Orientation of intermediate nucleotide states of indane dione spin-labeled myosin heads in muscle fibers.

We have used electron paramagnetic resonance to study the orientation of myosin heads in the presence of nucleotides and nucleotide analogs, to induce equilibrium states that mimic intermediates in the actomyosin ATPase cycle. We obtained electron paramagnetic resonance spectra of an indane dione spin label (InVSL) bound to Cys 707 (SH1) of the myosin head, in skinned rabbit psoas muscle fibers. This probe is rigidly immobilized on the catalytic domain of the head, and the principal axis of the probe is aligned nearly parallel to the fiber axis in rigor (no nucleotide), making it directly sensitive to axial rotation of the head. On ADP addition, all of the heads remained strongly bound to actin, but the spectral hyperfine splitting increased by 0.55 +/- 0.02 G, corresponding to a small but significant axial rotation of 7 degrees. Adenosine 5'-(adenylylim-idodiphosphate) (AMPPNP) or pyrophosphate reduced the actomyosin affinity and introduced a highly disordered population of heads similar to that observed in relaxation. For the remaining oriented population, pyrophosphate induced no significant change relative to rigor, but AMPPNP induced a slight but probably significant rotation (2.2 degrees +/- 1.6 degrees), in the direction opposite that induced by ADP. Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) relaxed the muscle fiber, completely dissociated the heads from actin, and produced disorder similar to that in relaxation by ATP. ATP gamma S plus Ca induced a weak-binding state with most of the actin-bound heads disordered. Vanadate had negligible effect in the presence of ADP, but in isometric contraction vanadate substantially reduced both force and the fraction of oriented heads. These results are consistent with a model in which myosin heads are disordered early in the power stroke (weak-binding states) and rigidly oriented later in the power stroke (strong-binding states), whereas transitions among the strong-binding states induce only slight changes in the axial orientation of the catalytic domain.     

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

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

Orientation of spin-labeled myosin heads in glycerinated muscle fibers.

We have used electron paramagnetic resonance (EPR) spectra to study spin labels selectively and rigidly attached to myosin heads in glycerinated rabbit psoas muscle fibers. Because the angle between the magnetic field and the principal axis of the probe determines the position of the EPR absorption line, spectra from labeled fibers oriented parallel to the magnetic field yielded directly the distribution of spin label orientations relative to the fiber axis. Two spin labels, having reactivities resembling iodoacetamide (IASL) and maleimide (MSL), were used. In rigor fibers with complete filament overlap, both labels displayed a narrow angular distribution, full width at half maximum approximately 15 degrees, centered at angles of 68 degrees (IASL) and 82 degrees (MSL). Myosin subfragments (heavy meromyosin and subfragment-1) were labeled and allowed to diffuse into fibers. The resulting spectra showed the same sharp angular distribution that was found for the labeled fibers. Thus is appears that virtually all myosin heads in a rigor fiber have the same orientation relative to the fiber axis, and this orientation is determined by the actomyosin bond. Experiments with stretched fibers indicated that the spin labels on the fraction of heads not interacting with actin filaments had a broad angular distribution. Addition of ATP to unstretched fibers under relaxing conditions produced orientational disorder, resulting in a spectrum almost indistinguishable from that of an isotropic distribution of probes. Addition of either an ATP analog (AMPPNP) or pyrophosphate produced partial disorder. That is a fraction of the probes remained sharply oriented as in rigor while a second fraction was in a disordered distribution similar to that of relaxed fibers.     

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%.     

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

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

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.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.     

Orientation of spin-labeled nucleotides bound to myosin in glycerinated muscle fibers.

Electron paramagnetic resonance (EPR) spectroscopy of paramagnetic derivatives of ATP has been used to probe the angular distribution of myosin in glycerinated muscle fibers. Three nucleotide spin labels have been prepared with the nitroxide free radical moiety attached, via an ester linkage to either: the 2' or 3' positions of the ribose unit of ATP (SL-ATP), the 2' position of 3' deoxy ATP (2'SL-dATP), or the 3' position of 2' deoxy ATP (3'SL-dATP). In muscle fibers, these nucleotides are quickly hydrolyzed to their diphosphate forms. All three diphosphate analogues bind to the nucleotide site of myosin with similar affinities: rabbit psoas fibers, 7 X 10(3)/M; insect flight muscle, 5 X 10(3)/M; and rabbit soleus muscle, 2 X 10(4)/M. Analysis of the spectra showed that the principal z-axis of the nitroxide attached to bound nucleotides was oriented with respect to the filament axis. The principal axes of 3'SL-dADP and 2'SL-dADP appeared to be preferentially aligned at mean angles of 67 degrees +/- 4 degrees and 55 degrees +/- 5 degrees, respectively. The distribution of probes about these angles can be described by Gaussians with widths of 16 degrees +/- 4 degrees and 13 degrees +/- 5 degrees, respectively. The spectrum of bound SL-ADP was a linear combination of the spectra of the two deoxy analogues. These orientations were the same in the three muscle types examined, indicating a high degree of homology in the nucleotide binding site. Applying static strains as high as 0.2 N/mm2 to muscle fibers caused no change in the orientation of myosin-bound, spin-labeled nucleotides. When muscle fibers were stretched to decrease actin and myosin filament overlap, bound SL-ADP produced EPR spectra indicative of probes with a highly disordered angular distribution. Sodium vanadate and SL-ATP caused fiber stiffness to decrease, and the EPR spectrum of the bound analogue indicated an increase in the fraction of disoriented probes with a concomitant decrease in the fraction of oriented probes. These findings indicate that when myosin is bound to actin its nucleotide site is highly oriented relative to the fiber axis, and when this interaction is removed the orientation of the nucleotide site becomes highly disordered.     

Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle

Thick filaments from leg muscle of tarantula, maintained under relaxing conditions (Mg-ATP and EGTA), were negatively stained and photographed with minimal electron dose. Particles were selected for three-dimensional image reconstruction by general visual appearance and by the strength and symmetry of their optical diffraction patterns, the best of which extend to spacings of 1/5 nm-1. The helical symmetry is such that, on a given layer-line, Bessel function contributions of different orders start to overlap at fairly low resolution and must therefore be separated computationally by combining data from different views. Independent reconstructions agree well and show more detail than previous reconstructions of thick filaments from Limulus and scallop. The strongest feature is a set of four long-pitch right-handed helical ridges (pitch 4 X 43.5 nm) formed by the elongated myosin heads. The long-pitch helices are modulated to give ridges with an axial spacing of 14.5 nm, lying in planes roughly normal to the filament axis and running circumferentially. We suggest that the latter may be formed by the stacking of a subfragment 1 (S1) head from one myosin molecule on an S1 from an axially neighbouring molecule. Internal features in the map indicate an approximate local twofold axis relating the putative heads within a molecule. The heads appear to point in opposite directions along the filament axis and are located very close to the filament backbone. Thus, for the first time, the two heads of the myosin molecule appear to have been visualized in a native thick filament under relaxing conditions.     

The T-SR junction in contracting single skeletal muscle fibers

The junction between the T system and sarcoplasmic reticulum (SR) of frog skeletal muscle was examined in resting and contracting muscles. Pillars, defined as pairs of electron-opaque lines bounding an electron- lucent interior, were seen spanning the gap between T membrane and SR. Feet, defined previously in images of heavily stained preparations, appear with electron-opaque interiors and as such are distinct from the pillars studied here. Amorphous material was often present in the gap between T membrane and SR. Sometimes the amorphous material appeared as a thin line parallel to the membranes; sometimes it seemed loosely organized at the sites where feet have been reported. Resting single fibers contained 39 +/- 14.3 (mean +/- SD; n = 9 fibers) pillars/micrometer2 of tubule membrane. Single fibers, activated by a potassium-rich solution at 4 degrees C, contained 66 +/- 12.9 pillars/micrometer2 (n = 8) but fibers contracting in response to 2 mM caffeine contained 33 +/- 8.6/micrometer2 (n = 5). Pillar formation occurs when fibers are activated electrically, but not when calcium is released directly from the SR; and so we postulate that pillar formation is a step in excitation-contraction coupling.     

Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high-resolution study with deuterated spin labels

We have used electron paramagnetic resonance (EPR) to determine the effects of ADP on the orientational distribution of nitroxide spin labels attached to myosin heads in skinned rabbit psoas muscle fibers. To maximize the specificity of labeling, we spin-labeled isolated myosin heads (subfragment 1) on a single reactive thiol (SH1) and diffused them into unlabeled muscle fibers. To maximize spectral and orientational resolution, we used perdeuterated spin labels, 2H-MSL and 2H-IASL, eliminating superhyperfine broadening and thus narrowing the line widths. Two different spin labels were used, with different orientation relative to the myosin head, to ensure that the results are not affected by unfavorable probe orientation. In rigor, a very narrow three-line spectrum was observed for both spin labels, indicating a narrow orientational distribution, as reported previously (Thomas & Cooke, 1980). ADP induced very slight changes in the spectrum, corresponding to very slight (but significant) changes in the orientational distribution. These changes were quantified by a digital analysis of the spectra, using a two-step simplex fitting procedure (Fajer et al., 1990). First, the magnetic tensor values and line widths were determined by fitting the spectrum of a randomly oriented sample. Then the spectrum of oriented fibers was fit to a model by assuming a Gaussian distribution of the tilt angle (theta) and twist angle (phi) of the nitroxide principal axes relative to the fiber axis. A single-Gaussian distribution resulted in inadequate fits, but a two-component model gave excellent results. ADP induces a small (less than 5 degrees) rotation of the major components for both spin labels, along with a similarly small increase of disorder about the average positions.     

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

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

Axial disposition of myosin heads in isometrically contracting muscles

Meridional x-ray diffraction diagrams, recorded with high angular resolution, from muscles contracting at the plateau of isometric tension show that the myosin diffraction orders are clusters of peaks. These clusters are due to pronounced interference effects between the myosin diffracting units on either side of the M-line. A theoretical analysis based on the polarity of the myosin (and actin) filaments shows that it is possible to extract phase information from which the axial disposition of the myosin heads can be determined. The results show that each head in a crown pair has a distinct structural disposition. It appears that only one of the heads in the pair stereospecifically interacts with the thin filament at any one time.     

Microsecond rotational motion of spin-labeled myosin heads during isometric muscle contraction. Saturation transfer electron paramagnetic resonance.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin heads in bundles of skinned muscle fibers, under conditions of rigor, relaxation, and isometric contraction. Experiments were performed on fiber bundles perfused continuously with an ATP-regenerating system. Conditions were identical to those we have used in previous studies of myosin head orientation, except that the fibers were perpendicular to the magnetic field, making the spectra primarily sensitive to rotational motion rather than to the orientational distribution. In rigor, the high intensity of the ST-EPR signal indicates the absence of microsecond rotational motion, showing that heads are all rigidly bound to actin. However, in both relaxation and contraction, considerable microsecond rotational motion is observed, implying that the previously reported orientational disorder under these conditions is dynamic, not static, on the microsecond time scale. The behavior in relaxation is essentially the same as that observed when myosin heads are detached from actin in the absence of ATP (Barnett and Thomas, 1984), corresponding to an effective rotational correlation time of approximately 10 microseconds. Slightly less mobility is observed during contraction. One possible interpretation is that in contraction all heads have the same mobility, corresponding to a correlation time of approximately 25 microseconds. Alternatively, more than one motional population may be present. For example, assuming that the spectrum in contraction is a linear combination of those in relaxation (mobile) and rigor (immobile), we obtained a good fit with a mole fraction of 78-88% of the heads in the mobile state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for cooperative interactions of myosin heads with thin filament in the force generation of vertebrate skeletal muscle fibers

To examine the possibility of cooperative interactions between the two myosin heads in muscle contraction, Ca2+-activated force development, K+-EDTA-and Mg2+-ATPase activities, muscle fiber stiffness, and the velocity of unloaded shortening were measured on partially p-phenylenedimaleimide (p-PDM)-treated glycerinated muscle fibers, which contained a mixture of myosin molecules with zero, one, and two of their heads inactivated, and the relationships among these values (expressed relative to the control values) were studied. It was found that the magnitude of the Ca2+-activated isometric force development was proportional to the square of both K+-EDTA- and Mg2+-ATPase activities and also to the square of muscle fiber stiffness. If the two myosin heads in the glycerinated fibers are assumed to react independently with p-PDM, the above results strongly suggest that each myosin molecule in the thick filaments can generate force only when its two heads do not react with p-PDM, muscle fiber stiffness is determined by the total number of native heads, and there is no cooperative interaction between the two myosin heads in catalyzing ATP hydrolysis.     

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.     

Curare-induced transformation of myosin pattern in developing skeletal muscle fibers

The effects of neuromuscular block on the pattern of distribution of myosin isozymes in developing skeletal muscle fibers was examined by immunocytochemistry. The homogeneous population of fibers in the anterior latissimus dorsi (ALD) of the 18-day chick embryo was converted by curare to a mosaic of at least two categories of fibers. Normally all fibers in this slow muscle reacted with antibodies against slow myosin (anti-ALD). They also reacted with an antibody specific for the alkali 1 light chain (anti-delta 1) but not the alkali 2 light chain (anti-delta 2) of fast myosin. After treatment with curare, which inhibits neuronal cell death and increases the number of axonal endings, ALD muscle fibers continued to react with anti-delta 1, but many now reacted with anti-delta 2 as well. The same fibers failed to react with anti-ALD. From this it can be concluded that the myosin in this population was converted to a type not normally present. The changes, therefore, are not merely a result of the preferential loss of a slow type of fiber, nor are they a result of delayed maturation. In contrast, curare had no apparent effect on the fast posterior latissimus dorsi (PLD). As in the normal muscle at 18 days, all fibers reacted strongly with anti-delta 1 and to variable degrees with anti-delta 2, and very few fibers reacted with anti-ALD. Our observations suggest that the dual response to antibodies against fast and slow myosin during development is not a necessary consequence of multiple axon terminals. We present evidence that curare induces the expression of a different myosin in the embryonic ALD, and we suggest that the selective transformation of the fiber population may be a manifestation of a change in composition of the motoneuron pool.     

Phosphorylating pathways and fatigue development in contracting Xenopus single skeletal muscle fibers

To investigate the differential contribution of oxidative and substrate-level phosphorylation to force production during repetitive, maximal tetanic contractions, single skeletal muscle fiber performance was examined under conditions of high-oxygen availability and anoxia. Tetanic force development (P) was measured in isolated, single type-1 muscle fibers (fast twitch; n = 6) dissected from Xenopus lumbrical muscle while being stimulated at increasing frequencies (0.25, 0.33, and 0.5 Hz), with each frequency lasting 2 min. Two separate work bouts were conducted, with the perfusate PO(2) being either 0 or 159 mmHg. No significant (P < 0. 05) difference was found in the initial peak tensions (P(0)) between the high (334 +/- 57 kPa) and the low (325 +/- 41 kPa) PO(2) treatment. No significant difference in P was observed between the treatments during the first 50 s. However, a significant difference in force production was observed between the high (P/P(0) = 0.96 +/- 0.02) and the low PO(2) condition (P/P(0) = 0.92 +/- 0.02) by 60 s of work. After 60 s, steady-state force production was maintained during the high compared with the low PO(2) condition until stimulation frequency was increased, at which point developed tension during the high PO(2) condition began to decline. Time to fatigue (P/P(0) = 0.3) was reached significantly sooner during the low (250 +/- 16 s) than the high PO(2) condition (367 +/- 28 s). These results demonstrate that during the first 50 s of 0.25-Hz contractions, substrate-level phosphorylation has the capacity to maintain force and ATP hydrolysis when oxidative phosphorylation is absent. This period was followed by an oxygen-dependent phase in which force generation was maintained during the high PO(2) condition (but not during the low PO(2) condition) until the onset of a final fatiguing phase, at which a calculated maximal rate of oxidative phosphorylation was reached.