Orthovanadate and Orthophosphate Inhibit Muscle Force via Two Different Pathways of the Myosin ATPase Cycle (original) (raw)
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Biophysical Journal, 2008
The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12°C, sarcomere length 2.5 mm). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 6 1.1 kPa nm À1 . Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 6 3.7 kPa nm À1 . An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r F ) is 23.5 6 1.0 s À1 in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 6 0.9 s À1 at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (.1 ms) delay between generation of stiffness and generation of force by the cross-bridges.
The Journal of Physiology, 2013
Force and shortening in muscle are caused by ATP-driven working strokes of myosin II motors, during their cyclic interactions with the actin filament in each half-sarcomere. Crystallographic studies indicate that the working stroke consists in an interdomain movement of the myosin motor associated with the release of inorganic phosphate (P i). • Here the coupling of the working stroke with the release of P i is studied in situ using fast half-sarcomere mechanics on skinned fibres from rabbit psoas. • The isotonic velocity transient following stepwise force reductions superimposed on isometric contraction measures the mechanical manifestation of the working stroke and its rate of regeneration. • The results indicate that the release of P i from the catalytic site of an actin-attached myosin motor can occur at any stage of the working stroke, and a myosin motor uses two consecutive actin monomers to maximize the power during shortening.
Biophysical Journal, 2007
The stiffness of the single myosin motor (e) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T 0 ) modulated by changing either [Ca 21 ] or temperature. The hs compliance is 43.0 6 0.8 nm MPa À1 in isometric contraction at saturating [Ca 21 ], whereas in rigor it is 28.2 6 1.1 nm MPa À1 . The equivalent compliance of myofilaments is 21.0 6 3.3 nm MPa À1 . Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5 6 1.7 kPa nm À1 in isometric contraction at saturating [Ca 21 ] (e 0 ), and in rigor (e r ) it rises to 138.9 6 21.2 kPa nm À1 . e, calculated from e r and the lattice molecular dimensions, is 1.21 6 0.18 pN nm À1 . e estimated, using a thermodynamic approach, from the relation of T 0 at saturating [Ca 21 ] versus the reciprocal of absolute temperature is 1.25 6 0.14 pN nm À1 , similar to that estimated for fibers in rigor. Consequently, the ratio e 0 /e r (0.33 6 0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca 21 ]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e 0 and e r increase by ;40%. e becomes ;1.7 pN nm À1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.
The Journal of Physiology, 2015
Muscle contraction is due to cyclical ATP-driven working strokes in the myosin motors while attached to the actin filament. Each working stroke is accompanied by the release of the hydrolysis products, orthophosphate and ADP. The rate of myosin-actin interactions increases with the increase in shortening velocity. r We used fast half-sarcomere mechanics on skinned muscle fibres to determine the relation between shortening velocity and the number and strain of myosin motors and the effect of orthophosphate concentration. r A model simulation of the myosin-actin reaction explains the results assuming that orthophosphate and then ADP are released with rates that increase as the motor progresses through the working stroke. The ADP release rate further increases by one order of magnitude with the rise of negative strain in the final motor conformation. r These results provide the molecular explanation of the relation between the rate of energy liberation and shortening velocity during muscle contraction.
Biophysical Journal, 1998
The mechanical behavior of skinned rabbit psoas muscle fiber contractions and in vitro motility of F-actin (V f ) have been examined using ATP, CTP, UTP, or their 2-deoxy forms (collectively designated as nucleotide triphosphates or NTPs) as contractile substrates. Measurements of actin-activated heavy meromyosin (HMM) NTPase, the rates of NTP binding to myosin and actomyosin, NTP-mediated acto-HMM dissociation, and NTP hydrolysis by acto-HMM were made for comparison to the mechanical results. The data suggest a very similar mechanism of acto-HMM NTP hydrolysis. Whereas all NTPs studied support force production and stiffness that vary by a factor 2 or less, the unloaded shortening velocity (V u ) of muscle fibers varies by almost 10-fold. 2-Deoxy ATP (dATP) was unique in that V u was 30% greater than with ATP. Parallel behavior was observed between V f and the steady-state maximum actin-activated HMM ATPase rate. Further comparisons suggest that the variation in force correlates with the rate and equilibrium constant for NTP cleavage; the variations in V u or V f are related to the rate of cross-bridge dissociation caused by NTP binding or to the rate(s) of product release.
The Journal of Physiology, 2005
Mechanical properties of skinned single fibres from rabbit psoas muscle have been correlated with biochemical steps in the cross-bridge cycle using a series of metal-nucleotide (Me•NTP) substrates (Mn 2+ or Ni 2+ substituted for Mg 2+ ; CTP or ITP for ATP) and inorganic phosphate. Measurements were made of the rate of force redevelopment following (1) slack tests in which force recovery followed a period of unloaded shortening, or (2) ramp shortening at low load terminated by a rapid restretch. The form and rate of force recovery were described as the sum of two exponential functions. Actomyosin-Subfragment 1 (acto-S1) Me•NTPase activity and Me•NDP release were monitored under the same conditions as the fibre experiments. Mn•ATP and Mg•CTP both supported contraction well and maintained good striation order. Relative to Mg•ATP, they increased the rates and Me•NTPase activity of cross-linked acto-S1 and the fast component of a double-exponential fit to force recovery by ∼50% and 10-35%, respectively, while shortening velocity was moderately reduced (by 20-30%). Phosphate also increased the rate of the fast component of force recovery. In contrast to Mn 2+ and CTP, Ni•ATP and Mg•ITP did not support contraction well and caused striations to become disordered. The rates of force recovery and Me•NTPase activity were less than for Mg•ATP (by 40-80% and 50-85%, respectively), while shortening velocity was greatly reduced (by ∼80%). Dissociation of ADP from acto-S1 was little affected by Ni 2+ , suggesting that Ni•ADP dissociation does not account for the large reduction in shortening velocity. The different effects of Ni 2+ and Mn 2+ were also observed during brief activations elicited by photolytic release of ATP. These results confirm that at least one rate-limiting step is shared by acto-S1 ATPase activity and force development. Our results are consistent with a dual rate-limitation model in which the rate of force recovery is limited by both NTP cleavage and phosphate release, with their relative contributions and apparent rate constants influenced by an intervening rapid force-generating transition.
Besides driving contraction of various types of muscle tissue, conventional (class II) myosins serve essential cellular functions and are ubiquitously expressed in eukaryotic cells. Three different isoforms in the human myosin complement have been identified as non-muscle class II myosins. Here we report the kinetic characterization of a human non-muscle myosin IIB subfragment-1 construct produced in the baculovirus expression system. Transient kinetic data show that most steps of the actomyosin ATPase cycle are slowed down compared with other class II myosins. The ADP affinity of subfragment-1 is unusually high even in the presence of actin filaments, and the rate of ADP release is close to the steady-state ATPase rate. Thus, non-muscle myosin IIB subfragment-1 spends a significantly higher proportion of its kinetic cycle strongly attached to actin than do the muscle myosins. This feature is even more pronounced at slightly elevated ADP levels, and it may be important in carrying out the cellular functions of this isoform working in small filamentous assemblies.
Biophysical Journal, 2013
Elevated levels of phosphate (P i) reduce isometric force, providing support for the notion that the release of P i from myosin is closely associated with the generation of muscular force. P i is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of P i on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of P i on the force-generating capacity of a small ensemble of myosin (~12 myosin heads) using a three-bead laser trap assay. In the absence of P i , myosin pulled the actin filament out of the laser trap an average distance of 54 5 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM P i , myosin generated only enough force to displace the actin filament by 13 5 1 nm, generating just 0.2 pN of force. The elevated P i also caused a >65% reduction in binding-event lifetime, suggesting that P i induces premature detachment from a strongly bound state. Definitive evidence of a P i-induced powerstroke reversal was not observed therefore, we determined if a branched kinetic model in which P i induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of P i on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that P i-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.
Acta Biochimica Polonica, 2002
In order to compare the ability of different isoforms of myosin essential light chain to interact with actin, the effect of the latter protein on the proteolytic susceptibility of myosin light chains (MLC-1S and MLC-1V-slow specific and same as ventricular isoform) from slow skeletal muscle was examined. Actin protects both slow muscle essential light chain isoforms from papain digestion, similarly as observed for fast skeletal muscle myosin (Nieznañska et al., 1998, Biochim. Biophys. Acta 1383: 71). The effect of actin decreases as ionic strength rises above physiological values for both fast and slow skeletal myosin, confirming the ionic character of the actin-essential light chain interaction. To better understand the role of this interaction, we examined the effect of synthetic peptides spanning the 10-amino-acid N-terminal sequences of myosin light chain 1 from fast skeletal muscle (MLC-1F) (MLCFpep: KKDVKKPAAA), MLC-1S (MLCSpep: KKDVPVKKPA) and MLC-1V (MLCVpep: KPEPKKDDAK) on the myofibrillar ATPase of fast and slow skeletal muscle. In the presence of MLCFpep, we observed an about 19% increase, and in the presence of MLCSpep about 36% increase, in the myofibrillar ATPase activity of fast muscle. On the other hand, in myofibrillar preparations from slow skeletal muscle, MLCSpep as well as MLCVpep caused a lowering of the ATPase activity by about 36%. The above results suggest that MLCSpep induces opposite effects on ATPase activity, depending on the