Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

The effect of the lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers. II. Elementary steps affected by the spacing change.

The actin-myosin lattice spacing of rabbit psoas fibers was osmotically compressed with a dextran T-500, and its effect on the elementary steps of the cross-bridge cycle was investigated. Experiments were performed at the saturating Ca (pCa 4.5-4.9), 200 mM ionic strength, pH 7.0, and at 20 degrees C, and the results were analyzed by the following cross-bridge scheme: [formula: see text] where A = actin, M = myosin head, S = MgATP, D = MgADP, and P = Pi = phosphate. From MgATP and MgADP studies on exponential process (C) and (D), the association constants of cross-bridges to MgADP (K0), MgATP (K1a), the rate constants of the isomerization of the AM S state (k1b and k-1b), and the rate constants of the cross-bridge detachment step (k2 and k-2) were deduced. From Pi study on process (B), the rate constants of the cross-bridge attachment (power stroke) step (k4- and k-4) and the association constant of Pi ions to cross-bridges (K5) were deduced. From ATP hydrolysis measurement, the rate constant of ADP-isomerization (rate-limiting) step (k6) was deduced. These kinetic constants were studied as functions of dextran concentrations. Our results show that nucleotide binding, the ATP-isomerization, and the cross-bridge detachment steps are minimally affected by the compression. The rate constant of the reverse power stroke step (k-4) decreases with mild compression (0-6.3% dextran), presumably because of the stabilization of the attached cross-bridges in the AM*DP state. The rate constant of the power stroke step (k4) does not change with mild compression, but it decreases with higher compression (> 6.3% dextran), presumably because of an increased difficulty in performing the power stroke. These results are consistent with the observation that isometric tension increases with a low level of compression and decreases with a high level of compression. Our results also show that the association constant K5 of Pi with cross-bridge state AM*D is not changed with compression. Our result further show that the ATP hydrolysis rate decreased with compression, and that the rate constants of the ADP-isomerization step (k6) becomes progressively less with compression. The effect of compression on the power stroke step and rate-limiting step implies that a large-scale molecular rearrangement in the myosin head takes place in these two slow reaction steps.     

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.     

Force generation and phosphate release steps in skinned rabbit soleus slow-twitch muscle fibers.

The force-generation and phosphate-release steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibers (STF) were investigated using sinusoidal analysis, and the results were compared with those of rabbit psoas fast-twitch fibers (FTF). Single fiber preparations were activated at pCa 4.40 and ionic strength 180 mM at 20 degrees C. The effects of inorganic phosphate (Pi) concentrations on three exponential processes, B, C, and D, were studied. Results are consistent with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and P is inorganic phosphate. The values determined are k4 = 5.7 +/- 0.5 s-1 (rate constant of isomerization step, N = 9, mean +/- SE), k-4 = 4.5 +/- 0.5 s-1 (rate constant of reverse isomerization), K4 = 1.37 +/- 0.13 (equilibrium constant of the isomerization), and K5 = 0.18 +/- 0.01 mM-1 (Pi association constant). The isomerization step (k4) in soleus STF is 20 times slower, and its reversal (k-4) is 20 times slower than psoas fibers. Consequently, the equilibrium constant of the isomerization step (K4) is the same in these two types of fibers. The Pi association constant (K5) is slightly higher in STF than in FTF, indicating that Pi binds to cross-bridges slightly more tightly in STF than FTF. By correlating the cross-bridge distribution with isometric tension, it was confirmed that force is generated during the isomerization (step 4) of the AMDP state and before Pi release in soleus STF.     

Role of MgATP and MgADP in the cross-bridge kinetics in chemically skinned rabbit psoas fibers. Study of a fast exponential process (C)

The role of the substrate (MgATP) and product (MgADP) molecules in cross-bridge kinetics is investigated by small amplitude length oscillations (peak to peak: 3 nm/cross-bridge) and by following amplitude change and phase shift in tension time courses. The range of discrete frequencies used for this investigation is 0.25-250 Hz, which corresponds to 0.6-600 ms in time domain. This report investigates the identity of the high frequency exponential advance (process C), which is equivalent to "phase 2" of step analysis. The experiments are performed in maximally activated (pCa 4.5-5.0) single fibers from chemically skinned rabbit psoas fibers at 20 degrees C and at the ionic strength 195 mM. The rate constant 2 pi c deduced from process (C) increases and saturates hyperbolically with an increase in MgATP concentration, whereas the same rate constant decreases monotonically with an increase in MgADP concentration. The effects of MgATP and MgADP are opposite in all respects we have studied. These observations are consistent with a cross-bridge scheme in which MgATP and MgADP are in rapid equilibria with rigorlike cross-bridges, and they compete for the substrate site on myosin heads. From our measurements, the association constants are found to be 1.4 mM-1 for MgATP and 2.8 mM-1 for MgADP. We further deduced that the composite second order rate constant of MgATP binding to cross-bridges and subsequent isomerization/dissociation reaction to be 0.57 x 10(6)M-1s-1.     

Kinetic and thermodynamic studies of the cross-bridge cycle in rabbit psoas muscle fibers.

The effect of temperature on elementary steps of the cross-bridge cycle was investigated with sinusoidal analysis technique in skinned rabbit psoas fibers. We studied the effect of MgATP on exponential process (C) to characterize the MgATP binding step and cross-bridge detachment step at six different temperatures in the range 5-30 degrees C. Similarly, we studied the effect of MgADP on exponential process (C) to characterize the MgADP binding step. We also studied the effect of phosphate (Pi) on exponential process (B) to characterize the force generation step and Pi-release step. From the results of these studies, we deduced the temperature dependence of the kinetic constants of the elementary steps and their thermodynamic properties. We found that the MgADP association constant (K0) and the MgATP association constant (K1) significantly decreased when the temperature was increased from 5 to 20 degrees C, implying that nucleotide binding became weaker at higher temperatures. K0 and K1 did not change much in the 20-30 degree C range. The association constant of Pi to cross-bridges (K5) did not change much with temperature. We found that Q10 for the cross-bridge detachment step (k2) was 2.6, and for its reversal step (k-2) was 3.0. We found that Q10 for the force generation step (Pi-isomerization step, k4) was 6.8, and its reversal step (k-4) was 1.6. The equilibrium constant of the detachment step (K2) was not affected much by temperature, whereas the equilibrium constant of the force generation step (K4) increased significantly with temperature increase. Thus, the force generation step consists of an endothermic reaction. The rate constant of the rate-limiting step (k6) did not change much with temperature, whereas the ATP hydrolysis rate increased significantly with temperature increase. We found that the force generation step accompanies a large entropy increase and a small free energy change; hence, this step is an entropy-driven reaction. These observations are consistent with the hypothesis that the hydrophobic interaction between residues of actin and myosin underlies the mechanism of force generation. We conclude that the force generation step is the most temperature-sensitive step among elementary steps of the cross-bridge cycle, which explains increased isometric tension at high temperatures in rabbit psoas fibers.     

The dependence of isometric tension, isometric ATPase activity, and shortening velocity of limulus muscle on the MgATP concentration.

The dependence of the isometric tension, the velocity of unloaded shortening, and the steady-state rate of MgATP hydrolysis on the MgATP concentration (range 0.01-5 mM MgATP) was studied in Ca-activated skinned Limulus muscle fibers. With increasing MgATP concentration the isometric tension increased to a peak at approximately 0.1 mM, and slightly decreased in the range up to 5 mM MgATP. The velocity of unloaded shortening depended on the MgATP concentration roughly according to the Michaelis-Menten law of saturation kinetics with a Michaelis-Menten constant Kv = 95 microM and a maximum shortening velocity of 0.07 muscle lengths s-1; the detachment rate of the cross-bridges during unloaded shortening was 24 s-1. The rate of MgATP splitting also depended hyperbolically on the MgATP concentration with a Michaelis-Menten constant Ka = 129 microM and a maximum turnover frequency of 0.5-1 s-1. The results are discussed in terms of a cross-bridge model based on a biochemical scheme of ATP hydrolysis by actin and myosin in solution.     

Elementary steps of the cross-bridge cycle in bovine myocardium with and without regulatory proteins.

The role of regulatory proteins in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed by gelsolin and the actin filament was reconstituted without tropomyosin or troponin. Further reconstitution was achieved by adding tropomyosin and troponin. The effects of MgATP and phosphate (Pi) on the rate constants of exponential processes were studied in control, actin filament-reconstituted, and thin filament-reconstituted myocardium at pCa < or = 4.66, pH 7.00, 25 degrees C. In control myocardium, the MgATP association constant was 9.1 +/- 1.3 mM(-1), and the Pi association constant 0.14 +/- 0.04 mM(-1). The equilibrium constant of the cross-bridge detachment step was 2.6 +/- 0.4, and the equilibrium constant of the force generation step was 0.59 +/- 0.04. In actin filament-reconstituted myocardium without regulatory proteins, the MgATP association constant was approximately the same, and the Pi association constant increased to 2.8x. The equilibrium constant of cross-bridge detachment decreased to 0.2x, but the equilibrium constant of the force generation step increased to 4x. These kinetic constants regained control values after reconstitution of the thin filament. These results indicate that tension/cross-bridge in the presence of regulatory proteins is approximately 1.5-1.7x of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers.

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.     

Measurement of the reversibility of ATP binding to myosin in calcium-activated skinned fibers from rabbit skeletal muscle. Oxygen exchange between water and ATP released to the solution

We have measured the rate constant for ATP release from myosin heads of Ca2+-activated, demembranated muscle fibers using the technique of phosphate-water oxygen exchange. Single rabbit psoas fibers were held in an activating solution in [18O]water ([MgATP] = 8 mM, ionic strength = 0.2 M, pH = 7.0, 24 degrees C). After about 20% hydrolysis of ATP, product Pi and remaining ATP were isolated, and the distribution of 18O in both molecules was analyzed using a mass spectrometer. The exchange in Pi was similar to that previously reported (Hibberd, M. G., Webb, M. R., Goldman, Y. E., and Trentham, D. R. (1985) J. Biol. Chem. 260, 3496-3501). The amount of 18O in ATP gave a rate constant of about 4 s-1 for ATP release, if it is assumed that each rate constant in the pathway of ATP hydrolysis has the same value for all myosin ATPase sites. However, the distribution of 18O in both released Pi and ATP is not well explained by a single pathway for ATP hydrolysis. We present a model that indicates how such distributions could arise from a range of values for the rate constants for Pi and ATP release from actomyosin, and this range is determined by differences in the amounts of strain in attached crossbridges. The kinetic information obtained from these isotope exchange experiments is compared to show that they give a compatible set of rate constants for actomyosin in fibers.     

The effect of partial extraction of troponin C on the elementary steps of the cross-bridge cycle in rabbit psoas muscle fibers.

The elementary steps of the cross-bridge cycle in which troponin C (TnC) was partially extracted were investigated by sinusoidal analysis in rabbit psoas muscle fibers. The effects of MgATP and phosphate on the rate constants of exponential processes were studied at 200 mM ionic strength, pCa 4.20, pH 7.00, and at 20 degrees C. The results were analyzed with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, S is MgATP, D is MgADP, and P is phosphate (Pi). When TnC was extracted so that the average remaining tension was 11% (range 8-15%), K1 (MgATP association constant) increased to 7x, k2 (rate constant of cross-bridge detachment) increased to 1.55x, k-2 (reversal of detachment) decreased to 0.27x, and K2 (= k2/k-2: equilibrium constant of cross-bridge detachment) increased to 6.6x, k4 (rate constant of force generation) decreased to 0.4x, k-4 (reversal of force generation) increased to 2x, K4 (= k4/k-4) decreased to 0.17x, and K5 (Pi association constant) did not change. The activation factor alpha, which represents the fraction of cross-bridges participating in the cycling, decreased from 1 to 0.14 with TnC extraction. The fact that K1 increased with TnC extraction implies that the condition of the thin filament modifies the contour of the substrate binding site on the myosin head and is consistent with the Fenn effect. The fact that alpha decreased to 0.14 is consistent with the steric blocking mechanism (recruitment hypothesis) and indicates that some of the cross-bridges disappear from the active cycling pool. The fact that the equilibrium constants changed is consistent with the cooperative activation mechanism (graded activation hypothesis) among thin-filament regulatory units that consist of troponin (TnC, Tnl, TnT), tropomyosin, and seven actin molecules, and possibly include cross-bridges.     

Kinetic mechano-chemical model of a muscle with a six-stage cross-bridge cycle]

A kinetic scheme of the mechano-chemical cycle of the cross-bridges and a mathematical model based on this scheme are proposed. The main assumptions accepted in the scheme are: the step of the inorganic phosphate release precedes the force-generating step of a cross-bridge; the rate-limiting step of the ATP hydrolysis is isomerization of the actomyosin-ADP complex. It is shown that the model well describes the mechanical and biochemical transients initiated by the temperature jump and flash photolysis of the caged compounds in skinned muscle fibres.     

Elementary steps of the cross-bridge cycle in fast-twitch fiber types from rabbit skeletal muscles

To understand the molecular mechanism underlying the diversity of mammalian skeletal muscle fibers, the elementary steps of the cross-bridge cycle were investigated in three fast-twitch fiber types from rabbit limb muscles. Skinned fibers were maximally Ca(2+)-activated at 20 degrees C and the effects of MgATP, phosphate (P, P(i)), and MgADP were studied on three exponential processes by sinusoidal analysis. The fiber types (IIA, IID, and IIB) were determined by analyzing the myosin heavy-chain isoforms after mechanical experiments using high-resolution SDS-PAGE. The results were consistent with the following cross-bridge scheme: where A is actin, M is myosin, D is MgADP, and S is MgATP. All states except for those in brackets are strongly bound states. All rate constants of elementary steps (k(2), 198-526 s(-1); k(-2), 51-328 s(-1); k(4), 13.6-143 s(-1); k(-4), 13.6-81 s(-1)) were progressively larger in the order of type IIA, type IID, and type IIB fibers. The rate constants of a transition from a weakly bound state to a strongly bound state (k(-2), k(4)) varied more among fiber types than their reversals (k(2), k(-4)). The equilibrium constants K(1) (MgATP affinity) and K(2) (=k(2)/k(-2), ATP isomerization) were progressively less in the order IIA, IID, and IIB. K(4) (=k(4)/k(-4), force generation) and K(5) (P(i) affinity) were larger in IIB than IIA and IID fibers. K(1) showed the largest variation indicating that the myosin head binds MgATP more tightly in the order IIA (8.7 mM(-1)), IID (4.9 mM(-1)), and IIB (0.84 mM(-1)). Similarly, the MgADP affinity (K(0)) was larger in type IID fibers than in type IIB fibers.     

Energetics of the calcium-transporting ATPase

A thermodynamic cycle for catalysis of calcium transport by the sarcoplasmic reticulum ATPase is described, based on equilibrium constants for the microscopic steps of the reaction shown in Equation 1 under a single set of experimental (formula; see text) conditions (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4): KCa = 5.9 X 10(-12) M2, K alpha ATP = 15 microM, Kint = 0.47, K alpha ADP = 0.73 mM, K'int = 1.7, K"Ca = 2.2 X 10(-6) M2, and Kp = 37 mM. The value of K"Ca was calculated by difference, from the free energy of hydrolysis of ATP. The spontaneous formation of an acylphosphate from Pi and E is made possible by the expression of 12.5 kcal mol-1 of noncovalent binding energy in E-P. Only 1.9 kcal mol-1 of binding energy is expressed in E X Pi. There is a mutual destabilization of bound phosphate and calcium in E-P X Ca2, with delta GD = 7.6 kcal mol-1, that permits transfer of phosphate to ADP and transfer of calcium to a concentrated calcium pool inside the vesicle. It is suggested that the ordered kinetic mechanism for the dissociation of E-P X Ca2, with phosphate transfer to ADP before calcium dissociation outside and phosphate transfer to water after calcium dissociation inside, preserves the Gibbs energies of these ligands and makes a major contribution to the coupling in the transport process. A lag (approximately 5 ms) before the appearance of E-P after mixing E and Pi at pH 6 is diminished by ATP and by increased [Pi]. This suggests that ATP accelerates the binding of Pi. The weak inhibition by ATP of E-P formation at equilibrium also suggests that ATP and phosphate can bind simultaneously to the enzyme at pH 6. Rate constants are greater than or equal to 115 s-1 for all the steps in the reaction sequence to form E-32P X Ca2 from E-P, Ca2+ and [32P]ATP at pH 7. E-P X Ca2 decomposes with kappa = 17 s-1, which shows that it is a kinetically competent intermediate. The value of kappa decreases to 4 s-1 if the intermediate is formed in the presence of 2 mM Ca2+. This decrease and inhibition of turnover by greater than 0.1 mM Ca2+ may result from slow decomposition of E-P X Ca3.     

Exchange of ATP for ADP on high-force cross-bridges of skinned rabbit muscle fibers

The contractile properties of rabbit skinned muscle fibers were studied at 1-2 degrees C in different concentrations of MgATP and MgADP. Double-reciprocal plots of maximum velocity against MgATP concentration at different MgADP concentrations all extrapolated to the same value. This finding suggests that MgATP and MgADP compete for the same site on the cross-bridge, and that the exchange of MgATP for MgADP occurs without a detectable step intervening. The K(m) for ATP was 0.32 mM. The K(i) for MgADP was 0.33 mM. Control experiments suggested that the tortuosity of diffusion paths within the fibers reduced the radial diffusion coefficients for reactants about sixfold. Increasing MgADP from 0.18 to 2 mM at 5 mM ATP or lowering MgATP from 10 to 2 mM at 0.18 mM MgADP, respectively, increased isometric force by 25% and 23%, increased stiffness by 10% and 20%, and decreased maximum velocity by 35% and 31%. Two mechanisms appeared to be responsible. One detained bridges in high-force states, where they recovered from a length step with a slower time course. The other increased the fraction of attached bridges without altering the kinetics of their responses, possibly by an increased activation resulting from cooperative effects of the detained, high-force bridges. The rigor bridge was more effective than the ADP-bound bridge in increasing the number of attached bridges with unaltered kinetics.     

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.     

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.     

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.     

Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady- state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.