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Two step mechanism of phosphate release and the mechanism of force generation in chemically skinned fibers of rabbit psoas muscle. 总被引:27,自引:8,他引:19
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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. 相似文献
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Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers. 总被引:4,自引:0,他引:4
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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. 相似文献
5.
Force generation and work production by covalently cross-linked actin-myosin cross-bridges in rabbit muscle fibers.
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To separate a fraction of the myosin cross-bridges that are attached to the thin filaments and that participate in the mechanical responses, muscle fibers were cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and then immersed in high-salt relaxing solution (HSRS) of 0.6 M ionic strength for detaching the unlinked myosin heads. The mechanical properties and force-generating ability of the cross-linked cross-bridges were tested with step length changes (L-steps) and temperature jumps (T-jumps) from 6-10 degrees C to 30-40 degrees C. After partial cross-linking, when instantaneous stiffness in HSRS was 25-40% of that in rigor, the mechanical behavior of the fibers was similar to that during active contraction. The kinetics of the T-jump-induced tension transients as well as the rate of the fast phase of tension recovery after length steps were close to those in unlinked fibers during activation. Under feedback force control, the T-jump initiated fiber shortening by up to 4 nm/half-sarcomere. Work produced by a cross-linked myosin head after the T-jump was up to 30 x 10(-21) J. When the extent of cross-linking was increased and fiber stiffness in HSRS approached that in rigor, the fibers lost their viscoelastic properties and ability to generate force with a rise in temperature. 相似文献
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Nonparallel isometric tension response of rabbit soleus skinned muscle fibers to magnesium adenosine triphosphate and magnesium inosine triphosphate
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B Krasner 《The Journal of general physiology》1979,74(2):261-274
The isometric tension response of single "skinned' rabbit soleus muscle fibers to MgATP and McITP in the absence of calcium was studied. [MgATP] or [MgITP] was varied in solutions of ionic strength 0.30 and temperature 20 degrees C. Steady-state tension that developed in MgATP or MgITP solutions was a biphasic bell-shaped function of log [MgATP] or log [MgITP] which increased from zero to maximum tension and then declined again to zero. Analysis of the data showed that, under comparable ionic conditions, percent tension vs. log [MgATP] and percent tension vs. log [MgITP] curves are not parallel. Instead, the percent tension vs. log [MgITP] curve is much broader. Additionally, under comparable ionic conditions maximum tension in MgITP solutions was higher than in MgATP solutions. In addition, in MgATP solutions, pH, [K+], and excess ATP were varied. Raising pH from 7 to 8, [K+] from 46 mM to 200 mM, or decreasing excess ATP from 2 to 0.5 mM all increased maximum tension. None of these factors, however, influenced the shape or position of the percent tension vs. log [MgATP] curve. 相似文献
7.
Compliance of thin filaments in skinned fibers of rabbit skeletal muscle. 总被引:19,自引:7,他引:12
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The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction. 相似文献
8.
M Iino H Takano-Ohmuro Y Kawana M Endo 《Biochemical and biophysical research communications》1992,185(2):713-718
Calpain treatment of rabbit skinned muscle fibers resulted in proteolysis of junctional foot protein or Ca2+ release channel of the sarcoplasmic reticulum. Electrophoretic and immunoblot analyses indicate that calpain cleaves off approximately 130 kDa peptide from the N-terminus. After such treatment, Ca2+ capacity of the sarcoplasmic reticulum remained normal and both Ca2+ and adenine nucleotide dependence of Ca2+-induced Ca2+ release mechanism were retained. However, the Ca2+-activated Ca2+ release rate was increased by two fold after the proteolysis. The results suggest the presence of functional domains in the junctional foot protein, and the N-terminus domain controls the activity of the Ca2+ channel without changing Ca2+ and nucleotide sensitivities. 相似文献
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Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle 总被引:4,自引:0,他引:4
M G Hibberd M R Webb Y E Goldman D R Trentham 《The Journal of biological chemistry》1985,260(6):3496-3500
The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release. 相似文献
10.
Cross-bridge scheme and force per cross-bridge state in skinned rabbit psoas muscle fibers. 总被引:9,自引:4,他引:5
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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. 相似文献
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Chloride-induced Ca release in skinned muscle fibers was studied by measuring isometric force transients and 45Ca loss from fiber to washout solutions. Skinned fibers prepared from muscles soaked in normal Ringer solution made large force transients in 120 mM Cl solution with 5 mM ATP and 1 mM Mg, but 3 mM Mg was inhibitory. Mg inhibition was antagonized by low temperature and by Cd, agents which slow active Ca uptake by the sarcoplasmic reticulum (SR). In low Mg++, Cl stimulated rapid 45Ca release from the SR in sufficient amounts to account for the force response. The increased 45Ca release was inhibited by EGTA, suggesting that release requires free Ca under these conditions. The 45Ca initially released was partially reaccumulated later. Reaccumulation was increased in higher Mg++. These results provide additional evidence that the Ca uptake rate is an important determinant of net release, and suggest that Mg++ acts primarily on this mechanism. Skinned fibers prepared from muscles soaked in low Cl solutions could give force responses to Cl solutions with 3 mM and 6 mM Mg. This observation suggests that the Cl stimulus varies with the [Cl] gradient across the internal membranes, and supports the hypothesis that applied Cl causes membrane depolarization. 相似文献
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Force generation and temperature-jump and length-jump tension transients in muscle fibers. 总被引:7,自引:2,他引:5
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Muscle tension rises with increasing temperature. The kinetics that govern the tension rise of maximally Ca(2+)-activated, skinned rabbit psoas fibers over a temperature range of 0-30 degrees C was characterized in laser temperature-jump experiments. The kinetic response is simple and can be readily interpreted in terms of a basic three-step mechanism of contraction, which includes a temperature-sensitive rapid preequilibrium(a) linked to a temperature-insensitive rate-limiting step and followed by a temperature-sensitive tension-generating step. These data and mechanism are compared and contrasted with the more complex length-jump Huxley-Simmons phases in which all states that generate tension or bear tension are perturbed. The rate of the Huxley-Simmons phase 4 is temperature sensitive at low temperatures but plateaus at high temperatures, indicating a change in rate-limiting step from a temperature-sensitive (phase 4a) to a temperature-insensitive reaction (phase 4b); the latter appears to correlate with the slow, temperature-insensitive temperature-jump relaxation. Phase 3 is absent in the temperature-jump, which excludes it from tension generation. We confirm that de novo tension generation occurs as an order-disorder transition during phase 2slow and the equivalent, temperature-sensitive temperature-jump relaxation. 相似文献
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The effect of ATP analogs on posthydrolytic and force development steps in skinned skeletal muscle fibers. 总被引:7,自引:1,他引:7
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ATP, 2-deoxy ATP (dATP), CTP, and UTP support isometric force and unloaded shortening velocity (Vu) to various extents (Regnier et al., Biophys. J. 74:3044-3058). Vu correlated with the rate of cross-bridge dissociation after the power stroke and the steady-state hydrolysis rate in solution, whereas force was modulated by NTP binding and cleavage. Here we studied the influence of posthydrolytic cross-bridge steps on force and fiber shortening by measuring isometric force and stiffness, the rate of tension decline (kPi) after Pi photogeneration from caged Pi, and the rate of tension redevelopment (ktr) after a sudden release and restretch of fibers. The slope of the force versus [Pi] relationship was the same for ATP, dATP, and CTP, but for UTP it was threefold less. ktr and kPi increased with increasing [Pi] with a similar slope for ATP, dATP, and CTP, but had an increasing magnitude of the relationship ATP < dATP < CTP. UTP reduced ktr but increased kPi. The results suggest that the rate constant for the force-generating isomerization increases with the order ATP < dATP < CTP < UTP. Simulations using a six-state model suggest that increasing the force-generating rate accounts for the faster kPi in dATP, CTP, and UTP. In contrast, ktr appears to be strongly affected by the rates of NTP binding and cleavage and the rate of the force-generating isomerization. 相似文献
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Strain-dependent modulation of phosphate transients in rabbit skeletal muscle fibers. 总被引:3,自引:0,他引:3
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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. 相似文献
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Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers 总被引:1,自引:1,他引:0
The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae. 相似文献
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Effect of joule temperature jump on tension and stiffness of skinned rabbit muscle fibers. 总被引:3,自引:1,他引:2
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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. 相似文献