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.     

The role of ATP in energy-deprivation contractures in unloaded rat ventricular myocytes

Energy-deprivation contractures were investigated in unloaded rat ventricular myocytes. Application of 2 mM cyanide in the presence of 10 mM 2-deoxyglucose (metabolic blockade) led to a rapid shortening "contracture" (maximum speed 1.5 +/- 0.2% control cell length/s). Cells shortened to a constant length of 69 +/- 1.6% of the control length. Removal of cyanide caused cells to shorten further ("recontracture"), before relaxing towards the control length. Cells shortened to 57 +/- 2.0% during the recontracture. Similar behaviour was observed in zero extracellular [Ca2+]. Cells permeabilized with saponin (0.1% w/v) responded to the removal of ATP from the bathing solution, and to readdition of ATP, as intact cells did to complete metabolic blockade and its removal. In these permeabilized cells, the extent and speed of contracture shortening were similar at pCa = 7 and pCa greater than 9. When the bath concentration of ATP ([ ATP]b) was lowered to zero, shortening stopped at about 70% of the control length. However, when [ATP]b was lowered to an intermediate level (4-20 microM), cells contracted to lengths as short as 30% of the control length. Similarly, when [ATP]b was restored from zero to an intermediate concentration (4-20 microM), recontracture shortening continued without relaxation. The peak speed of this Ca2(+)-independent shortening showed a sigmoidal dependence on pMgATP (pMgATP0.5 = 4.0). Phosphocreatine (10 mM) shifted the ATP dependence of Ca2(+)-independent shortening to lower [ATP]b (pMgATP0.5 = 5.0), suggesting that gradients of [ATP] could exist between the bath and the myofilaments. Ca2(+)-independent shortening was inhibited by the chemical phosphatase 2,3-butanedione monoxime (BDM), although BDM did not relax cells from the shortened state during energy deprivation. Using a simple model, we show that the results can be explained by cross-bridge cycling occurring independently of Ca2+ over a "window" range of [MgATP] (0.1-100 microM). Therefore, when [MgATP] falls, cross-bridge cycling occurs and the cell shortens. As [MgATP] falls to very low levels ([ MgATP] less than 1 microM), shortening ceases as the rate of cross-bridge cycling declines. Recontracture occurs on restoring ATP production, because stiffness falls and Ca2(+)-independent cross-bridge cycling initially increases. As [MgATP] rises above 100 microM, Ca2(+)-independent cross-bridge cycling ceases and the cell relaxes towards the control length. We conclude that energy-deprivation contractures, and recontractures, can result from changes in [MgATP] and do not necessarily require changes in [Ca2+]i.     

Contraction of rabbit skinned skeletal muscle fibers at low levels of magnesium adenosine triphosphate

The contractile properties of skinned single fibers from rabbit psoas muscle were investigated under conditions of low MgATP and no Ca2+ (i.e., less than 10(-8) M). At 1 microM MgATP, fibers shortened at a maximum velocity of 660 +/- 420 A/half sarcomere/s (n = 9), compared with 34,000 A/half sarcomere/s measured during maximum Ca2+-activation at 1 mM MgATP (Moss, R. L., 1982. J. Muscle Res. Cell. Motil ., 3:295-311). The observed dependence of Vmax on pMgATP between 7.0 and 5.3 was similar to that of actomyosin ATPase measured previously by Weber, A., R. Herz , and I. Reiss (1969, Biochemistry, 8:2266-2270). Isometric tension was found to vary with pMgATP in a manner much like that reported by Reuben , J. P., P. W. Brandt, M. Berman , and H. Grundfest (J. Gen. Physiol. 1971. 57:385-407). A simple cross-bridge model was developed to simulate contractile behaviour at both high and low levels of MgATP. It was found that the pMgATP dependence of Vmax and ATPase could be successfully modeled if the rate of detachment of the cross-bridge was made proportional to the concentration of MgATP. In the model, the similar dependence of Vmax and ATPase on pMgATP was derived from the fact that in this range of pMgATP every pass of a cross-bridge by an actin site resulted in an attachment-detachment cycle, and every such cycle caused hydrolysis of one molecule of ATP.     

The inhibition of muscle contraction by adenosine 5' (beta, gamma-imido) triphosphate and by pyrophosphate.

We have studied the inhibition of the contraction of glycerinated rabbit psoas muscle caused by ligands that bind to the ATPase site of myosin. Two ligands, adenosine 5' (beta, gamma-imido) triphosphate (AMPPNP) and pyrophosphate (PPi), decreased the force and stiffness developed in isometric contractions and the velocity of shortening of isotonic contractions. The force exerted by isometric fibers was measured as a function of MgATP in the presence and absence of a constant concentration of the ligands. As the MgATP concentration decreased, the inhibition of tension caused by the ligand increased, reaching approximately 50% at 25 microM MgATP and either 2 mM MgPPi or 2 mM MgAMPPNP. The maximum velocity of shortening was also measured as a function of MgATP concentration in the presence of 1 and 2 mM MgPPi and 2.5 and 5 mM MgAMPPNP. Both ligands acted as pure competitive inhibitors with Ki = 3.0 mM for PPi and 5.1 mM for MgAMPPNP. These data show that both ligands are weak inhibitors of the contraction of fibers. The results provided information on the energetics of actin-myosin-ligand states that occur in the portion of the cross-bridge cycle where MgATP binds to myosin. A simple analysis of the inhibition of velocity suggests that MgAMPPNP binds to the actomyosin complex at this step of the cycle with an effective affinity constant of approximately 2 X 10(2) M-1.     

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.     

Calcium regulation of tension redevelopment kinetics with 2-deoxy-ATP or low [ATP] in rabbit skeletal muscle.

The correlation of acto-myosin ATPase rate with tension redevelopment kinetics (k(tr)) was determined during Ca(+2)-activated contractions of demembranated rabbit psoas muscle fibers; the ATPase rate was either increased or decreased relative to control by substitution of ATP (5.0 mM) with 2-deoxy-ATP (dATP) (5.0 mM) or by lowering [ATP] to 0.5 mM, respectively. The activation dependence of k(tr) and unloaded shortening velocity (Vu) was measured with each substrate. With 5.0 mM ATP, Vu depended linearly on tension (P), whereas k(tr) exhibited a nonlinear dependence on P, being relatively independent of P at submaximum levels and rising steeply at P > 0.6-0.7 of maximum tension (Po). With dATP, Vu was 25% greater than control at Po and was elevated at all P > 0.15Po, whereas Po was unchanged. Furthermore, the Ca(+2) sensitivity of both k(tr) and P increased, such that the dependence of k(tr) on P was not significantly different from control, despite an elevation of Vu and maximal k(tr). In contrast, lowering [ATP] caused a slight (8%) elevation of Po, no change in the Ca(+2) sensitivity of P, and a decrease in Vu at all P. Moreover, k(tr) was decreased relative to control at P > 0.75Po, but was elevated at P < 0.75Po. These data demonstrate that the cross-bridge cycling rate dominates k(tr) at maximum but not submaximum levels of Ca(2+) activation.     

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

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

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

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

Substrate and product dependence of force and shortening in fast and slow smooth muscle

To explore the molecular mechanisms responsible for the variation in smooth muscle contractile kinetics, the influence of MgATP, MgADP, and inorganic phosphate (P(i)) on force and shortening velocity in thiophosphorylated "fast" (taenia coli: maximal shortening velocity Vmax = 0.11 ML/s) and "slow" (aorta: Vmax = 0.015 ML/s) smooth muscle from the guinea pig were compared. P(i) inhibited active force with minor effects on the V(max). In the taenia coli, 20 mM P(i) inhibited force by 25%. In the aorta, the effect was markedly less (< 10%), suggesting differences between fast and slow smooth muscles in the binding of P(i) or in the relative population of P(i) binding states during cycling. Lowering of MgATP reduced force and V(max). The aorta was less sensitive to reduction in MgATP (Km for Vmax: 80 microM) than the taenia coli (Km for Vmax: 350 microM). Thus, velocity is controlled by steps preceding the ATP binding and cross-bridge dissociation, and a weaker binding of ATP is not responsible for the lower V(max) in the slow muscle. MgADP inhibited force and V(max). Saturating concentrations of ADP did not completely inhibit maximal shortening velocity. The effect of ADP on Vmax was observed at lower concentrations in the aorta compared with the taenia coli, suggesting that the ADP binding to phosphorylated and cycling cross-bridges is stronger in slow compared with fast smooth muscle.     

Kinetic studies on mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

The substrate kinetics and the role of free Mg(2+) and free ATP were studied in membrane-bound F(1)-ATPase from crayfish (Orconectes virilis) gills. It was shown that the MgATP complex was the true substrate for the ATPase activity with a K(m) value of 0.327 mM. In the absence of bicarbonate, the maximum azide-sensitive activities in the presence and absence (<18 microM) of free ATP were 0.878 and 0.520 micromol P(i)/mg protein/min, respectively, while the maximum bicarbonate-stimulated activity in absence of free ATP was 1.486 micromol P(i)/mg protein/min. Free ATP was a competitive inhibitor (K(i)=0.77 mM) and free Mg(2+) was a mixed inhibitor (K(i)=0.81 mM, K(i)'=5.89 mM). However, free ATP also acted as an activator. Lineweaver-Burk plots for MgATP hydrolysis at high free Mg(2+) concentrations exhibited an apparent negative cooperativity, which was not the case for high free ATP levels. These results suggest that, although free ATP inhibited the enzyme by binding to catalytic sites, it stimulated ATPase activity by binding to non-catalytic sites and promoted the dissociation of inhibitory MgADP from the catalytic site.     

Effect of denervation on ATP consumption rate of diaphragm muscle fibers.

Denervation (DNV) of rat diaphragm muscle (DIAm) decreases myosin heavy chain (MHC) content in fibers expressing MHC(2X) isoform but not in fibers expressing MHC(slow) and MHC(2A). Since MHC is the site of ATP hydrolysis during muscle contraction, we hypothesized that ATP consumption rate during maximum isometric activation (ATP(iso)) is reduced following unilateral DIAm DNV and that this effect is most pronounced in fibers expressing MHC(2X). In single-type-identified, permeabilized DIAm fibers, ATP(iso) was measured using NADH-linked fluorometry. The maximum velocity of the actomyosin ATPase reaction (V(max) ATPase) was determined using quantitative histochemistry. The effect of DNV on maximum unloaded shortening velocity (V(o)) and cross-bridge cycling rate [estimated from the rate constant for force redevelopment (k(TR)) following quick release and restretch] was also examined. Two weeks after DNV, ATP(iso) was significantly reduced in fibers expressing MHC(2X), but unaffected in fibers expressing MHC(slow) and MHC(2A). This effect of DNV on fibers expressing MHC(2X) persisted even after normalization for DNV-induced reduction in MHC content. With DNV, V(o) and k(TR) were slowed in fibers expressing MHC(2X), consistent with the effect on ATP(iso). The difference between V(max) ATPase and ATP(iso) reflects reserve capacity for ATP consumption, which was reduced across all fibers following DNV; however, this effect was most pronounced in fibers expressing MHC(2X). DNV-induced reductions in ATP(iso) and V(max) ATPase of fibers expressing MHC(2X) reflect the underlying decrease in MHC content, while reduction in ATP(iso) also reflects a slowing of cross-bridge cycling rate.     

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.     

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP

The chemomechanicalcoupling mechanism in striated muscle contraction was examined bychanging the nucleotide substrate from ATP to CTP. Maximum shorteningvelocity [extrapolation to zero force from force-velocity relation(V_max) andslope of slack test plots (V₀)], maximumisometric force (P_o), power, andthe curvature of the force-velocity curve[a/P_o(dimensionless parameter inversely related to the curvature)] weredetermined during maximumCa²⁺-activated isotoniccontractions of fibers from fast rabbit psoas and slow rat soleusmuscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTPas the nucleotide substrate. In addition to a decrease in the maximumCa²⁺-activated force in both fibertypes, a change from 4 mM ATP to 10 mM CTP resulted in a decrease inV_max in psoasfibers from 3.26 to 1.87 muscle length/s. In soleus fibers,V_max was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by thechange in nucleotide as the result of a three- to fourfold decrease inthe curvature of the force-velocity relationship. The results areinterpreted in terms of the Huxley model of muscle contraction as anincrease in f₁and g₁ coupled toa decrease in g₂(where f₁ is therate of cross-bridge attachment and g₁ andg₂ are rates ofdetachment) when CTP replaces ATP. This adequately accounts for theobserved changes in P_o,a/P_o,and V_max.However, the two-state Huxley model does not explicitly reveal thecross-bridge transitions that determine curvature of the force-velocityrelationship. We hypothesize that a nucleotide-sensitive transitionamong strong-binding cross-bridge states followingP_i release, but before the release of the nucleotide diphosphate, underlies the alterations ina/P_o reported here.

     

Investigation of soluble mitochondrial ATPase by the reacting enzyme sedimentation method.

Soluble mitochondrial ATPase from bovine heart (factor F1) loses its activity during ATP hydrolyses. The inactivation is accelerated by moderate pressure, which is generated in an ultracentrifuge cell. The rate of inactivation slows down if the concentration of the substrate (MgATP) is diminished. ATP hydrolysis proceeds at an almost constant rate if the substrate concentration is as low as 0.05 mM. One intersubunit cross-link formed by dimethylsuberimidate per molecule of factor F1, prevents its inactivation during the ATPase reaction both without pressure and in an ultracentrifuge. Sedimentation coefficients measured by the reacting enzyme centrifugation method of both unmodified factor F1 at a low (about 0.05 mM MgATP) substrate concentration and of its dimethylsuberimidate cross-linked form in the presence of 10 mM MgATP, were determined to be s20, w = 12.4 +/- 0.4 S. The value is the same as that obtained by the conventional boundary sedimentation method in the absence of the substrate. This result testifies to the fact that the conformation of reacting factor F1 in solution is similar to that of the enzyme in the absence of the substrate.     

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.     

Specific enhancement of the cardiac myofibrillar ATPase by bound creatine kinase.

The kinetic influence of bound creatine kinase (CK) on the Ca(2+)-activated myosin ATPase was evaluated. ATPase rates were measured from 0.8 microM to 3.2 mM MgATP. Under control conditions, the apparent KmATP was 79.9 +/- 13.3 microM. In contrast, the addition of 12.2 mM phosphocreatine (PCr) decreased the apparent KmATP to a value of 13.6 +/- 1.4 microM. To determine if this reduction was merely the result of an ATP maintenance system, ATP was regenerated using either phosphoenolpyruvate and pyruvate kinase (PEP-PK), or PCr and soluble bovine cardiac CK. Data obtained with PEP + PK indicated an apparent KmATP of 65.5 +/- 7.3 microM. To study the effects of exogenous CK, the endogenous CK was irreversibly inhibited with 1 mM iodoacetamide. The kinetics of the ATPase were then examined by adding soluble CK to the incubation medium. Under these conditions, the KmATP was 56.4 +/- 0.86 microM. Therefore, these two ATP regeneration systems could not duplicate the effects of endogenous CK. The reduction of the apparent KmATP by endogenous CK was not the result of an altered inhibition by MgADP. MgADP inhibition was determined to be non-competitive, with a Ki of 5.0 +/- 0.1 mM. These data suggest that the observed kinetic effects reflect the proximity of the enzymes in the myofibrillar bundle, thus emphasizing the importance of bound CK for the localized regeneration of MgATP utilized by the myosin ATPase.     

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.     

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.     

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis.

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strain-dependent cross-bridge cycle for muscle. II. Steady-state behavior.

Quantitative predictions of steady-state muscle properties from the strain-dependent cross-bridge for muscle are presented. With a stiffness of 5.4 x 10(-4) N/m per head, a throw distance of 11 nm, and three allowed actin sites/head, isometric properties and their dependence on phosphate and nucleotide levels are well described if the tension-generating step occurs before phosphate release. At very low ATP levels, rigorlike states with negative strain are predicted. The rate-limiting step for cycling and ATP consumption is strain-blocked ADP release for isometric and slowly shortening muscle. Under rapid shortening, ATP hydrolysis on detached heads is the rate-limiting step, and the ratio of bound ATP to bound ADP.Pi increases by a factor of 7. At large positive strains, bound heads must be forcibly detached from actin to account for tension in rapid extension, but forced detachment in shortening has no effect without destroying isometric attached states. Strain-blocked phosphate release as proposed produces modest inhibition of the ATPase rate under rapid shortening, sufficient to give a maximum for one actin site per helix turn. Alternative cross-bridge models are discussed in the light of these predictions.