Showing papers in "Journal of Muscle Research and Cell Motility in 1984"

Length dependence of changes in sarcoplasmic calcium concentration and myofibrillar calcium sensitivity in striated muscle fibres

Abstract: It has long been known that the contractile performance of striated muscle is strongly dependent upon the length of the muscle (e.g. Ramsey & Street, 1940) and the sliding filament theory of muscle contraction offers a ready explanation for many features of the isometric length-force relation of fully activated skeletal muscle fibres (Gordon et al., 1966b). Over the past decade, however, it has become increasingly more apparent that differences in the mechanical behaviour of striated muscle at different lengths, particularly if submaximally activated, often cannot be satisfactorily explained on the basis of filament overlap alone. There is growing evidence that changing the length of a muscle fibre alters not only the physical overlap of the filaments (i.e. the striation spacing or sarcomere length) but also modifies other processes involved in the activation of contraction. In particular it appears that both the characteristics of the sarcoplasmic Ca 2+ concentration change, which occurs upon excitation of the fibre, and the apparent sensitivity of the contractile proteins to Ca 2+ can vary with muscle length. This article will concern itself principally with the length dependence of these two aspects of contractile activation.

Kinetics of acto-S1 interaction as a guide to a model for the crossbridge cycle

Abstract: Recent experiments on the kinetics of the interaction between myosin subfragment 1 (S1) and F-actin in solution are summarized. It is concluded that, at every step of the ATPase cycle, the association between the two proteins takes place in two stages. The equilibrium constant of the second step and thus the affinity of S1 for actin changes from step to step during the enzymatic reaction.

Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

Abstract: We have investigated the conditions for isolation of Ca2+-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml−1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g−1 artery wet weight) were made up of actin, tropomyosin and a 120 000Mr protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca2+. Up to 80% inhibition was observed in the absence of Ca2+. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca2+ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000Mr protein.

The effect of myosin sulphydryl modification on the mechanics of fibre contraction.

Abstract: Glycerinated rabbit psoas fibres have been modified with paramagnetic probes (IASL and MSL) which react selectively with the reactive sulphydryl on the myosin head. The extent of SH-1 modification was monitored by extracting myosin and measuring its ATPase activity in the presence of EDTA and of Ca2+. The isometric tension, stiffness, maximum velocity of contraction (slack test), and the force-velocity relation was measured as a function of the degree of SH-1 modification. Reaction of up to 50% of SH-1, i.e. 50% reduction in the K+-EDTA ATPase activity of extracted myosin, produced little change (<10%) in any of the fibre parameters. Modification of 75% of the SH-1 sites produced small decreases (15–30%) in the magnitude of all parameters, while reaction of more than 90% of SH-1 required long reaction times and produced decreases of 40–75%. In all cases the velocities of contraction decreased in parallel with the decrease in tension, while the decrease in stiffness was less pronounced. We conclude that a large fraction of muscle fibre SH-1 groups can be modified without greatly affecting the mechanical performance of the fibre. At least a portion of the decrease in fibre parameters that is observed at high levels of SH-1 modification can be attributed to modification of other sulphydryls by the probes. The reaction of both SH-1 and nonspecific sulphydryls abolishes myosin ATPase activity, and can account for approximately one half of the decrease in fibre parameters that is observed at high degrees of sulphydryl modification. We conclude that the modification of SH-1 does not greatly affect the function of a myosin head in the filament array of a fibre. This is in contrast to results obtainedin vitro where SH-1 modification alters several rates in the interaction of myosin with ATP and decreases the actin-activated ATPase activity of myosin subfragments.

Creatine kinase as an intracellular regulator

Abstract: Several recent studies have demonstrated the presence of creatine kinase and of phosphorylcreatine in a variety of cells besides striated muscle and brain cells. The total creatine kinase and phosphagen levels in these cells encompass a wide range of values. The available data are collected in this article to demonstrate that the variation of the enzyme and phosphagen concentrations is not random but that the two are interrelated. With both the major isoenzymes of creatine kinase, namely the muscle type and the brain type, the basal levels of phosphorylcreatine follow closely the cellular creatine kinase levels. A hypothesis is presented in which the enzyme itself is the major determinant of phosphorylcreatine content by virtue of its ability to act as an intracellular binding protein for creatine derived from extracellular fluid, and also for cellular ADP. The proposed mechanism further predicts that in cells that contain high levels of actin and thus sequester the cytoplasmic free ADP (e.g. most muscle cells), a high level of creatine kinase can effectively regulate the myokinase reaction by its ability to bind ADP. The net effect of such regulation is to conserve the adenine nucleotide pools in the cell. The evolutionary advantage of these two regulatory functions of creatine kinase in terms of energy conservation is discussed.

Heterogeneity of T-tubule geometry in vertebrate skeletal muscle fibres.

Abstract: Average dimensions of transverse tubules were obtained from electron micrographs of thin sections of mammalian and amphibian skeletal muscle fibres and the effect of transverse tubule geometry on the electrical characteristics of the fibres has been considered. The preparations examined were toad sartorius, mouse soleus, rat extensor digitorum longus, soleus and sternomastoid muscles. The T-tubule dimensions varied considerably between the different preparations and the average volume to surface ratio of the transverse tubule in amphibian fibres (8.1 nm) was generally greater than that in mammalian fibres (3.0–6.2 nm). The small volume to surface ratio of the mammalian transverse tubule would tend to reduce the electrical space constant of the transverse tubule system and reduce the rate of cross-sectional activation of the fibres during a twitch contraction. The area of transverse tubule membrane in junctional contact with the sarcoplasmic reticulum was determined and was found to be greater in mammalian fibres than in amphibian fibres. The relative areas of junctional contact, along a unit length of transverse tubule, were the same in rat extensor digitorum longus and soleus fibres.

Tryptic digestion of scallop S1: evidence for a complex between the two light-chains and a heavy-chain peptide

Abstract: When scallop S1(+LC) (formerly called CaMg S1) is digested by trypsin, the heavy chain degrades while the two light chains remain complexed to each other and a peptide fragment of the heavy chain The three components of the complex comigrate during electrophoresis under nondissociating conditions and can be purified by chromatography and concentrated by precipitation with ammonium sulphate in the presence of millimolar calcium ions The truncated regulatory light chain remains associated with the binary complex consisting of the peptide and essential light chain as long as divalent cations are present; in the presence of EDTA it dissociates This behaviour of the light chains-peptide complex mimics that of the intact molecule

Chemical energy usage and myosin light chain phosphorylation in mammalian skeletal muscle

Abstract: The purpose of this study was to ascertain whether phosphorylation of the regulatory light chain of myosin plays a role in modulating the rate of chemical energy usage in mammalian skeletal muscle. There was no change in the average rate of chemical energy usage with duration of isometric stimulation in the rat extensor digitorum longus (EDL), even though the degree of light chain phosphorylation increased from 5% at rest to above 60% after 7 s of stimulation. When the initial degree of phosphorylation was increased to 73% by prestimulation of the muscle, there was still no change in the chemical energy usage under isometric conditions. In contrast, under the conditions used, the mouse EDL showed changes in the average rate of energy usage that depended upon both tetanus duration and stimulation history. However, there was no consistent relationship between phosphorylation of the light chain and average rate of chemical energy usage. These results suggest that while there are factors which can change crossbridge cycling rate in mammalian skeletal muscle, phosphorylation of the regulatory light chain of myosin is neither necessary nor sufficient to cause such changes.

The characterization of vanadate-trapped nucleotide complexes with spin-labelled myosins

Abstract: The properties of spin-labelled myosin, prepared from rabbit skeletal and scallop adductor muscle, on forming a long-lived complex with ADP and vanadate (M.†ADP.Vi), have been investigated. In the case of an iodoacetamide-based label attached to rabbit myosin or subfragment 1, M.†ADP.Vi formation is characterized by a marked increase in the mobility of the probe, similar to that seen during steady-state ATPase activity. Hence, this complex appears to be a good analogue of the M**ADP.Pi state. The kinetics of M.†ADP.Vi formation were determined by following the electron paramagnetic resonance (e.p.r.) signal with time and were analysed according to the scheme: After correction for Vi polymerization,K'4=3.2×10−4M,k'−3=8.7×10−3s−1 andk'3=1.5×10−4s−1. The major effect of spin-labelling the reactive SH1 thiol is to increasek'3, so that M.†ADP.Vi dissociates over a period of hours rather than days. In contrast, a maleimide-based spin-label attached to rabbit myosin does not exhibit a large change in mobility, on formation of the M.†ADP.Vi complex. However, the small change observed in both the conventional and saturation transfer spectra questions the assumption that this probe is completely insensitive to librational motion during ATPase activity. The immobilized spectrum of the iodoacetamide-based spin label attached to scallop myosin is insensitive to M.†ADP.Vi formation in the presence or absence of Ca2+. Under these conditions, the label appears to reflect gross head motion and hence this observation lends no support to the idea that, in the myosin-linked regulatory system, Ca2+ operates by controlling the flexibility of the subfragment 1-subfragment 2 joint.

Crystallization of intramembrane particles in rabbit sarcoplasmic reticulum vesicles by vanadate.

Abstract: Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle appear in freeze-fracture as 0.15–0.2 µm vesicles. The concave fracture surface (P-face) contains a dense population of 8.5 nm particles that were previously identified as the Ca2+-transport ATPase. The convex surface (E-face) is mostly smooth, displaying an occasional particle but no complementary arrays of pits. Incubation of the vesicles at 4°C in calcium-free solutions containing 5 mM Na3VO4 induces the formation of two-dimensional crystalline arrays of the Ca2+, Mg2+-ATPase, accompanied by structural changes visible by freeze-etch electron microscopy. Most vesicles elongate into tubules 60–80 nm in diameter and the 8.5 nm intramembrane particles of the P-face become regularly organized into parallel ridges. The ridges are coiled around the tubules in right-handed helices, oriented at 50–60° angle to the long axis of the tubules. The particles repeat along the rows at about 5.5 nm and the rows repeat at 10.5–11.0. Occasionally the ridges seem to break up into 8.5 nm particles. Parallel furrows are visible on the (convex) E-face of the tubules. In high resolution replicas, the furrows are resolved into rows of pits that are complementary images of the ridges. Deep etching and rotary shadowing reveal oblique crests on the protoplasmic surface, consisting of dimeric particles close to 8.5 × 5.5 nm in size, in which each monomer can frequently be resolved into two structural domains. These data suggest that vanadate induces a conformational change in the Ca2+-transport ATPase, with crystallization of the intramembrane particles.

Biological structures and coupled flows

Abstract: It is easy to become complacent in one's theoretical ideas and never take time to think deeply about the kinds of explanation that are possible for muscle contraction, or whatever one's obsession may be. Taking a broader point of view can help to dispel this complacency, and here,is a book .that tries to do so, and shows that there are plenty of adventurous ideas in circulation. It is the photoreproduced typescript report of the proceedings of a symposium held in June 1982 to commemorate the 10th anniversary of the death of Aharon Katzir-Katchalsky. As befits a memorial to such a scientific polymath, the aim of the symposium was broad: to discuss 'directional movement in biological systems: how it is coupled to the driving force, and the structural asymmetry and polarities which are needed to drive it. Such processes include cytoplasmic streaming, active transport of ions across membranes, as well as muscle contraction' and the formation of biological structures. Of course a synthesis of ideas across this broad front was not achieved, nor does it appear to be in sight. As usual the contributors restricted themselves to considering just one of these phenomena. Thus the section on the dynamics of biostructure formation, which is a lively one, and fascinating for an outsider, does not seem to share any theoretical basis with, say, the study of active transport of ions. Some of the ideas current in the study of transport processes are however used also in considering muscle contraction. This has of course been previously emphasized by, for example, T. L. Hill in 1977" and in the present volume the article by S. R. Caplan on the reason for linearity between forces and flows in a coupled system near equilibrium is a stimulant to thinking about muscle contraction, as well as the transport processes to which Caplan has related these ideas. T. L. Hill contributes a carefully explained theoretical treatment of the energetics of 'treadmilling' of microtubules or actin filaments and how mechanical work might be done by these processes, which are perhaps involved in various types of cell motility. Some of the other contributions which I particularly enjoyed were: Carolyn Cohen's thoughtful essay on thin filament regulation in which she emphasizes the importance of considering the dynamic behaviour of the proteins; the explanation by A. Oplatka, R. Levy and J. E. Freidman of why they consider muscle as a kind of rocket engine; Robert

The effect of the ATP analogue AMPPNP on the structure of crossbridges in vertebrate skeletal muscles: X-ray diffraction and mechanical studies.

Abstract: Adenylylimidodiphosphate (AMPPNP), a nonhydrolysable analogue of ATP, has been used to arrest the crossbridge cycle of muscular contraction in one of its hypothetical intermediate states. Whole frog sartorius muscles were chemically demembranated, and it was found possible to cycle such skinned muscles reversibly between the relaxed and rigor states. The effect of binding of AMPPNP on the structure and spatial arrangement of the crossbridges of such muscles was studied using low-angle X-ray diffraction, with simultaneous recording of the mechanical effects, starting from the rigor state. Saturating concentrations of MgAMPPNP produce a characteristic decrease of about 50% in the original rigor isometric tension with a concomitant increase in muscle length by 0.13%. The equatorial X-ray diffraction pattern is modified in the following way: the lattice dimensions and the intensity of the (10) equatorial reflection do not change, while the intensity of the (11) equatorial reflection increases slightly. These observations of very small equatorial changes could be explained by assuming that in these muscles (as distinct from others such as rabbit psoas) the analogue does not produce a significant degree of detachment of crossbridges; that is, there are only AMPPNP-modified attached ones. The changes in the meridional X-ray diffraction pattern are more pronounced: the meridional reflection at 14.5 nm decreases in intensity, and the meridional reflection at 7.2 nm increases considerably: the intensity of all the actin-based off-meridional layer-lines decreases. There are no signs of the characteristic relaxed layer-lines, and the changes in the layer-line intensities are probably due to there being a single population of AMPPNP-modified attached crossbridges, rather than a mixture of attached and detached crossbridges. Thus the AMPPNP X-ray pattern, both equatorially and meridionally, is somewhat similar to the rigor one, indicating that most of the crossbridges remain attached. On the other hand, the fact that there are some changes in the layer-line intensities of the AMPPNP frog pattern, without the appearance of any signs of a relaxed equatorial pattern, indicates that the attached crossbridges are in a structural state that is different from rigor, one is not seeing, apparently, simply a mixture of rigor and relaxed states. Our tentative interpretation of this result is that there may be a structural change in the crossbridge near to the junction with S2, with less significant changes occurring in the parts of the crossbridge close to actin.

The membrane capacity of mammalian skeletal muscle fibres

Abstract: Membrane capacity was measured as a function of fibre diameter in mammalian skeletal muscle fibres under normal conditions and under conditions designed to reduce the membrane chloride conductance, i.e. in solutions in which choride ions were replaced by sulphate or methylsulphate ions, or in normal Krebs solutions containing 2,4-dichlorophenoxyacetic acid (2.5mm). The experiments were done on rat sternomastoid, extensor digitorum longus and soleus muscle fibres. The average membrane capacity of fibres in each muscle was greater than normal when chloride conductance was reduced and the slope of the relationship between membrane capacity and fibre diameter increased. The results were consistent with the hypothesis that the space constant of the transverse tubule system in mammalian fibres is normally short because the transverse tubule membrane has a high chloride conductance. The experimental results imply that the space constant of the transverse tubule system was less than 40 µm for fibres in normal Krebs solution and greater than 100 µm for fibres with low membrane chloride conductance. The space constant was calculated using measured geometrical parameters of the transverse tubule, and measured membrane conductance, and the values were close to 20 µm for fibres in normal Krebs solution and between 50 and 120 µm for fibres with low chloride conductance.

The origin of muscle stem cells in rat triceps surae regenerating after mincing

Abstract: Rat triceps surae was minced and orthotopically autografted. Twitch time to peak, maxima tetanic tension, lactate dehydrogenase activity and total creatine concentration were measured in muscles regenerating for 30, 60 and 90 days. If the minces were frozen and thawed before grafting, muscle regeneration was suppressed. If they were further heated before grafting, muscle regeneration was also suppressed. If one half of the mince was either frozen and thawed or frozen, thawed and heated, and then recombined with the remaining half, muscle regeneration was delayed. However, at 90 days, ‘intensive properties’ (twitch time to peak, maximum tetanic tension, total creatine concentration and lactate dehydrogenase activity) of regenerates obtained from such partially treated minces were similar to those of regenerates obtained from untreated minces although their ‘extensive properties’ (weight and maximal tetanic force) were approximately halved. The extent of regeneration depends on the mass of untreated mince autografted and thus, presumably, on the number of viable muscle stem cells initially present in the mince.

Ca2+-activated force-generating properties of mammalian skeletal muscle fibres: histochemically identified single peeled rabbit fibres

Abstract: Single peeled (sarcolemma removed) rabbit skeletal muscle fibres, identified histochemically from their myofibrillar ATPase and oxidative staining pattterns, were characterized according to their Ca2+-activated steady-state force-generating properties at normal intracellular pH (7.0) and under acidotic (pH 6.5) conditions. Maximum force-generating capacity of each fibre was assessed by measuring steady-state isometric force generation at saturating Ca2+ concentration at both pH values. The Ca2+ sensitivity of each fibre was ascertained by determining the percentage of maximum force generated at each of several subsaturating Ca2+ concentrations at both pH values. Fibres were selected from soleus, tibialis anterior and adductor magnus muscles. At subsaturating Ca2+ concentrations only two functional groups of fibres were distinguishable, corresponding to the histochemical classifications type I and type II. Type I fibres were more sensitive to Ca2+ and less depressed by acidosis than type II fibres in the subsaturating range of Ca2+ concentrations. At saturating Ca2+ concentrations, the acidotic depression of maximum force was significantly less for type I fibres than type II nonoxidative fibres regardless of their muscle of origin. Type II oxidative fibre maximum force properties depended upon the muscle of origin and demonstrated subgroups of these fibres that were different from type II nonoxidative fibres and similar to type I fibres.

The structure and disposition of crossbridges in deep-etched fish muscle.

Abstract: Deep-etching and rotary-shadowing techniques were used to describe crossbridges in fish (Chanda ranga) muscle, relaxed and in iodoacetate rigor conditions. Three major fracture planes from rigor muscle were studied using stereomicroscopy and Fourier image analysis.

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

Abstract: 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-naphthalene-sulphonate (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 degrees C was 7.5 X 10(-3)S-1. The association rate constant, which was independent of the RLC concentration, was 5 X 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 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.

Capillary endothelial cell migration: loss of stress fibres in response to retina-derived growth factor.

Abstract: While the migration of capillary endothelial cells is believed central to the process of new blood vessel developmentin vivo, the biochemical basis for endothelial motility is unknown. Herein, we demonstrate that retina-derived growth factor (RDGF), a mitogen for endothelial cells (EC), stimulates the migration of microvascular endotheliumin vitro. The addition of RDGF directly to the culture medium causes an increase in the random movement (chemokinesis) of the EC as measured by the phagokinetic assay. Release of the factor as a gradient results in a stimulation of the directed migration (chemotaxis) of the microvascular EC. This increased EC migration is associated with a shift in morphology of the stimulated cells from a rounded to a more polarized shape. Concomitant with the RDGF-stimulated migration is a dramatic decrease in stress fibre staining visualized by immunofluorescence microscopy using affinity-purified antibodies to actin and myosin.

Sarcomere and filament lengths in passive muscle fibres with wavy myofibrils.

Abstract: Longitudinal compression of isolated skeletal muscle fibres ofRana pipiens caused waves to appear sharply at a critical striation spacing which was slightly less than the slack length measured at the same point. Both slack length and critical length varied between fibres and along the length of one fibre, being shortest near the tendons. The critical length varied from 1.93 to 2.11 µm. The troponin periodicity (P diff) was measured in embedded material by light diffraction of calibrated electron micrographs. Comparison between the troponin periodicities in a fibre made wavy at one end and stretched at the other showed that longitudinal compression did not cause shortening of the thin filaments. Comparison betweenP diff and the troponin periodicity of fresh muscle provided an estimate of the artefact mainly caused by shrinkage during specimen preparation. It varied from 3 to 11%. The gaps between the ends of the thin filaments in the M-line region were estimated from sarcomere length (corrected for shrinkage) and the assumedin vivo values for total thin-filament length or the length between the last troponin lines (1.975 µm and 1.925 µm respectively). The estimates were confirmed by a few direct measurements of thin-filament length and periodicity. Sarcomere length varied from fibre to fibre, from 1.91 to 2.12 µm, except at the inside of bends in wedge-shaped sarcomeres where it fell to 1.86 µm in some cases. This indicates that in one fibre the tips of the thin filaments overlapped at the level of the last troponin lines, while, at the other extreme, the tips of the thin filaments only just reached the bare zone of the thick filaments. The origin of the resistance to sliding and the force which restores an actively shortened fibre to its slack length are discussed. While there may be a well-defined barrier to sliding at the point where the troponins of opposite polarity meet, there must also be an additional length-dependent resistance to account for the appearance of waves at longer sarcomere lengths. The formation of waves is interpreted as a buckling phenomenon in which a longitudinal compressive force is applied to the myofibrils which have a finite stiffness against bending and a finite elastic restraint against lateral displacement. The bending stiffness is largely and perhaps entirely accounted for by contributions from (1) the stiffness of the individual filaments and (2) the stiffness of myofibrils calculated from their Young's modulus.

Anti-troponin-T monoclonal antibody crossreacts with all muscle types

Abstract: Monoclonal antibodies to troponin-T were produced by the hybridoma technique. Culture supernatants were initially screened using an enzyme-linked immunoabsorbent assay (ELISA). Positive clones were subcloned twice and further characterized. One of these, 7/H3:C9:D10, produced antibodies against troponin-T; immunoblotting experiments indicated its specificity for only troponin-T when challenged with a variety of striated muscle myofibrillar proteins. Indirect immunofluorescence staining with the antibody shows specific I-band staining in both adult and embryonic skeletal and cardiac muscle of various vertebrate species. In addition, intense but diffuse cytoplasmic staining was seen in chicken gizzard smooth muscle. Our results suggest that troponin-T contains an antigenic determinant that is common to both striated and smooth muscle.

Modification of crossbridge states by ethylene glycol in insect flight muscle.

Abstract: Substitution of ethylene glycol for part of the solvent water changes the mechanical properties, structure and nucleotide binding of glycerol-extracted flight muscle fibres from the waterbugLethocerus. On addition of ethylene glycol the rigor tension falls, rapidly and reversibly. With increasing glycol concentration the effect saturates at a non-zero tension. The isotonic stiffness is unchanged on adding ethylene glycol. Adding MgAMPPNP (adenylylimidodiphosphate) to a muscle fibre in 50% ethylene glycol causes a further rapid tension fall; above 100 µm AMPPNP the tension reaches zero. The isotonic stiffness of restretched muscle is then close to that of a relaxed fibre. Removal of MgAMPPNP from the bathing medium has no immediate mechanical effect. After several hours the isotonic stiffness rises to some extent; on removal of the glycol both tension and stiffness rise to rigor values within one minute.3H-Labelled AMPPNP binds to muscle fibres in 50% ethylene glycol in a similar amount to the number of myosin heads present. The binding is tighter than that in aqueous solution and the nucleotide is only released very slowly. Upon removal of the ethylene glycol nucleotide is rapidly released. X-ray diffraction of muscle in 50% ethylene glycol reveals a highly ordered structure, in which both the 14 nm and the 38 nm layer lines are sharply sampled and are of intermediate values between rigor and relaxation. The two inner equatorial peaks are also of intermediate values. On adding MgAMPPNP the pattern resembles that of relaxed muscle. Upon removal of the nucleotide the pattern does not revert towards rigor but on removal of glycol it does. These results are interpreted in terms of changes within the myosin heads and their array within the filament lattice.

Structural studies of the waves in striated muscle fibres shortened passively below their slack length.

Abstract: Isolated skeletal muscle fibres ofRana pipiens were shortened below their slack length by longitudinal compression in a gelatine block, and examined by light and electron microscopy. Waves appeared sharply when the striation spacing (S) reached a critical value (about 2 µm) and increased in height with further compression down toS = 1.6 µm while the resting band pattern was maintained. The waves were plane, helical or irregular, with wave lengths of 5–15 striations. The Z lines usually ran perpendicular to the direction of the myofibrils to form wedge-shaped sarcomeres. The bending occurred mainly in the I band. The thin filaments ran stiffly for about 30 nm from the Z line and then bent toward the A band. The thick filaments bent very slightly, particularly at their tips. The edges of the A band were indistinct, and there were no dense lines at the A–I junction.

A possible mechanism of length activation in insect fibrillar flight muscle

Abstract: When insect fibrillar flight muscle is stretched it becomes more active, as indicated by an increase in oscillatory work output and ATPase activity. Using glycerol-extracted muscle it is demonstrated that this effect is periodic with a repeat of around 3% length change, independent of temperature and ionic strength. This corresponds to 38 nm per half sarcomere, the same repeat as the actin helix. Since there is also a period of 38 nm in the myosin helix the periodic change of activity with muscle length could be caused by filament sliding. This may be the basis of the mechanism of length activation.

Geometrical constraints affecting crossbridge formation in insect flight muscle.

Abstract: Computer-modelling studies have explored how rigor crossbridge interactions in insect flight muscle are affected by using a four-stranded helical thick filament and by restricting each myosin to forming one crossbridge with only one actin filament. Crossbridges searching over an axial range of ±7.2 nm, and within an azimuthal range around actin of ±45°, can simulate the actin-labelling patterns observed in thin electron microscope sections well. However, the number of crossbridges attached between any myosin filament and an adjacent actin filament depends on their relative axial and azimuthal positions, and can vary by a factor of two. The relative position that maximized the number of attached bridges also produced the best modelling of the ‘double chevron’ appearance of two crossbridge pairs attaching within target zones every 38.6 nm, as seen in thin longitudinal sections, and the ‘flared X’ of crossbridges extending to four out of six surrounding actins at each crossbridge level seen in thin cross-sections. Micrographs show that excellent lattice register of rigor crossbridges in longitudinal sections does not depend on lateral register of thick or thin filament ends. Our modelling suggests how the crossbridge lattice may be generated by filaments becoming mutually annealed to the axial and azimuthal positions at which most crossbridges can attach, at which time the actin filaments are arranged at the diad positions of the P64 crystalline lattice. When the actin filaments are so oriented, in a P64 lattice, two crossbridges on adjacent actin filaments will slew toward the same point on the myosin filament, producing the flared X appearance of origin from a common stem and a single myosin, even if they originate from distinct points and separate molecules.

The 3′-untranslated sequence of human skeletal muscle α-actin mRNA

Abstract: A human actin cDNA clone pGF3 isolated from a fetal skeletal muscle cDNA library is described. The insert cDNA is homologous to skeletal muscle α-actin as judged by restriction mapping and nucleotide sequencing. The recombinant contains a substantial portion of the coding and the complete 3′-untranslated region. Comparison of the 3′ ends of human and rat skeletal muscle and human cardiac α-actins reveals little homology between different types of actin genes in man but marked conservation of this region in the skeletal muscle actins of man and rat.

Fine structures in the light diffraction pattern of striated muscle.

Abstract: Single skeletal muscle fibres of frog were illuminated with a He-Ne, argon-ion or rhodamine 6G dye laser. The fine structures lying within the diffraction columns moved parallel to the fibre axis without changing their pattern when either the wavelength or the incident angle of the laser beam was varied, or when the fibre was stretched slightly. However, their pattern remained nearly constant when the fibre was submerged in hypotonic or hypertonic solution. As the illumination of about 1 mm or 0.1 mm width scanned along the length of the fibre, new structures emerged while others faded away giving rise to the notion that the diffraction columns were moving in the direction of the scan. A decrease in the illumination width caused the structures lying on the periphery of the diffraction column to disappear and the width of the remaining structures to increase. Measurements rule out the existence of large diffraction planes in these muscles. In addition, they indicate that the fine structures come from the diffraction of the whole rather than independent components of the illuminated volume. The origin of the fine structures is explained by two diffraction models.

The maximum velocity of shortening during the early phases of the contraction in frog single muscle fibres

Abstract: The maximum velocity of shortening (V max) was determined at preset times during the development and the plateau of isometric tetani in single fibres isolated from the tibialis anterior muscle of the frog. Experiments were performed at low temperature (3.6–6° C) and at about 2.25 µm sarcomere length. The controlled velocity release method was used.V max was measured by determining the lowest velocity of release required to keep the tension at zero. Extreme care was taken in dissection and mounting of the fibres in order to make the passive series compliance very small. The value ofV max at the end of the latent period for the development of isometric tension (at 4.5° C about 10 ms after the beginning of the stimulus volley) was already the same as later during either the tension rise or at the plateau of isometric tetani. These results show that the value ofV max of intact fibres is independent of time and activation subsequent to the latent period, and suggest that the cycling rate of the crossbridges may thus attain its steady-state value just at the end of the isometric latent period.

The rate of polymerization of rabbit skeletal muscle actin is enhanced by polyethylene glycol.

Abstract: The effect of polyethylene glycol on the kinetics of actin polymerization was determined by monitoring the enhancement in the fluorescence of pyrenyl-labelled actin. The polymerization of actin at 15 mM KCl was in addition followed by viscometry and light scattering. All three methods showed that the overall rate of polymerization of actin increased 3-4-fold when the concentration of polyethylene glycol was increased from 0 to 6% (w w−1). A further increase in polyethylene glycol concentration to 10% (w w−1) caused a relatively small contribution to the increase in the rate of polymerization. The enhancement of the overall rate of polymerization by polyethylene glycol was also reflected in a significant decrease in the lag time observed when the time course of polymerization was followed by viscometry and light scattering. The steady-state value of fluorescence enhancement and critical concentration of actin were also influenced by polyethylene glycol and the results showed that the extent of polymerization was increased by an increase in the concentration of polyethylene glycol in solution. The effect of polyethylene glycol on both rate and extent of polymerization persisted at physiological salt concentration (150mm KCl, 2mm MgCl2). Since the rate of elongation was affected only to a small extent by polyethylene glycol, we propose that its main effect is on nucleation.

Distance between nucleotide site and cysteine-373 of G-actin by resonance energy transfer measurements.

Abstract: The distance between the nucleotide site and the reactive cysteine-373 of G-actin was determined from resonance energy transfer measurements by using 1,N 6-ethenoadenosine triphosphate (eATP) as the donor and 4-[N-(iodoacetoxy)ethylN methyl]amino 7 nitrobenz 2 oxa 1,3 diazole covalently attached to the sulphydryl group as acceptor. The quenching of the lifetime of bound donor in the presence of attached acceptor arose predominantly from transfer of excitation energy. The polarization spectrum of free eATP in glycerol revealed that the minimum value of its fundamental anisotropy is 0.32 at 340 nm, indicating that the maximum value of the angle between the absorption and emission dipoles of the ethenoadenosine moiety is 21°. The polarization result indicates that the bound nucleotide is depolarized and has considerable motional freedom. This motion is restricted and unlikely to be rapid or isotropic during the time interval of energy transfer. The attached acceptor is highly immobile, however. The range of the donor-acceptor distance is 24–45 A. This range was not affected by polymerization. In the absence of independent structural information it is not possible to assign a single value to the donor-acceptor separation.