Strain sensitivity and turnover rate of low force cross-bridges in contracting skeletal muscle fibers in the presence of phosphate.

Inorganic phosphate (Pi) decreases the isometric tension of skinned skeletal muscle fibers, presumably by increasing the relative fraction of a low force quaternary complex of actin, myosin, ADP, and Pi (A.M.ADP.Pi). At the same time, Pi gives rise to a fast relaxing mechanical component as detected by oscillations at 500 Hz. To characterize the dynamic properties of this A.M.ADP.Pi complex, the effect of Pi on the tension response to stretch was investigated with rabbit psoas fibers. A ramp stretch applied in the presence of 20 mM Pi increased tension more than in the control solution (0 mM Pi) but reduced the fast relaxing component to the control level. Thus, a stretch seems to convert the low force, fast relaxing A.M.ADP.Pi complex to a high force, slow relaxing form. However, the Pi-induced enhancement of the tension response was not observed until the fibers were stretched more than 0.4% of their length, suggesting that a critical cross-bridge extension of approximately 4 nm is required for this conversion. The rate constant of the attachment/detachment of this low force complex was estimated from the velocity dependence of the enhancement. It was approximately 10 s-1, in marked contrast to the A.M.ADP.Pi complex under low salt, relaxed conditions (approximately 10,000 s-1). The enhancement of the tension response was not observed when isometric tension was reduced by lowering free calcium, implying that calcium and Pi affect different steps in the actomyosin ATPase cycle during contraction.     

Millisecond-scale biochemical response to change in strain

Muscle fiber contraction involves the cyclical interaction of myosin cross-bridges with actin filaments, linked to hydrolysis of ATP that provides the required energy. We show here the relationship between cross-bridge states, force generation, and Pi release during ramp stretches of active mammalian skeletal muscle fibers at 20°C. The results show that force and Pi release respond quickly to the application of stretch: force rises rapidly, whereas the rate of Pi release decreases abruptly and remains low for the duration of the stretch. These measurements show that biochemical change on the millisecond timescale accompanies the mechanical and structural responses in active muscle fibers. A cross-bridge model is used to simulate the effect of stretch on the distribution of actomyosin cross-bridges, force, and Pi release, with explicit inclusion of ATP, ADP, and Pi in the biochemical states and length-dependence of transitions. In the simulation, stretch causes rapid detachment and reattachment of cross-bridges without release of Pi or ATP hydrolysis.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

Suppression of muscle contraction by vanadate. Mechanical and ligand binding studies on glycerol-extracted rabbit fibers

The suppression of tension development by orthovanadate (Vi) was studied in mechanical experiments and by measuring the binding of radioactive Vi and nucleotides to glycerol-extracted rabbit muscle fibers. During active contractions, Vi bound to the cross-bridges and suppressed tension with an apparent second-order rate constant of 1.34 X 10(3) M-1s-1. The half-saturation concentration for tension suppression was 94 microM Vi. The incubation of fibers in Vi relaxing or rigor solutions prior to initiation of active contractions had little effect on the initial rise of active tension. The addition of adenosine diphosphate (ADP) and Vi to fibers in rigor did not cause relaxation. Suppression of tension only developed during cross-bridge cycling. After slow relaxation from rigor in 1 mM Vi and low (50 microM) MgATP concentration (0 Ca2+), radioactive Vi and ADP were trapped within the fiber. This finding indicated the formation of a stable myosin X ADP X Vi complex, as has been reported in biochemical experiments with isolated myosin. Vi and ADP trapped within the fibers were released only by subsequent cross-bridge attachment. Vi and ADP were preferentially trapped under conditions of cross-bridge cycling in the presence of ATP rather than in relaxed fibers or in rigor with ADP. These results indicate that in the normal cross-bridge cycle, inorganic phosphate (Pi) is released from actomyosin before ADP. The resulting actomyosin X ADP intermediate can bind Vi and Pi. This intermediate probably supports force. Vi behaves as a close analogue of Pi in muscle fibers, as it does with isolated actomyosin.     

Vanadate and phosphate ions reduce tension and increase cross-bridge kinetics in chemically skinned heart muscle

Tension development, immediate stiffness and ATPase of chemically skinned myocardial strips were measured in solutions with varying concentrations of phosphate (Pi) or vanadate (predominantly H2VO4 at pH 7) ion. Vanadate and Pi decreased stiffness in proportion to tension. The results show that, like Pi, vanadate accelerates the turnover rate of cross-bridges, but is effective at about 1/500 the concentration required for the Pi effect. Both Pi and vanadate increased the energy cost of isometric tension maintenance (that is, the ratio of ATPase to tension) and increased the velocity of delayed tension development following quick stretch of the chemically skinned myocardial strips. The results also show that changes in the rate of rise of delayed tension during stretch activation probably reflect changes in the kinetics of the biochemical cycle of the cross-bridges.     

Strain-dependent modulation of phosphate transients in rabbit skeletal muscle fibers.

When inorganic phosphate (Pi) is photogenerated from caged Pi during isometric contractions of glycerinated rabbit psoas muscle fibers, the released Pi binds to cross-bridges and reverses the working stroke of cross-bridges. The consequent force decline, the Pi-transient, is exponential and probes the kinetics of the power-stroke and Pi release. During muscle shortening, the fraction of attached cross-bridges and the average strain on them decreases (Ford, L. E., A.F. Huxley, and R.M. Simmons, 1977. Tension responses to sudden length change in stimulated frog muscle fibers near slack length. J. Physiol. (Lond.). 269:441-515; Ford, L. E., A. F. Huxley, and R.M. Simmons, 1985. Tension transients during steady state shortening of frog muscle fibers. J. Physiol. (Lond.). 361:131-150. To learn to what extent the Pi transient is strain dependent, muscle fibers were activated and shortened or lengthened at a fixed velocity during the photogeneration of Pi. The Pi transients observed during changes in muscle length showed three primary characteristics: 1) during shortening the Pi transient rate, Kpi, increased and its amplitude decreased with shortening velocity; Kpi increased linearly with velocity to > 110 s-1 at 0.3 muscle lengths per second (ML/s). 2) At a specific shortening velocity, increases in [Pi] produce increases in Kpi that are nonlinear with [Pi] and approach an asymptote. 3) During forced lengthening Kpi and the amplitude of the Pi transient are little different from the isometric contractions. These data can be approximated by a strain-dependent three-state cross-bridge model. The results show that the power stroke's rate is strain-dependent, and are consistent with biochemical studies indicating that the rate-limiting step at low strains is a transition from a weakly to a strongly bound cross-bridge state.     

Effects of Ca2+ on the kinetics of phosphate release in skeletal muscle.

The process of phosphate dissociation during the muscle cross-bridge cycle has been investigated by photoliberation of inorganic phosphate (Pi) within skinned fibers of rabbit psoas muscle. This permitted a test of the idea that Ca2+ controls muscle contraction by regulating the Pi release step of the cycle. Photoliberation of Pi from structurally distinct "caged" Pi precursors initiated a rapid tension decline of up to 12% of active tension, and this was followed by a slower tension decline. The apparent rate constant of the fast phase, kPi, depended on both [Pi] and [Ca2+], whereas the slow phase generally occurred at 2-4 s-1. At maximal Ca2+, kPi increased in a nonlinear manner from 43 +/- 2 s-1 to 118 +/- 7 s-1, as Pi was raised from 0.9 to 12 mM. This was analyzed in terms of a three-state kinetic model in which a force-generating transition is coupled to Pi dissociation from the cross-bridge. As Ca(2+)-activated tension was reduced from maximal (Pmax) to 0.1 Pmax, (i) kPi decreased by up to 2.5-fold, (ii) the relative amplitude of the rapid phase increased 2-fold, and (iii) the relative amplitude of the slow phase increased about 6-fold. Changes in the rapid phase are compatible with Ca2+ influencing an apparent equilibrium constant for the force-generating transition. By comparison, kPi was faster than the rate constant of tension redevelopment, ktr, and was influenced less by Ca2+. Ca2+ effects on the caged Pi transient cannot account for the large effects of Ca2+ on actomyosin ATPase rates or cross-bridge cycling kinetics but may be a manifestation of reciprocal interactions between the thin filament and force-generating cross-bridges, and may represent Ca2+ regulation of the distribution of cross-bridges between non-force-and force-generating states.     

Changes in the ATPase activity of insect fibrillar flight muscle during calcium and strain activation probed by phosphate-water oxygen exchange

During ATP hydrolysis by Ca2+-activated chemically skinned fibers from the flight muscle of the giant waterbug Lethocerus indicus, there is extensive phosphate-water oxygen exchange. For unstrained fibers the pattern of exchange shows that there is more than one pathway for hydrolysis, due to the ATPase activity of cross-bridges. Multiple pathways are an established property of both vertebrate actomyosin and fibers. The pattern of exchange can be fitted by two pathways: one with low exchange because the step(s) controlling Pi release are rapid, the other with high exchange and slow Pi release. The high-exchange pathway is responsible for most of the increase in ATPase activity on Ca2+ activation. On strain activation, only the high-exchange pathway is present, accounting for all the ATPase increase and responsible for force generation. In fully activated fibers, the cross-bridges which hydrolyze ATP and generate force behave uniformly with respect to oxygen exchange. The exchange pattern shows that the rate of Pi release changes dramatically over a very narrow strain increase. Step(s) controlling Pi release are at least partially rate-limiting for the overall ATPase reaction. The results are discussed in relation to models for strain activation and the identity of force-generating states.     

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.06 s(-1)) than tonic (0.29 +/- 0.03 s(-1)) smooth muscles. Thiophosphorylation of regulatory light chains increased and strain decreased the release rate approximately twofold. The calculated (k-ADP/k+ADP) dissociation constant, Kd of unstrained, unphosphorylated cross-bridges for ADP was 0.6 microM for rabbit bladder and 0.3 microM for femoral artery. The rates of ADP release from rigor bridges and reported values of Pi release (corresponding to the steady-state adenosine triphosphatase (ATPase) rate of actomyosin (AM)) from cross-bridges during a maintained isometric contraction are similar, indicating that the ADP-release step or an isomerization preceding it may be limiting the adenosine triphosphatase rate. We conclude that the strain- and dephosphorylation-dependent high affinity for and slow ADP release from smooth muscle myosin prolongs the fraction of the duty cycle occupied by strongly bound actomyosin.ADP state(s) and contributes to the high economy of force.     

Are weakly binding bridges present in resting intact muscle fibers?

Several experimental results (Schoenberg, M. 1988. Biophys. J. 54:135-148) have shown that the force response of relaxed skinned muscle fibers to fast stretches arises from the presence of cross-bridges rapidly cycling between attached and detached states. These bridges were identified with the M.ATP<-->AM.ATP and M.ADP.Pi<-->AM.ADP.Pi states seen in solution and are commonly referred to as weakly binding bridges. In this paper we have investigated the possibility that weakly binding bridges are also present in resting intact muscle fibers. The force response to fast stretches can be accounted for by assuming the presence in the fiber of a viscous and a viscoelastic passive component arranged in parallel. None of these components has the properties previously attributed to weakly binding bridges. This shows that in intact resting fibers there is no mechanical evidence of attached cross-bridges, suggesting that, under physiological conditions, either the M.ATP or M.ADP.Pi states have a negligibly small affinity for actin or the AM.ATP and AM.ADP.Pi cross-bridge states are unable to bear tension and contribute to fiber stiffness.     

Regulation of force development studied by photolysis of caged ADP in rabbit skinned psoas fibers.

The present study examined the effects of Ca(2+) and strongly bound cross-bridges on tension development induced by changes in the concentration of MgADP. Addition of MgADP to the bath increased isometric tension over a wide range of [Ca(2+)] in skinned fibers from rabbit psoas muscle. Tension-pCa (pCa is -log [Ca(2+)]) relationships and stiffness measurements indicated that MgADP increased mean force per cross-bridge at maximal Ca(2+) and increased recruitment of cross-bridges at submaximal Ca(2+). Photolysis of caged ADP to cause a 0.5 mM MgADP jump initiated an increase in isometric tension under all conditions examined, even at pCa 6.4 where there was no active tension before ADP release. Tension increased monophasically with an observed rate constant, k(ADP), which was similar in rate and Ca(2+) sensitivity to the rate constant of tension re-development, k(tr), measured in the same fibers by a release-re-stretch protocol. The amplitude of the caged ADP tension transient had a bell-shaped dependence on Ca(2+), reaching a maximum at intermediate Ca(2+) (pCa 6). The role of strong binding cross-bridges in the ADP response was tested by treatment of fibers with a strong binding derivative of myosin subfragment 1 (NEM-S1). In the presence of NEM-S1, the rate and amplitude of the caged ADP response were no longer sensitive to variations in the level of activator Ca(2+). The results are consistent with a model in which ADP-bound cross-bridges cooperatively activate the thin filament regulatory system at submaximal Ca(2+). This cooperative interaction influences both the magnitude and kinetics of force generation in skeletal muscle.     

The effect of polyethylene glycol on the mechanics and ATPase activity of active muscle fibers

We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke.     

Probing actomyosin interactions with 2,4-dinitrophenol

Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.     

ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants

We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Cross-bridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site. First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached. Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PPi) do, it was possible, by using ADP as a competitive inhibitor of PPi or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges. We found that adding 175 microM ADP to 4 mM PPi or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM. This suggests that ADP is binding to attached cross-bridges with a dissociation constant of approximately 60 microM. This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-S1) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-S1 in solution (Johnson, R.E., and P. H. Adams. 1984. FEBS Letters. 174:11-14; Schoenberg, M., and E. Eisenberg. 1985. Biophysical Journal. 48:863-871; Biosca, J.A., L.E. Greene, and E. Eisenberg. 1986. Journal of Biological Chemistry. 261:9793-9800).     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.     

Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers.

The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AM*ADP.Pi state (A = actin, M = myosin). Since the state which leads into the AM*ADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AM*ADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AM*ADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

A non-cross-bridge stiffness in activated frog muscle fibers

Force responses to fast ramp stretches of various amplitude and velocity, applied during tetanic contractions, were measured in single intact fibers from frog tibialis anterior muscle. Experiments were performed at 14 degrees C at approximately 2.1 microm sarcomere length on fibers bathed in Ringer's solution containing various concentrations of 2,3-butanedione monoxime (BDM) to greatly reduce the isometric tension. The fast tension transient produced by the stretch was followed by a period, lasting until relaxation, during which the tension remained constant to a value that greatly exceeded the isometric tension. The excess of tension was termed "static tension," and the ratio between the force and the accompanying sarcomere length change was termed "static stiffness." The static stiffness was independent of the active tension developed by the fiber, and independent of stretch amplitude and stretching velocity in the whole range tested; it increased with sarcomere length in the range 2.1-2.8 microm, to decrease again at longer lengths. Static stiffness increased well ahead of tension during the tetanus rise, and fell ahead of tension during relaxation. These results suggest that activation increased the stiffness of some sarcomeric structure(s) outside the cross-bridges.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.