temperature range of 0-40 degrees C and irreversible unfolding of these proteins structure at temperatures 40-70 degrees C have been studied by the fluorescence and light scattering methods. The results obtained permit stating that myosin and its active subfragments form associates at the concentrations above 0.3 microM. Hydrophobic interactions between definite sites of S-1 are likely to be primarily responsible for the association. The complex profile of S-1 melting curve at high ionic strength indicates the existence of three structural domains in the heavy chain of the myosin head.     

Structural properties of frog muscle myosin.

Frog myosin is a labile molecule, undergoing irreversible aggregation and rapid loss of ATPase; however, a procedure is described which provides highly purified myosin, with stable solubility and enzymatic properties, from skeletal muscle of Rana catesbeiana. Frog myosin contains heavy chains and light chains 1, 2, and 3. Light chain 3 is present in excess over light chain 1, and light chain 2 may occur as either, or both, of 2 closely migrating bands. On two-dimensional electrophoresis, light chain 1 generates an isoelectric component with pK 5.60; light chain 2 generates a complex pattern with 3 or 4 major components; and light chain 3 generates 2 major components with pK 5.00 and 4.92. The same subunit composition is obtained for frogs acclimated at 25 and 5 degrees C; however, proteolytic artifacts may occur in myosin preparations purified in the absence of protease inhibitors, especially in warm-acclimated frogs.     

Tension responses of frog skeletal muscle to ramp and step length changes

Tension responses to ramp shortening of varying speed in whole muscle or single fibres from the plateau of an isometric tetanus, revealed at least two distinct phases. There was a fast initial drop in tension followed by a change of slope and a definite inflexion on the tension record. As the velocity of the imposed length change was increased, the inflexion point appeared at a lower tension. Similar inflexions were not observed during ramp releases to an elastic band or a segment of semitendinosus tendon. The tension records obtained with moderately fast ramp length changes to contracting muscle reflect the T1 and T2 phases of the tension transients.     

Structural changes in myosin during contraction and the state of ATP in the intact frog muscle

The reactivity of myosin to [¹⁴C]-labeled N-ethylmaleimide ([¹⁴C] NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [¹⁴C] NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [¹⁴C] NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [¹⁴C] NEM into myosin and that upon the irreversible loss of ATP the rate increases. ³¹P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.     

Shape and length of myosin heads.

There is controversy concerning the shape and length of myosin heads. In the present paper we try to analyse the data and to draw clear conclusions in this field. When the myosin heads are isolated (S1) from the rest of the molecule, their length is approximately 12 nm and their shape is close to that of a prolate ellipsoid with an axial ratio approximately 2.3 (in solution) or close to that of a comma when attached to F-actin (with a length of 12-13 nm). When the myosin heads are observed on a whole molecule, their length is approximately 19 nm and they are pear-shaped. Here we suggest that all these observations are compatible. We believe that, for a whole myosin molecule, a large part of the head-rod joint (S1/S2 joint) is measured with the head, owing to a particularly heavy staining or shadowing of this joint. On the other hand, S1 is probably built up of a head part plus the S1/S2 joint, which is not revealed by the usual techniques (hydrodynamics, X-ray and neutron scattering). Finally, the comma shape would be related to a flexible part in the head region of S1, which is significantly bent when S1 is attached to F-actin, but which would be less bent for S1 in solution. A similar bending also occurs in crystalline S1.     

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.     

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.     

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.     

Orientational dynamics of indane dione spin-labeled myosin heads in relaxed and contracting skeletal muscle fibers.

We have used electron paramagnetic resonance (EPR) spectroscopy to study the orientation and rotational motions of spin-labeled myosin heads during steady-state relaxation and contraction of skinned rabbit psoas muscle fibers. Using an indane-dione spin label, we obtained EPR spectra corresponding specifically to probes attached to Cys 707 (SH1) on the catalytic domain of myosin heads. The probe is rigidly immobilized, so that it reports the global rotation of the myosin head, and the probe's principal axis is aligned almost parallel with the fiber axis in rigor, making it directly sensitive to axial rotation of the head. Numerical simulations of EPR spectra showed that the labeled heads are highly oriented in rigor, but in relaxation they have at least 90 degrees (Gaussian full width) of axial disorder, centered at an angle approximately equal to that in rigor. Spectra obtained in isometric contraction are fit quite well by assuming that 79 +/- 2% of the myosin heads are disordered as in relaxation, whereas the remaining 21 +/- 2% have the same orientation as in rigor. Computer-simulated spectra confirm that there is no significant population (> 5%) of heads having a distinct orientation substantially different (> 10 degrees) from that in rigor, and even the large disordered population of heads has a mean orientation that is similar to that in rigor. Because this spin label reports axial head rotations directly, these results suggest strongly that the catalytic domain of myosin does not undergo a transition between two distinct axial orientations during force generation. Saturation transfer EPR shows that the rotational disorder is dynamic on the microsecond time scale in both relaxation and contraction. These results are consistent with models of contraction involving 1) a transition from a dynamically disordered preforce state to an ordered (rigorlike) force-generating state and/or 2) domain movements within the myosin head that do not change the axial orientation of the SH1-containing catalytic domain relative to actin.     

Spectrophotometric measurements of metabolically induced pH changes in frog skeletal muscle.

A means of measuring pH spectrophotometrically with two pH-sensitive indicators, neutral red (NR) and bromcresol purple (BCP), is presented. Theoretical calculations and experimental measurements of pH in solution correspond in a satisfactory manner for both dyes. Spectrophotometric determinations of pH were made of dye-equilibrated frog sartorius muscles. A more accurate indication of shifting intracellular pH (pH_i) was obtained with NR than with BCP, since only the spectra of NR-equilibrated muscles were insensitive to changes in external pH over the range 7.0–6.0. Muscles were stimulated in oxygenated Ringer's and the latent phase of acidification correlated with lactic acid production by analysis of tissue frozen after spectral determination of ΔpH_i. Buffering capacities were calculated to be 0.041 to 0.048 g equiv per liter of strong acid or base per resultant ΔpH for a pH_i range covering 0.25 unit.     

An x-ray diffraction study on the ADP-induced conformational change in skeletal muscle myosin

Effects of ADP on the conformation of myosin cross-bridges were studied in x-ray diffraction experiments on single skinned fibers of frog skeletal muscle by photorelease of ADP from caged-ADP. The experiments were performed at the third-generation synchrotron radiation facility SPring-8 with a time resolution of 5 ms. The intensity of the third-order meridional reflection from myosin filaments (at 1/14.4 nm(-1)) increased promptly after the ADP release with a time constant smaller than 5 ms, which was similar to that of tension decline. The results show that ADP binding induces a conformational change of myosin in skeletal muscle fibers.     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Spectrophotometric studies on the pH of frog skeletal muscle. PH change during and after contractile activity

The spectral characteristics of the pH-sensitive dyes neutral red (NR) and bromcresol purple (BCP) were utilized for studies of the changing intracellular pH (pHi) of sartorius muscles from Rana pipiens, both during the course of an isometric twitch and during recovery metabolism subsequent to a train of twitches. The information from the two dissimilar dyes correlated to confirm the methodology. Neither the fast realkalinization observed during a twitch nor the slow alkalizing phase of recovery metabolism was affected in an obvious manner when phosphocreatine (PC) hydrolysis was blocked by 1-fluoro-2,4-dinitrobenzene (FDNB). Iodoacetic acid (IAA) did inhibit the slow acidic phase of recovery metabolism. The conclusion is made that alkalizing reactions other than PC breakdown must be considered as operative at these levels of activity. Hypertonic solutions altered twitch tension and time course without altering the pHi shifts observed until approximately 75% of the twitch amplitude was abolished. Multiple effects of hypertonic solutions as the muscle approach tonic equilibrium are proposed.     

Tension response in skinned Ca-activated skeletal muscle fibers of the frog to temperature jump following stepwise changes in the fiber length

Joulean temperature jump from 4-7 degrees to 20-25 degrees completed in 0.2 ms was applied to suspended in the air chemically skinned Ca-activated (pCa = 5.5-6) skeletal muscle fibres of the frog 2 ms after stepwise length changes (duration 0.3 ms, amplitudes --6. +3 nm per half sarcomere). The temperature jump induced a biphasic rise of tension, as was described earlier. Neither the time constant of the 2nd slow phase, nor maximum tension after the temperature jump were dependent on the length step amplitude. The amplitude and time constant of the 1st phase (1.2-0.28 ms) decreased after the fibre release. It shows that the 1st phase of the tension rise induced by the temperature jump is due to conformation in cross-bridges attached to thin filaments.     

Phosphoinositides in frog skeletal muscle: a quantitative analysis

The contents of major phospholipids per g of wet wt. in frog skeletal muscle are: 5.3 mumol PC; 1.4 mumol PE; 1 mumol SM; 0.4 mumol PtdIns; 0.3 mumol CL; and 0.13 mumol PS. The quantities of polyphosphoinositides per g of wet wt. are: 181 nmol PtInsP; 28 nmol PtdInsP2; and 8 nmol lyso-PtdInsP2. The specific activity of labelling of the total muscle ATP attained by external incubation with [32P]Pi was found to be 57 dpm/nmol x g muscle wet wt. PtdInsP2, the highest labelled polyphosphoinositide, showed a specific activity of 64,000 dpm/nmol per g muscle wet wt., suggesting that high specific activity ATP may be compartmentalized in the local environment of the triads and used as a substrate by the PtdIns and PtInsP kinase in that region. PtdInsP2 which is the immediate precursor for the release of InsP3, is found at a significant concentration and strategically located for its postulated role as a substrate for the action of phosphoinositidase C. The presence of a novel endogenous polyphosphoinositide, lyso-PtdInsP2, in animal tissues is reported for the first time. Electrical stimulation leads towards a rapid catabolization of polyphosphoinositides revealed by reductions in the 3H- and 32P-labelling, suggesting that muscle excitation is associated with the activation of breaking down of polyphosphoinositides.     

Structural studies of rabbit skeletal muscle myosin light chain kinase with monoclonal antibodies

Monoclonal antibodies directed against rabbit skeletal muscle myosin light chain kinase have been used to study the domains of this kinase. Specificity of nine monoclonal antibodies against rabbit skeletal muscle myosin light chain kinase was demonstrated by immunoblot analysis and immunoadsorption of kinase activity. None of the antibodies reacted by immunoblot analysis with either chicken skeletal or rabbit smooth muscle myosin light chain kinases. Epitope mapping of trypsin-digested rabbit skeletal muscle myosin light chain kinase showed that antibodies 2a, 9a, 9b, 12a, 12b, 16a, and 16b are directed against the 40-kDa catalytic domain. In addition, these seven antibodies reacted with sites that are clustered within a 14-kDa fragment of the kinase generated by Staphylococcus aureus V8 protease digestion. Two monoclonal antibodies, 14a and 19a, reacted with two distinct epitopes located within the inactive, asymmetric trypsin fragment. Six of nine monoclonal antibodies (2a, 9a, 9b, 12a, 12b, and 14a) inhibited kinase activity. Kinetic analyses demonstrated that antibodies 2a, 12a, and 14a inhibited kinase activity competitively with respect to myosin phosphorylatable light chain; 2a, 12a, and 14a exhibit noncompetitive inhibition with respect to calmodulin. These data suggest that monoclonal antibodies 2a, 12a, and 14a bind at or adjacent to the active site of the kinase.     

Recovery of action potentials and twitches after K-contractures in frog skeletal muscle

To give information about intracellular Ca2+ translocation during and after K-contractures in vertebrate skeletal muscle fibers, we examined recovery of action potentials and twitches after interruption and spontaneous relaxation of K-contractures at low temperature (3 degrees C) that greatly reduced the rate of Ca2+ reuptake by the sarcoplasmic reticulum. On membrane repolarization interrupting K-contractures, the amplitude of both action potentials and twitches recovered quickly, while the falling phase of action potential was markedly slowed at first to prolong its refractory period, so that repetitive stimulation (20 Hz) did not produce a complete tetanus. Meanwhile, on membrane repolarization after spontaneous relaxation of K-contractures, the action potentials were markedly reduced in amplitude and prolonged in duration at first, also resulting in prolonged refractory period. These results are discussed in connection with Ca2+ absorption to the surface and transverse tubule membranes, producing changes in action potential kinetics.