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.     

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 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.     

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.     

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.     

Relaxation from rigor of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of ATP-induced rigor cross-bridge detachment were studied by initiating relaxation in chemically skinned trabeculae of the guinea pig heart using photolytic release of ATP in the absence of calcium ions (pCa > 8). The time course of the fall in tension exhibited either an initial plateau phase of variable duration with little change in tension or a rise in tension, followed by a decrease to relaxed levels. The in-phase component of tissue stiffness initially decreased. The rate then slowed near the end of the tension plateau, indicating transient cross-bridge rebinding, before falling to relaxed levels. Estimates of the apparent second-order rate constant for ATP-induced detachment of rigor cross-bridges based on the half-time for relaxation or on the half-time to the convergence of tension records to a common time course were similar at 3 x 10(3) M-1 s-1. Because the characteristics of the mechanical transients observed during relaxation from rigor were markedly similar to those reported from studies of rabbit psoas fibers in the presence of MgADP (Dantzig, J. A., M. G. Hibberd, D. R. Trentham, and Y. E. Goldman. 1991. Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol. 432:639-680), direct measurements of MgADP using [3H]ATP in cardiac tissue in rigor were made. Results indicated that during rigor, nearly 18% of the cross-bridges in skinned trabeculae had [3H]MgADP bound. Incubation of the tissue during rigor with apyrase, an enzyme with both ADPase and ATPase activity, reduced the level of [3H]MgADP to that measured following a 2-min chase in a solution containing 5 mM unlabeled MgATP. Apyrase incubation also significantly reduced the tension and stiffness transients, so that both time courses became monotonic and could be fit with a simple model for cross-bridge detachment. The apparent second-order rate constant for ATP-induced rigor cross-bridge detachment measured in the apyrase treated tissue at 4 x 10(4) M-1 s-1 was faster than that measured in untreated tissue. Nevertheless, this rate was still over an order of magnitude slower than the analogous rate measured in previous studies of isolated cardiac actomyosin-S1. These results are consistent with the hypothesis that the presence of MgADP bound cross-bridges suppresses the inhibition normally imposed by the thin filament regulatory system in the absence of calcium ions and allows cross-bridge rebinding and force production during relaxation from rigor.     

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.     

Observation of two orientations from rigor cross-bridges in glycerinated muscle fibers

The fluorescence polarization from rhodamine labels specifically attached to the fast-reacting thiol of the myosin cross-bridge in glycerinated muscle fibers has been measured to determine the angular distribution of the cross-bridges in different physiological states of the fibers as a function of temperature. To investigate the fibers at temperatures below 0 degree C, we have added glycerol to the bathing solution as an anti-freezing agent. We find that the fluorescence polarization from the rhodamine probe detects distinct angular distributions of the cross-bridges in isometric-active, rigor, MgADP, and low ionic strength relaxed fibers at 4 degrees C. We also find that the rigor cross-bridges in the presence of glycerol can maintain at least two distinct orientations relative to the actin filament, one dominant at temperatures T greater than 2 degrees C and another dominant at T less than -10 degrees C. MgADP cross-bridges in the presence of glycerol maintain approximately the same orientation for all temperatures investigated. The rigor cross-bridge orientation at T less than -10 degrees C is similar to both the MgADP cross-bridge orientation in the presence of glycerol and the active muscle cross-bridge orientation at 4 degrees C. These findings show that the rigor cross-bridge in the presence of glycerol has at least two distinct orientations while attached to actin: one of them dominant at high temperature, the other dominant at low temperature or when MgADP is present. The latter orientation resembles that present in isometric-active fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Two step mechanism of phosphate release and the mechanism of force generation in chemically skinned fibers of rabbit psoas muscle.

The elementary steps of contraction in rabbit fast twitch muscle fibers were investigated with particular emphasis on the mechanism of phosphate (Pi) binding/release, the mechanism of force generation, and the relation between them. We monitor the rate constant 2 pi b of a macroscopic exponential process (B) by imposing sinusoidal length oscillations. We find that the plot of 2 pi b vs. Pi concentration is curved. From this observation we infer that Pi released is a two step phenomenon: an isomerization followed by the actual Pi release. Our results fit well to the kinetic scheme: [formula: see text] where A = actin, M = myosin, S = MgATP (substrate), D = MgADP, P = phosphate, and Det is a composite of all the detached and weakly attached states. For our data to be consistent with this scheme, it is also necessary that step 4 (isomerization) is observed in process (B). By fitting this scheme to our data, we obtained the following kinetic constants: k4 = 56 s-1, k-4 = 129 s-1, and K5 = 0.069 mM-1, assuming that K2 = 4.9. Experiments were performed at pCa 4.82, pH 7.00, MgATP 5 mM, free ATP 5 mM, ionic strength 200 mM in K propionate medium, and at 20 degrees C. Based on these kinetic constants, we calculated the probability of each cross-bridge state as a function of Pi, and correlated this with the isometric tension. Our results indicate that all attached cross-bridges support equal amount of tension. From this, we infer that the force is generated at step 4. Detailed balance indicates that 50-65% of the free energy available from ATP hydrolysis is transformed to work at this step. For our data to be consistent with the above scheme, step 6 must be the slowest step of the cross-bridge cycle (the rate limiting step). Further, AM*D is a distinctly different state from the AMD state that is formed by adding D to the bathing solution. From our earlier ATP hydrolysis data, we estimated k6 to be 9 s-1.     

Effects of magnesium pyrophosphate on mechanical properties of skinned smooth muscle from the guinea pig taenia coli.

Effects of the non-hydrolyzable nucleotide analogue magnesium pyrophosphate (MgPPi) on cross-bridge properties were investigated in skinned smooth muscle of the guinea pig Taenia coli. A "high" rigor state was obtained by removing MgATP at the plateau of an active contraction. Rigor force decayed slowly towards an apparent plateau of approximately 25-35% of maximal active force. MgPPi markedly increased the rate of force decay. The initial rate of the force decay depended on [MgPPi] and could be described by the Michaelis-Menten equation with a dissociation constant of 1.6 mM. The decay was irreversible amounting to approximately 50% of the rigor force. Stiffness decreased by 20%, suggesting that the major part of the cross-bridges were still attached. The results can be interpreted as "slippage" of PPi-cross-bridges to positions of lower strain. The initial rate of MgPPi-induced force decay decreased with decreasing ionic strength in the range 45-150 mM and was approximately 25% lower in thiophosphorylated fibers. MgADP inhibited the MgPPi-induced force decay with an apparent Ki of 2 microM. The apparent Km of MgATP for the maximal shortening velocity in thiophosphorylated fibers was 32 microM. This low Km of MgATP suggests that steps other than MgATP-induced detachment are responsible for the low shortening velocity in smooth muscle. No effects were observed of 4 mM MgPPi on the force-velocity relation, suggesting that cross-bridges with bound MgPPi do not constitute an internal load or that binding of MgPPi is weaker in negatively strained cross-bridges during shortening.     

Effects of MgATP, MgADP, and Pi on actin movement by smooth muscle myosin.

To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.     

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.     

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.     

The smooth muscle cross-bridge cycle studied using sinusoidal length perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (, Pharmacol. Rev. 20:197-272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P(i)) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P(i), force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P(i) release. For the aorta, elevating P(i) decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P(i) state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased approximately 50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P(i) release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.     

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.     

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.     

Probing cross-bridge angular transitions using multiple extrinsic reporter groups.

15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation.