Role of myosin light chains

Summary All conventional myosin IIs, whether isolated from skeletal, smooth, or invertebrate muscle sources, have two heads attached to an extended 16 nm -helical coiled-coil tail. The head can be divided into a globular motor domain of 770 amino acids that contains the catalytic and actin binding sites, and a neck region of 70 amino acids which binds one essential and one regulatory light chain (ELC and RLC). The neck region with its associated LCs plays both structural and regulatory roles. While the mechanism and extent of regulation by the LCs varies for different myosins, the structural role may be a more fundamental feature of myosin II motors. Our understanding of the neck region has advanced rapidly in recent years primarily because of two types of information: (1) the high resolution structures of the LC binding domain from the thick-filament regulated scallop myosin (Xie et al., 1994) and of the head of unregulated skeletal myosin (Rayment et al., 1993), and (2) the ability to remove and/or mutate portions of both the heavy and light chains for analysis by in vitro motility assays.     

Formation of new quasi-crystalline ordered aggregates by gizzard myosin

Summary Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 ihm KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl₂ and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-Si and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2m KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg²⁺ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 him MgCl₂, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.     

8-Anilino-1-naphthalenesulphonate,a fluorescent probe for the regulatory light chain binding site of scallop myosin

Summary Regulatory light chain (RLC) dissociation from scallop myofibrils, myosin or its subfragments was accompanied by an increase in binding of the hydrophobic fluorophore, 8-anilino-1-naphthalenesulphonate (ANS) to the denuded proteins. The binding was monitored by the large increase in fluorescence emission at 460 nm when excited directly at 380 nm or via energy transfer from nearby tryptophan residues at 295 nm. ANS thus provides a convenient probe for following the kinetics of RLC dissociation in the presence of EDTA and its association in the presence of divalent metal ions. The observed RLC dissociation rate constant for myosin at 20° C was 7.5×10^–3 s^–1. The association rate constant, which was independent of the RLC concentration, was 5×10^–3 s^–1. Subfragment 1, prepared by digestion of myosin in the presence of divalent metal ions to protect the light chains [S1(+LC)], showed reversible ANS binding qualitatively similar to the parent molecule. However when prepared in the presence of EDTA, subfragment 1 lacked RLC [S1(–LC)], its heavy chain molecular weight was reduced by about 4000 and it lacked the ANS binding region attributed to the RLC site. The tryptic digestion pattern of S1(+LC) and S1(–LC) suggested that the 4000 difference peptide is at the C-terminus. Tryptic digestion of S1(+LC) has been shown to lead to the production of a regulatory peptide, comprising the two light chains and a heavy chain fragment, which displayed reversible ANS binding on addition of EDTA. Evidence is presented which suggests that this domain is at the C-terminus of subfragment 1.     

Regulation of nonmuscle myosins by heavy chain phosphorylation

Functional activities of many nonmuscle myosin isoforms are (or are postulated to be) regulated by heavy chain phosphorylation. Depending on the myosin isoform, the serine or threonine residues located within the head (myosin I or myosin VI) or within the C-terminal tail domains (myosin II or myosin V) can be phosphorylated by more or less specific endogenous kinases. In some isoforms phosphorylation can occur both in the head and tail domains, as it has been found for myosin III. There are also isoforms that can be regulated both by the heavy and regulatory light chain phosphorylation, as for the example myosin II from slide mold Dictyostelium discoideum. The goal of this review was to describe recent findings on regulation of myosin I, myosin II, myosin III, myosin V and myosin VI isoforms by their heavy chain phosphorylation including the short charcteristics of the relevant kinases. The biological aspects of the phosphorylation are also discussed.     

Temperature dependence of the force-generating process in single fibres from frog skeletal muscle

Generation of force and shortening in striated muscle is due to the cyclic interactions of the globular portion (the head) of the myosin molecule, extending from the thick filament, with the actin filament. The work produced in each interaction is due to a conformational change (the working stroke) driven by the hydrolysis of ATP on the catalytic site of the myosin head. However, the precise mechanism and the size of the force and length step generated in one interaction are still under question. Here we reinvestigate the endothermic nature of the force-generating process by precisely determining, in tetanised intact frog muscle fibres under sarcomere length control, the effect of temperature on both isometric force and force response to length changes. We show that raising the temperature: (1) increases the force and the strain of the myosin heads attached in the isometric contraction by the same amount (∼70 %, from 2 to 17 °C); (2) increases the rate of quick force recovery following small length steps (range between −3 and 2 nm (half-sarcomere)⁻¹) with a Q ₁₀ (between 2 and 12 °C) of 1.9 (releases) and 2.3 (stretches); (3) does not affect the maximum extent of filament sliding accounted for by the working stroke in the attached heads (10 nm (half-sarcomere)⁻¹). These results indicate that in isometric conditions the structural change leading to force generation in the attached myosin heads can be modulated by temperature at the expense of the structural change responsible for the working stroke that drives filament sliding. The energy stored in the elasticity of the attached myosin heads at the plateau of the isometric tetanus increases with temperature, but even at high temperature this energy is only a fraction of the mechanical energy released by attached heads during filament sliding.     

The effect of pyrophosphate on the reaction of myosin with 2, 4, 6-trinitrobenzene sulphonate

Summary Myosin was reacted with 2, 4, 6-trinitrobenzene sulphonate (TNBS) in the presence or absence of Mg-pyrophosphate. The reaction led to trinitrophenylation of lysyl residues which could be divided on the basis of the reaction into three classes: (i) two rapidly reacting lysyl residues (RLR), one residing on each head of myosin, whose rate of reaction depends on the presence of Mg-pyrophosphate; (ii) two lysyl residues which react with intermediate rate (ILR) and reside on the rod segment of myosin; and (iii) the remaining lysyl residues of myosin which react slowly with TNBS. The rate of the trinitrophenylation of RLR was followed spectrophotometrically and enzymatically, measuring an absorbance change at 345 nm, and also changes in K⁺(EDTA)-, Mg²⁺- and Ca²⁺-activated ATPase activities, respectively. According to analysis of the kinetics of the reaction, Mg-pyrophosphate inhibited the rate of trinitrophenylation in both heads of myosin, not in one head only as was suggested by Miyanishiet al. (J. Biochem Tokyo 85; 1979). Myosin heads (myosin subfragment-1, S-1) were prepared by digesting myosin trinitrophenylated in the absence and presence of Mg-pyrophosphate with chymotrypsin. S-1, with trinitrophenylated RLR, was separated from non-trinitrophenylated S-1 by DEAE cellulose column-chromatography. The trinitrophenylated S-1 had a high Mg²⁺- and a low K⁺(EDTA)-activated ATPase while the non-trinitrophenylated species had the usual high K⁺(EDTA)- and low Mg²⁺-ATPase activity. This result excluded the possibility suggested by Miyanishiet al., that the myosin head, which is resistant to trinitrophenylation in the presence of Mg-pyrophosphate, did not possess K⁺(EDTA)-activated ATPase activity. The presence of Mg-pyrophosphate during trinitrophenylation substantially affected the enzymic characteristics of the modified myosin. The myosin trinitrophenylated in the presence of Mg-pyrophosphate had a higher K⁺(EDTA)- and a lower Mg²⁺-ATPase activity.SH₁ (Cys-707) also probably becomes a target of the reaction if myosin is trinitrophenylated in the presence of Mg-pyrophosphate. This is deduced from the following findings: (i) the addition of dithiothreitol after trinitrophenylation partially reversed the loss in the K⁺(EDTA)-ATPase activity; and (ii) the specific alkylation of the SH₁ thiol by 1, 5-IAEDANS prior to trinitrophenylation prevented the effect of dithiothreitol on the ATPase activity of myosin. The results indicated that Mg-pyrophosphate induced structural changes in the myosin molecule which influenced the course and possibly the target(s) of trinitrophenylation.     

Structural changes induced in scallop heavy meromyosin molecules by Ca2+ and ATP

Summary We have used physicochemical and ultrastructural methods to investigate the effects of Ca²⁺ and ATP on the structure of purified heavy meromyosin (HMM) from the striated adductor muscle of the scallop, a species with myosin-linked regulation. Using papain as a structural probe, we found that, in the presence of ATP, the head/tail junction was five times more susceptible to digestion at high levels of Ca²⁺ than at low levels.wBy HPLC gel filtration, two fractions of scallop HMM with different Stokes radii were detected in the presence of ATP at low Ca²⁺, while at high Ca²⁺ a single peak with the larger Stokes radius predominated. Electron microscopy of rotary-shadowed HMM suggested that molecules with the smaller Stokes radius had their heads bent back towards their tails, while those with the larger radius had heads pointing away from the tail. The number of molecules with their heads bent back decreased at high Ca²⁺ levels. The data also showed that in the absence of ATP or at high salt, HMM molecules behaved similarly to those in the presence of ATP at high Ca²⁺.These results suggest that scallop myosin heads can exist in two conformations (heads down towards the tail and heads up away from the tail) and that the equilibrium between these two conformations is altered by the concentrations of salt, ATP and Ca²⁺. However, the equilibrium between the two forms appears to be too slow to be involved in regulating contraction. The heads-down configuration may instead be related to the inactive, folded (10S) form of scallop myosin and possibly involved in filament assembly during development.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 1996     

A-band architecture in vertebrate skeletal muscle: polarity of the myosin head array

Despite extensive knowledge of many muscle A-band proteins (myosin molecules, titin, C-protein (MyBP-C)), details of the organization of these molecules to form myosin filaments remain unclear. Recently the myosin head (crossbridge) configuration in a relaxed vertebrate muscle was determined from low-angle X-ray diffraction (Hudson et al. (1997), J Mol Biol 273: 440–455). This showed that, even without C-protein, the myosin head array displays a characteristic polar pattern with every third 143 Å-spaced crossbridge level particularly prominent. However, X-ray diffraction cannot determine the polarity of the crossbridge array relative to the neighbouring actin filaments; information crucial to a proper understanding of the contractile event. Here, electron micrographs of negatively-stained goldfish A-segments and of fast-frozen, freeze-fractured plaice A-bands have been used to determine the resting myosin head polarity relative to the M-band. In agreement with the X-ray data, the prominent 429 Å-spaced striations are seen outside the C-zone, where no non-myosin proteins apart from titin are thought to be located. The head orientation is with the concave side of the curved myosin heads (containing the entrance to the ATP-binding site) facing towards the M-band and the convex surface (containing the actin-binding region at one end) facing away from the M-band.     

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca²⁺ activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.     

Role of gizzard myosin light chains in calcium binding

Summary The contraction of molluscan and vertebrate smooth muscles is regulated by myosin. Although the myosin and its associated two subunits, the regulatory light chain and the essential light chain, constitute the Ca²⁺ regulatory system in both types of muscles, the mechanisms by which Ca²⁺ signal is transduced are quite different. In molluscan muscles, the direct binding of Ca²⁺ to the regulatory system triggers muscle contraction. In vertebrate smooth muscles, however, phosphorylation of the regulatory light chain is the major triggering mechanism. We measured Ca²⁺ binding in gizzard myosin and in hybrids of scallop myosin containing gizzard regulatory light chain or in hybrids of scallop regulatory domain containing gizzard essential light chain. Isolated chicken gizzard myosin did not bind Ca²⁺ in the range of pCa 8.0 to 5.0 in the presence of 2mM MgCl₂, supporting the lack of the specific Ca²⁺-binding site in gizzard myosin. Phosphorylation of the regulatory light chain did not generate a specific Ca²⁺-binding site. The hybrid scallop myosin containing gizzard regulatory light chain showed a similar Ca²⁺ binding as native scallop myosin with a one to one stoichiometry of Ca²⁺ to myosin head saturating at about pCa 6.0 at pH 7.6. In contrast, the hybrid scallop regulatory domain containing gizzard essential light chain did not bind Ca²⁺ either at pCa 6.0 or at pCa 8.0. Control preparations reconstituted with scallop essential light chains bound 0.69 mol per mol Ca²⁺ at pCa 6.0 with no binding at pCa 8.0. These results indicate that the inability of gizzard essential light chain to form the specific Ca²⁺-binding site is a major reason for the lack of the specific Ca²⁺-binding site in gizzard myosin.     

Differences in myosin head arrangement on relaxed thick filaments from Lethocerus and rabbit muscles

Relaxed thick filaments from insect asynchronous flight muscle appear different from those of other striated muscles, both in sections and as separated, negatively-stained structures. Unlike relaxed filaments of scallops, chelicerate arthropods, or vertebrate striated muscle, all of which display a predominantly helical arrangement of surface myosin heads, insect asynchronous flight muscle filaments appear striped, with cross-striations or shelves at spacings of 14.5 nm. Using a bifunctional agent to cross-link the active sites of nearest-neighbour myosin heads we previously demonstrated that the helical arrays on the surfaces of scallop, arthropod, fish and frog filaments are produced by the association of two oppositely-oriented myosin heads, each of which originates from an axially sequential molecule within the same helical strand. The effect of similarly cross-linking nearest-neighbour heads with the bifunctional agent 3,3′- dithio-bis[3′(2′)-O-(6- propionylamino)hexanoyl]adenosine 5′-triphosphate in the presence of vanadate on the solubility of thick filaments separated from Lethocerus indirect flight muscle (an insect asynchronous flight muscle) and rabbit psoas muscle was examined. After incubation on high salt, treated rabbit filaments retained their length and surface myosin, while untreated filaments and those with severed cross-links dissolved, indicating that the myosin head arrangement on rabbit filaments is similar to those previously studied. Treated indirect flight muscles filaments, how ever, separated into distinct segments of variable lengths, usually multiples of 150 nm, while untreated filaments and those with severed cross-links dissolved completely. This implies that intermolecular associations on indirect flight muscles filaments most likely occur between circumferentially-adjacent heads within each crown, but originating from different helical strands. We interpret this difference in the relaxed orientations of splayed myosin heads on the two types of filament as reflecting a difference in functional requirements at the onset of, or during, contractile activity This revised version was published online in July 2006 with corrections to the Cover Date.     

Bacteriophage φW-14: The contribution of covalently bound putrescine to DNA Packing in the Phage Head

Bacteriophage φW-14 is unusual because its DNA contains 12 mol% of the hypermodified pyrimidine, α-putrescinylthymine. The φW-14 virion is similar in morphology to T4, except that the φW-14 head is isometric rather than prolate, there is no collar-whisker structure associated with the neck, the tail fibers are short (～15 nm), and the base plate terminates in small plates or knobs rather than spikes. The contractile tail sheath of φW-14 appears to have a right-handed helical arrangement of subunits with a pitch in the extended form of ～20 nm. The “stacked disk” appearance of the tail sheath visible on negatively stained particles has a periodicity of 3–4 nm. The protein shell of the head has a similar thickness (2–3 nm) to that of T4. The φW-14 virion contains at least 17 different polypeptide species. Based on measurements from electron micrographs of negatively stained phage particles on the same grid square, the volume of the φW-14 head was estimated to be approximately 72% that of the T4 head. Surprisingly, however, the lengths of the DNA molecules released from φW-14 and T4 heads by osmotic shock were 59.6 ± 1.9 and 62.1 ± 2.4 μm, respectively. am42 is an amber mutant of φW-14 in which there is only 5 mol% putThy in the DNA made in the nonpermissive host. am42 virions are morphologically normal, but the length of the DNA released from these virions is only 53.1 ± 3.1 μm. We conclude that φW-14 DNA is packed much more compactly than T4 DNA into a virion of similar morphology and comparable complexity and that the tight packing is a consequence of, and dependent upon, the presence of putThy in φW-14 DNA.     

Visualization of domains in native and nucleotide-trapped myosin heads by negative staining

Summary Electron microscopy of negatively stained vertebrate skeletal muscle myosin molecules has revealed substructure suggestive of globular domains in the head portions of the molecule. This head substructure has been examined after both low and high electron dose. The results suggest it is probably not an artefact of radiation damage. The most common appearance is of one or two stain-filled clefts which run roughly perpendicular to the long axis of the head, giving rise to the appearance of two or three domains in a line. A large domain is located at the end of the head, while two smaller domains are arranged between this and the head-tail junction. The size of the large distal domain (about 10 nm long and about 7 nm wide at its widest point) is similar in heads showing either two or three domains.Stable analogues of M. ATP and M. ADP.Pi, the predominant complexes present during hydrolysis of ATP by myosin, were prepared by crosslinking the two reactive SH groups (SH₁ and SH₂) in the myosin head heavy chain with N,N-p-phenylenedimaleimide (pPDM) in the presence of ADP, and by forming a complex with vanadate ion and ADP. At this resolution ( 2 nm) the heads of these modified molecules did not appear markedly different from those of the untreated protein, although there was a small increase in the number of straight as opposed to curved heads after cross-linking withpPDM.Abbreviations pPDM N,N-p-phenylenedimaleimide - PAR 4-(2-pyridylazo)-resorcinol - Vi vanadate ion     

Crossbridge and filament compliance in muscle: implications for tension generation and lever arm swing

The stiffness of myosin heads attached to actin is a crucial parameter in determining the kinetics and mechanics of the crossbridge cycle. It has been claimed that the stiffness of myosin heads in the anterior tibialis muscle of the common frog (Rana temporaria) is as high as 3.3 pN/nm, substantially higher than its value in rabbit muscle (~1.7 pN/nm). However, the crossbridge stiffness measurement has a large error since the contribution of crossbridges to half-sarcomere compliance is obtained by subtracting from the half-sarcomere compliance the contributions of the thick and thin filaments, each with a substantial error. Calculation of its value for isometric contraction also depends on the fraction of heads that are attached, for which there is no consensus. Surprisingly, the stiffness of the myosin head from the edible frog, Rana esculenta, determined in the same manner, is only 60% of that in Rana temporaria. In our view it is unlikely that the value of such a crucial parameter could differ so substantially between two frog species. Since the means of the myosin head stiffness in these two species are not significantly different, we suggest that the best estimate of the stiffness of the myosin heads for frog muscle is the average of these data, a value similar to that for rabbit muscle. This would allow both frog and rabbit muscles to operate the same low-cooperativity mechanism for the crossbridge cycle with only one or two tension-generating steps. We review evidence that much of the compliance of the myosin head is located in the pliant region where the lever arm emerges from the converter and propose that tension generation (“tensing”) caused by the rotation and movement of the converter is a separate event from the passive swinging of the lever arm in its working stroke in which the strain energy stored in the pliant region is used to do work.     

SH-1 modification of rabbit myosin interferes with calcium regulation

Summary The reactive thiol of the myosin head, SH-1, can be selectively labelled in glycerinated rabbit muscle fibres. This residue has been used as an attachment site for either fluorescent or spectroscopic probes which report on head movements and orientations in various functional states of muscle. We have specifically modified SH-1in vitro, using purified rabbit myosin and conditions similar to those employed in the labelling of muscle fibres (low ionic strength [40mM NaCl] at 4°C), with stoichiometric amounts of either [¹⁴C]-iodoacetamide, 5-(2((iodoacetyl)amino)ethyl) aminonaphthalene-1-sulphonic acid (IAEDANS), or 4-(2-iodoacetamido-2,2,6,6-tetramethyl piperidinooxyl (IASL). The specificity of modification was determined by measuring the well-defined alterations in the high salt ATPase activities of myosin and by localizing both IAAm and IAEDANS to the 20-kDa C-terminal subfragment 1 (S1) which contains SH-1. The low ionic strength actin-activated Mg²⁺-ATPase of SH-1-modified rabbit myosin was measured in the presence of the thin filament regulatory, complex, troponin-tropomyosin. A significant increase in this activity in the absence of calcium, concomitant with a decrease in activity in the presence of calcium, was observed as the extent of SH-1 modification was incrementally increased from zero to one mole of label bound per mole of SH-1. The elevated myosin Mg²⁺-ATPase, which results from SH-1 modification, does not account for the increased actin-activated Mg²⁺-ATPase in resting conditions (i.e. in the absence of calcium). Thein vitro actin-activated Mg²⁺-ATPase activities become equal in both active and resting conditions when one mole of SH-1 is modified per mole of myosin head. These results demonstrate that SH-1 is located in a region of the myosin head which plays a part in the calcium-sensitive regulation of the actin-activated Mg²⁺-ATPase by troponin-tropomyosin. These studies also indicate that SH-1-labelled preparations may not be suitable for the analysis of myosin head motion and/or orientation in the resting state.     

Effect of electrical stimulation and exercise on the phosphorylation state of myosin light chains from fish skeletal muscle

This paper investigates the phosphorylation of fish muscle myosin following electrical stimulation and exercise. Purified myosin isolated from the fast myotomal muscle of rainbow trout (Salmo gairdneri) shows three light chains on SDS polyacrylamide gels of molecular weights 16,000 (LC1) 18,000 (LC2) and 24,000 (LC3). The 18,000 dalton light chain (LC2) is capable of being phosphorylated and dephosphorylated in vitro by respectively rabbit myosin light chain kinase and phosphatase.The native phosphorylation state of fast muscle myosin has been investigated in rainbow trout and dogfish (Scyliorhinus canicula) using a technique involving freeze-clamping in liquid nitrogen (–159°C) and extraction of a light chain fraction in 5 M guanidine-HCl/ethanol. The two forms of the LC2 light chain, can be resolved in 8 M urea polyacrylamide gels at pH 8.6.Fast muscle from anaesthetised trout contained 0.16 moles phosphorylated LC/mole LC2. Electrical stimulation of isolated muscle under various conditions (1–10 s isometric tetani, 5–500 Hz) did not result in a significant increase in the phosphorylated form of the LC2 light chain. Qualitatively similar results were obtained for isolated fast muscles from dogfish (Scyliorhinus canicula). In five trout subject to strenuous exercise, two fish showed a slight increase in myosin phosphorylation (0.32 moles P LC/mole LC2) and three, no significant change (0.20 moles P LC/mole LC2).The lack of correlation between the phosphorylation state of LC2 light chain and electrical stimulation indicates that, unlike rabbit and frog skeletal muscle myosin, phosphorylation is not an integral part of the excitation-contraction cycle in fish muscle.     

A self-induced translation model of myosin head motion in contracting muscle I. Force-velocity relation and energy liberation

Summary In our previous model, it was assumed that the two heads of myosin act co-operatively in producing force for the sliding of actin filaments relative to myosin filaments. We eliminate the assumption of co-operativity in the present model, following the conclusion by Harada and co-workers that a co-operative interaction between the two heads of myosin is not essential in producing actin filament movement. We assume that (1) a myosin head activated by ATP hydrolysis binds to the thin filament at a definite angle and does not do the power stroke, i.e. does not change its orientation during attachment, (2) a potential of force acting on the myosin head is induced around the thin filament when an ATP-activated myosin head binds to an actin molecule in the thin filament, and (3) the potential remains for a while after detachment of the myosin head and statistically controls the direction of thermal motion of the myosin head, so that the myosin head translates toward the Z-line as a statistical average.We did calculations on these assumptions with a mean tension approximation and got the following results, (a) The calculated force-velocity relation in muscle contraction is in fairly good agreement with experimental observation, including the give phenomenon that lengthening velocity becomes very large for a force about twice the isometric tension. (b) The calculated rate of energy liberation during muscle contraction as a function of load on muscle is in good agreement with experimental results. (c) The calculated distance over which a myosin molecule moves along the thin filament during one ATP hydrolysis can be more than 60 nm under unloaded conditions.