Muscle cross-bridge kinetics in rigor and in the presence of ATP analogues.

Recently we reported preliminary mechanical experiments on freshly skinned rabbit psoas fibers that suggested that while almost all of the cross-bridges are attached to actin in the presence of 4 mM adenyl-5'-yl-imidodiphosphate (AMP-PNP) (ionic strength, 0.13 M), there is an equilibrium between the attached and detached states, so that, in the presence of 4 mM AMP-PNP, fibers should not be able to maintain tension (Schoenberg, et al., 1984, in Contractile Mechanisms in Muscle, Pollack and Sugi, editors., Plenum Publishing Corp., NY). Since this suggestion was at variance with published results of Clarke and Tregear (1982, FEBS [Fed. Eur. Biochem. Soc.] Lett, 143:217), we reinvestigated the ability of rabbit psoas fibers to support tension following a 2-nm stretch in rigor and in the presence of the nucleotide analogues, PPi and AMP-PNP, for analogue concentrations ranging from 0.25 to 4 mM. We find that, whereas in rigor there is very little tension decay following a stretch, in 4 mM nucleotide analogue solution, the force generated by stretch quickly decays to zero. The force decay is not exponential; rather, it can be described by rate constants that range from approximately 0.1 to 100 s-1 in 4 mM PPi, and 0.01 to 10 s-1 in 4 mM AMP-PNP. This large range of decay rate constants may be partially related to the dependence of either analogue binding or cross-bridge dissociation upon strain, since we find that the rate constants for force decay decrease with decreasing size of stretch or with decrease of analogue concentration below the maximum studied (4 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].     

Binding of ADP and ATP analogs to cross-linked and non-cross-linked acto X S-1

We previously determined the binding constants of ADP, adenylyl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to acto . myosin subfragment 1 (acto X S-1) by measuring the dissociation of acto X S-1 as a function of ATP analog concentration (Greene, L.E., and Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548). In the present study, we reinvestigated this question by measuring the extent to which these ATP analogs inhibit the acto X S-1 ATPase activity using both cross-linked actin X S-1 and non-cross-linked proteins. No significant difference was found between the cross-linked and non-cross-linked acto X S-1 complexes in their affinity for either ADP or AMP-PNP. The binding constant of ADP to acto X S-1 determined by the inhibition method was in excellent agreement with that obtained previously by the dissociation method, both techniques giving values of about 7 X 10(3) M-1. However, this was not the case for AMP-PNP and PPi, with the inhibition method giving about 10-fold weaker binding constants than those determined previously by the dissociation method. Upon redoing our dissociation experiments over a wider range of actin concentrations than we used previously, we now find that the dissociation method gives much weaker values for the binding constants of PPi and AMP-PNP to acto X S-1, i.e. values on the order of 4 X 10(2) M-1. The very weak binding of these ATP analogs to acto X S-1 makes it difficult to obtain these values with great accuracy. Nevertheless, they seem to be in good agreement with the binding constants determined by the inhibition method. The weak binding of AMP-PNP and PPi to acto X S-1 is consistent with the recent fiber studies of Pate and Cooke (Pate, E., and Cooke, R. (1985) Biophys. J. 47, 773-780) and Schoenberg and Eisenberg (Schoenberg, M., and Eisenberg, E. (1986) Biophys. J. 48, 863-872).     

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.     

Ca2+-sensitive cross-bridge dissociation in the presence of magnesium pyrophosphate in skinned rabbit psoas fibers.

We find that at 6 degrees C in the presence of 4 mM MgPPi, at low or moderate ionic strength, skinned rabbit psoas fibers exhibit a stiffness and an equatorial x-ray diffraction pattern similar to that of rigor fibers. As the ionic strength is increased in the absence of Ca2+, both the stiffness and the equatorial x-ray diffraction pattern approach those of the relaxed state. This suggests that, as in solution, increasing ionic strength weakens the affinity of myosin cross-bridges for actin, which results in a decrease in the number of cross-bridges attached. The effect is Ca2+-sensitive. Assuming that stiffness is a measure of the number of cross-bridge heads attached, in the absence of Ca2+, the fraction of attached cross-bridge heads varies from approximately 75% to approximately 25% over an ionic strength range where ionic strength in solution weakens the binding constant for myosin subfragment-1 binding to unregulated actin by less than a factor of 3. Therefore, this phenomenon appears similar to the cooperative Ca2+-sensitive binding of S1 to regulated actin in solution (Greene, L. E., and E. Eisenberg, 1980, Proc. Natl. Acad. Sci. USA, 77:2616). By comparing the binding constants in solution and in the fiber under similar conditions, we find that the "effective actin concentration," that is, the concentration that gives the same fraction of S1 molecules bound to actin in solution as cross-bridge heads are bound to actin in a fiber, is in the millimolar range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dissociation of the actin.subfragment 1 complex by adenyl-5'-yl imidodiphosphate, ADP, and PPi

The ability of adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP, and PPi to dissociate the actin.myosin subfragment 1 (S-1) complex was studied using an analytical ultracentrifuge with UV optics, which enabled the direct determination of the dissociated S-1. At mu = 0.22 M, pH 7.0, 22 degrees C, with saturating nucleotide present, ADP weakens the binding of S-1 to actin about 40-fold (K congruent to 10(5) M-1), while both AMP-PNP and PPi weakens the binding about 400-fold (K congruent to 10(4) M-1). This 10-fold stronger dissociating effect of AMP-PNP and PPi compared to ADP correlates with our data showing that the binding of AMP-PNP and PPi to S-1 is about 10-fold stronger than the binding of ADP. In contrast, the binding constants of ADP, AMP-PNP, and PPi to acto.S-1 are nearly identical (K congruent to 5 x 10(3) M-1). At 4 degrees C, AMP-PNP has only a 3-fold stronger dissociating effect than ADP and, similarly, our data suggest that the binding of AMP-PNP and ADP to S-1 is quite similar at 4 degrees C. AMP-PNP and PPi are, therefore, somewhat better dissociating agents than ADP, but the difference among these three ligands is quite small. These data also show that actin and nucleotide bind to separate but interacting sites on S-1 and that the S-1 molecules bind independently along the F-actin filament with a binding constant of about 1 x 10(7) M-1 at 22 degrees C and physiological ionic strength.     

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.     

Binding of ADP and 5'-adenylyl imidodiphosphate to rabbit muscle myofibrils

The binding of [3H]ADP and [3H]adenyl-5'-yl-imidodiphosphate ([3H]AMP-PNP) to rabbit skeletal myofibrils was measured at 25 and 7 degrees C, mu = 0.12 M, using [14C]mannitol as a volume marker. We found that ADP bound to myosin heads in overlap with a binding constant of about 10(4) M-1, similar to the value we previously obtained in vitro with acto.S-1. The binding of AMP-PNP to myosin heads was measured both in and out of overlap. The affinity of AMP-PNP to the heads out of overlap was similar to that obtained in vitro with S-1 alone. The binding of AMP-PNP to the myosin heads in overlap was much weaker. We could fit these data with a binding constant of about 1 x 10(3) M-1, assuming a single population of cross-bridges and 1 mol of AMP-PNP bound per mol of myosin head. This value was reduced by a factor of 2 when we corrected for nonspecific binding. It was also possible to fit the data assuming two equal populations of cross-bridges with one of the populations binding AMP-PNP about 5-fold more strongly than the other population. Therefore, for at least half of the cross-bridges in overlap, the binding of AMP-PNP is almost as weak as the value of 3 x 10(2) M-1 we previously measured for the acto.S-1 complex in vitro (Biosca, J. A., Greene, L. E., and Eisenberg, E. (1986) J. Biol. Chem. 261, 9793-9800).     

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.     

Cross-bridge behavior in rigor muscle

Properties of the rigor state in muscle can be explained by a simple cross-bridge model, of the type which has been suggested for active muscle, in which detachment of cross-bridges by ATP is excluded. Two attached cross-bridge states, with distinct force vs. distortion relationships, are required, in addition to a detached state, but the attached cross-bridge states in rigor muscle appear to differ significantly from the attached cross-bridge states in active muscle. The stability of the rigor force maintained in muscle under isometric conditions does not require exceptional stability of the attached cross-bridges, if the positions in which attachment of cross-bridges is allowed are limited so that the attachment of cross-bridges in positions which have minimum free energy is excluded. This explanation of the stability of the rigor state may also be applicable to the maintenance of stable rigor waves on flagella.     

Equilibrium muscle cross-bridge behavior. Theoretical considerations.

We have developed a model for the equilibrium attachment and detachment of myosin cross-bridges to actin that takes into account the possibility that a given cross-bridge can bind to one of a number of actin monomers, as seems likely, rather than to a site on only a single actin monomer, as is often assumed. The behavior of this multiple site model in response to constant velocity, as well as instantaneous stretches, was studied and the influence of system parameters on the force response explored. It was found that in the multiple site model the detachment rate constant has considerably greater influence on the mechanical response than the attachment rate constant. It is shown that one can obtain information about the detachment rate constants either by examining the relationship between the apparent stiffness and duration of stretch for constant velocity stretches or by examining the force-decay rate constants following an instantaneous stretch. The main effect of the attachment rate constant is to scale the mechanical response by influencing the number of attached cross-bridges. The significance of the modeling for the interpretation of experimental results is discussed.     

Backward movements of cross-bridges by application of stretch and by binding of MgADP to skeletal muscle fibers in the rigor state as studied by x-ray diffraction

The effects of the applied stretch and MgADP binding on the structure of the actomyosin cross-bridges in rabbit and/or frog skeletal muscle fibers in the rigor state have been investigated with improved resolution by x-ray diffraction using synchrotron radiation. The results showed a remarkable structural similarity between cross-bridge states induced by stretch and MgADP binding. The intensities of the 14.4- and 7.2-nm meridional reflections increased by approximately 23 and 47%, respectively, when 1 mM MgADP was added to the rigor rabbit muscle fibers in the presence of ATP-depletion backup system and an inhibitor for muscle adenylate kinase or by approximately 33 and 17%, respectively, when rigor frog muscle was stretched by approximately 4.5% of the initial muscle length. In addition, both MgADP binding and stretch induced a small but genuine intensity decrease in the region close to the meridian of the 5.9-nm layer line while retaining the intensity profile of its outer portion. No appreciable influence was observed in the intensities of the higher order meridional reflections of the 14.4-nm repeat and the other actin-based reflections as well as the equatorial reflections, indicating a lack of detachment of cross-bridges in both cases. The changes in the axial spacings of the actin-based and the 14.4-nm-based reflections were observed and associated with the tension change. These results indicate that stretch and ADP binding mediate similar structural changes, being in the correct direction to those expected for that the conformational changes are induced in the outer portion distant from the catalytic domain of attached cross-bridges. Modeling of conformational changes of the attached myosin head suggested a small but significant movement (about 10-20 degrees) in the light chain-binding domain of the head toward the M-line of the sarcomere. Both chemical (ADP binding) and mechanical (stretch) intervensions can reverse the contractile cycle by causing a backward movement of this domain of attached myosin heads in the rigor state.     

Effect of ionic strength on skinned rabbit psoas fibers in the presence of magnesium pyrophosphate.

The effect of ionic strength on the kinetics of myosin cross-bridges in the presence of the ATP analogue PP, has been examined. It was found that increasing ionic strength from moderate values (mu approximately 100 mM) to high values (mu approximately 200 mM) has three effects. It causes a big decrease in the half time for the force decay after a small stretch, it causes a significant decrease in the sigmoidicity of the nucleotide analogue concentration dependence of the "apparent rate constant" of force decay after a small stretch, and it causes a big decrease in the range of rate constants necessary to describe the multiexponential force decay. It causes the last of these by causing a much larger increase in the slowest rate constants of the decay than in the fastest rate constants. The results suggest that whereas the behavior of cross-bridges in the presence of ATP is well-described by the simple independent-head equilibrium cross-bridge model of Schoenberg (1985. Biophys. J. 48:467-475), cross-bridges in the presence of the ATP analogue PPi require the more complicated double-headed equilibrium cross-bridge model of Anderson and Schoenberg (1987. Biophys. J. 52: 1077-1082) to describe their behavior.     

The interaction of adenyl-5'-yl imidodiphosphate and PPi with actomyosin

We previously studied the equilibrium binding of ADP, adenyl-5'-yl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to actomyosin-subfragment 1 (acto.S-1) and found that AMP-PNP and PPi bind considerably more weakly to acto.S-1 than does ADP. In this study, we investigated the pre-steady-state kinetics of the binding of AMP-PNP and PPi to acto.S-1 and of S-1.AMP-PNP and S-1.PPi to actin to determine if the pre-steady-state kinetic data are consistent with our previous equilibrium data. We find that the kinetic data are consistent with the equilibrium data and agree with a model in which acto.S-1 forms a collision intermediate with the ATP analog, followed by a slower conformational change to a ternary complex that rapidly dissociates into actin and the S-1.ATP analog. Although this scheme fits the AMP-PNP as well as the PPi data, we find that the isomerization of the collision intermediate to the ternary complex is approximately 10 times faster in the presence of PPi than in the presence of AMP-PNP, which is consistent with previous physiological studies (Schoenberg, M., and Eisenberg, E. (1985) Biophys. J. 48, 863-872).     

Effects of AMPPNP on the orientation and rotational dynamics of spin-labeled muscle cross-bridges.

We have used electron paramagnetic resonance (EPR) to investigate the orientation, rotational motion, and actin-binding properties of rabbit psoas muscle cross-bridges in the presence of the nonhydrolyzable nucleotide analogue, 5'-adenylylimido-diphosphate (AMPPNP). This analogue is known to decrease muscle tension without affecting its stiffness, suggesting an attached cross-bridge state different from rigor. We spin-labeled the SH1 groups on myosin heads and performed conventional EPR to obtain high-resolution information about the orientational distribution, and saturation transfer EPR to measure microsecond rotational motion. At 4 degrees C and 100 mM ionic strength, we find that AMPPNP increases both the orientational disorder and the microsecond rotational motion of myosin heads. However, computer analysis of digitized spectra shows that no new population of probes is observed that does not match either rigor or relaxation in both orientation and motion. At 4 degrees C, under nearly saturating conditions of 16 mM AMPPNP (Kd = 3.0 mM, determined from competition between AMPPNP and an ADP spin label), 47.5 +/- 2.5% of myosin heads are dynamically disoriented (as in relaxation) without a significant decrease in rigor stiffness, whereas the remainder are rigidly oriented as in rigor. The oriented heads correspond to actin-attached heads in a ternary complex, and the disoriented heads correspond to detached heads, as indicated by EPR experiments with spin-labeled subfragment 1 (S1) that provide independent measurements of orientation and binding. We take these findings as evidence for a single-headed cross-bridge that is as stiff as the double-headed rigor cross-bridge. The data are consistent with a model in which, in the presence of saturating AMPPNP, one head of each cross-bridge binds actin about 10 times more weakly, whereas the remaining head binds at least 10 times more strongly, than extrinsic S1. Thus, although there is no evidence for heads being attached at nonrigor angles, the attached cross-bridge differs from that of rigor. The heterogeneous behavior of heads is probably due to steric effects of the filament lattice.     

Effect of ethylene glycol and Ca2+ on the binding of Mg2+ x adenyl-5'-yl imidodiphosphate to rabbit skeletal myofibrils

The binding of Mg2+ X adenyl-5'-yl imidodiphosphate (Mg2+ X AMP-PNP) to rabbit skeletal myofibrils has been measured in aqueous solution and in 50% ethylene glycol in the presence and absence of Ca2+. In water, the observed binding was weak with less than half the calculated myosin active sites filled even at 1 mM Mg2+ X AMP-PNP. In 50% ethylene glycol, the binding is at least 100-fold tighter and extrapolates to the expected number of binding sites. This is contrasted to the small change seen for Mg2+ X ADP binding between the same sets of conditions. This difference between Mg2+ X AMP-PNP and Mg2+ X ADP is attributed to the strong coupling of Mg2+ X AMP-PNP binding to dissociation of myosin cross-bridges. The Ca2+ sensitivity of Mg2+ X AMP-PNP binding in 50% ethylene glycol is taken as further evidence of the thermodynamic coupling of Mg2+ X AMP-PNP binding to cross-bridge dissociation. In addition, the binding of Mg2+ X AMP-PNP in 50% ethylene glycol is biphasic while Mg2+ X ADP binding under the same conditions is not. The biphasic Mg2+ X AMP-PNP binding could be caused by either the presence of two or more classes of cross-bridges or by negative cooperativity, but the presence of only a single class of Mg2+ X ADP-binding sites implies that if multiple classes of sites are involved, they do not simply differ in steric hindrance or accessibility of the binding site as a whole. The importance of using purified AMP-PNP in the study of actomyosin X AMP-PNP complexes is discussed.     

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.     

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.     

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.