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.     

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.     

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.     

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.     

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.     

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.     

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.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers. A steady-state and transient kinetic study

The effect of varying concentrations of Pi and Ca2+ on isometric force and on the rate of force development in skinned rabbit psoas muscle fibers has been investigated. Steady-state results show that the three parameters that define the force-pCa relation (Po, pK, and n) all vary linearly with log [Pi]. As [Pi] increases, Po and pK decrease while n increases. The kinetics of force generation in isometrically contracting fibers were studied by laser flash photolysis of caged phosphate. The observed rate of the resulting tension transient, kPi, is 23.5 +/- 1.7 s-1 at 10 degrees C, 0.7 mM Pi, and is independent of [Ca2+] over the range pCa 4.5-7.2. By contrast, kTR, the rate of tension redevelopment following a period of isotonic shortening, is sensitive to [Ca2+] and is slower than kPi (kTR = 13.6 +/- 0.2 s-1 at pCa 4.5, 0.7 mM Pi). The results show that [Ca2+] does not directly affect the Pi release or force-generating steps of the cross-bridge cycle and show that the observed rate of force development depends on how the measurement is made. The data can be interpreted in terms of a model in which strong cross-bridges activate the thin filament, this activation being modulated by Ca2+ binding to troponin.     

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.     

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.     

Effect of joule temperature jump on tension and stiffness of skinned rabbit muscle fibers.

The effects of a temperature jump (T-jump) from 5-7 degrees C to 26-33 degrees C were studied on tension and stiffness of glycerol-extracted fibers from rabbit psoas muscle in rigor and during maximal Ca2+ activation. The T-jump was initiated by passing an alternating current pulse (30 kHz, up to 2.5 kV, duration 0.2 ms) through a fiber suspended in air. In rigor the T-jump induces a drop of both tension and stiffness. During maximal activation, the immediate stiffness dropped by (4.4 +/- 1.6) x 10(-3)/1 degree C (mean + SD) in response to the T-jump, and this was followed by a monoexponential stiffness rise by a factor of 1.59 +/- 0.14 with a rate constant ks = 174 +/- 42 s-1 (mean +/- SD, n = 8). The data show that the fiber stiffness, determined by the cross-bridge elasticity, in both rigor and maximal activation is not rubber-like. In the activated fibers the T-jump induced a biexponential tension rise by a factor of 3.45 +/- 0.76 (mean +/- SD, n = 8) with the rate constants 500-1,000 s-1 for the first exponent and 167 +/- 39 s-1 (mean +/- SD, n = 8) for the second exponent. The data are in accordance with the assumption that the first phase of the tension transient after the T-jump is due to a force-generating step in the attached cross-bridges, whereas the second one is related to detachment and reattachment of cross-bridges.     

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.     

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.     

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.     

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.     

Effects of pH on myofibrillar ATPase activity in fast and slow skeletal muscle fibers of the rabbit.

In permeabilized single fibers of fast (psoas) and slow (soleus) muscle from the rabbit, the effect of pH on isometric myofibrillar ATPase activity and force was studied at 15 degrees C, in the pH range 6.4-7.9. ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH, present in the bathing solution. NADH absorbance at 340 nm was determined inside a measuring chamber. To measure ATP turnover in single soleus fibers accurately, a new measuring chamber (volume 4 microliters) was developed that produced a sensitivity approximately 8 times higher than the system previously used. Under control conditions (pH 7.3), the isometric force was 136 and 115 kN/m2 and the ATP turnover was 0.43 and 0.056 mmol per liter fiber volume per second in psoas and soleus fibers, respectively. Over the pH range studied, isometric force increased monotonically by a factor 1.7 for psoas and 1.2 for soleus fibers. In psoas the isometric ATPase activity remained constant, whereas in soleus it slightly decreased with increasing pH. The pH dependency of relative tension cost (isometric ATPase activity divided by force) was practically identical for psoas and soleus fibers. In both cases it decreased by about a factor 0.57 as pH increased from 6.4 to 7.9. The implications of these findings are discussed in terms of cross-bridge kinetics. For both fiber types, estimates of the reaction rates and the distribution of cross-bridges and of their pH dependencies were obtained. A remarkable similarity was found between fast- and slow-twitch fibers in the effects of pH on the reaction rate constants.     

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.     

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.     

A birefringence study of changes in myosin orientation during relaxation of skinned muscle fibers induced by photolytic ATP release.

The birefringence of isolated skinned fibers from rabbit psoas muscle was measured continuously during relaxation from rigor produced by photolysis of caged ATP at sarcomere length 2.8-2.9 microns, ionic strength 0.1 M, 15 degrees C. Birefringence, the difference in refractive index between light components polarized parallel and perpendicular to the fiber axis, depends on the average degree of alignment of the myosin head domain with the fiber axis. After ATP release birefringence increased by 5.8 +/- 0.7% (mean +/- SE, n = 6) with two temporal components. A small fast component had an amplitude of 0.9 +/- 0.2% and rate constant of 63 s-1. By the completion of this component, the instantaneous stiffness had decreased to about half the rigor value, and the force response to a step stretch showed a rapid (approximately 1000 s-1) recovery phase. Subsequently a large slow birefringence component with rate constant 5.1 s-1 accompanied isometric force relaxation. Inorganic phosphate (10 mM) did not affect the fast birefringence component but accelerated the slow component and force relaxation. The fast birefringence component was probably caused by formation of myosin.ATP or myosin.ADP.Pi states that are weakly bound to actin. The average myosin head orientation at the end of this component is slightly more parallel to the fiber axis than in rigor.