Structure of the actin-myosin complex produced by crosslinking in the presence of ATP.

The structure of the actin-myosin complex during ATP hydrolysis was studied by covalently crosslinking myosin subfragment 1 (S1) to F-actin in the presence of nucleotides (especially ATP) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The fluorescence energy transfer was measured between N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine and 6-(iodoacetamide)fluorescein bound to the SH1 thiol of S1 and the Cys374 thiol of actin. The covalent acto-S1, produced by crosslinking in the absence of nucleotide or in the presence of ADP, showed transfer efficiency of 0.50 to 0.52 and intersite distance of 4.5 to 4.7 nm, which were equal to those obtained with non-crosslinked acto-S1 in the absence of nucleotide. However, the covalent acto-S1, produced by crosslinking in the presence of either 5'-adenylyl imidodiphosphate (AMPPNP) at high ionic strength or ATP, showed a significant decrease in the efficiency to 0.26 to 0.34 and hence an increase in the distance to 5.2 to 5.5 nm. These results suggest that AM-ATP and/or AM-ADP-P (formed during ATP hydrolysis) and AM-AMPPNP have a very different conformation from AM and AM-ADP (in which A is actin and M is myosin).

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers.

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.

Interaction of nonpolymerizable actins with myosin.

Polymerization of G-actin in the presence of salt and phalloidin was blocked by treatment of G-actin with m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) (designated as m-actin). The actin dimer produced by chemical crosslinking of F-actin with N,N'-p-phenylenedimaleimide did not polymerize and was still dimeric or tetrameric after further treatment with MBS (designated as d-actin). The m- and d-actins retained the ability to bind to myosin heads with apparent dissociation constants of 3-8 x 10(-6) and 3-5 x 10(-7) M, respectively. d-Actin formed a 1:1 actin monomer-myosin head complex. However, m-actin formed a 2:1 m-actin-head complex, suggesting the presence of at least two latent actin-binding sites on a myosin head. ATP weakens only 2- to 6-fold the binding of these complexes. One of two m-actins on a myosin head was replaced by d-actin. Native F-actin blocked the binding of both m- and d-actins to myosin heads in the presence and absence of ATP, although the affinities of myosin head for MBS-treated actins and F-actin are similar in the presence of ATP. These results suggest that there are at least three actin binding sites on a myosin head: one is responsible for binding of F-, m-, and d-actins, the second for binding of F- and m-actins, and the third for binding of F-actin at least in the presence of ATP. F-Actin binding to the third site may in some way block the first and second binding sites.

Chemical crosslinking of myosin subfragment-1 to F-actin in the presence of nucleotide.

F-Actin and myosin subfragment-1 (S-1) were covalently crosslinked in the absence and presence of nucleotides, adenyl-5'-yl imidodiphosphate (AMPPNP), ATP, and ADP, at various KCl concentrations (0-0.5 M), using a zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The rate of production of the covalent acto-S-1 complex was almost proportional to the amount of the acto-S-1(-nucleotide) complex existing in the reaction medium. However, the Mg-ATPase activity of the covalent acto-S-1 complex thus produced was affected by the presence of nucleotides. When S-1 was crosslinked to F-actin in the absence of nucleotide at 0-0.5 M KCl, the Mg-ATPase activity of S-1 was enhanced from 0.1-0.3 to 12-15 s-1. The Mg-ATPase activity of covalent complex produced in the presence of AMPPNP at 0 M KCl was 13 s-1 which was equal to that produced in the absence of nucleotide. The activity decreased with increasing KCl concentration of the crosslinking medium, reaching 6 s-1 at 0.5 M KCl. Covalent acto-S-1 complex was also produced when acto-S-1-ADP-P was formed during ATP hydrolysis. The Mg-ATPase activity of the covalent acto-S-1 complex crosslinked during ATP hydrolysis at 0 M KCl was about 5 s-1 which was about half of that obtained in the absence of nucleotide, and it decreased to about 2 s-1 at 0.15 M KCl of crosslinking medium. In contrast, the acto-S-1-ADP complex gave a covalent complex which was indistinguishable in its extent and Mg-ATPase activity from that obtained in the absence of nucleotide. These results suggest that the structures of acto-S-1-AMPPNP and acto-S-1-ADP-P complexes are different from those of acto-S-1 and acto-S-1-ADP complexes.

Factors affecting the transient tension change after applying stepwise length change to glycerol-treated muscle fibers. Effects of temperature, divalent cations, and modification with p-chloromercuribenzoate.

The dynamic properties of cross-bridge movement were investigated in glycerol-treated muscle fibers under various conditions by analyzing tension responses to two types of length change. First, the fiber bundles were stretched linearly with time for 0.3 s from the rest length (L0) by 2.5% of L0, suddenly released, then fixed at L0 (sudden release of the slow stretch). Second, they were stretched for 0.01 s by 2.5% of L0, then held at the plateau length (a quick stretch). 1. The transient tension responses following both length changes were divided into three phases: (i) very quick recovery of tension (0 approximately 0.05 s), (ii) quick recovery (0.05 approximately 0.3-0.4 s), and (iii) gradual recovery (0.3-0.4 s approximately several seconds). 2. The effects of activating conditions on the rates of the quick phases (0 approximately 0.3-0.4 s) were not associated with those on the nucleoside triphosphatase [EC 3.6.1.3] rates: the rates of the quick phases increased with increase in temperature and Mg2+-ATP concentration, with decrease in Ca2+ concentration, and also on replacement of Mg2+-ATP by Mg2+-ITP or Mn2+-ATP. Only a small amount of ADP, 0.07 mol per mol of myosin (Fig. 24 in the preceding paper), was liberated during the quick recovery phases. 3. The remaining slow tension recovery was concluded to be associated with one cycle of ATP splitting, and progressed very smoothly. This suggests that most of the cross-bridges do not exist in a synchronously dissociated state during one cycle of ATP splitting.

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.

Effect of tryptic digestion of myosin subfragment-1 on its binding to F-actin.

We investigated the effect of tryptic digestion of S-1 (95K) into three segments (27K, 50K, and 20K) on the binding of F-actin with S-1, using an ultracentrifugal separation method and a light-scattering method. The tryptic digestion of S-1 decreased the affinity of S-1 for F-actin both in the absence of nucleotide and the presence of AMPPNP or ATP, suggesting that the peptide cutting impairs the structures participating in the binding of S-1 or S-1-nucleotide complex with F-actin. Although nucleotides markedly weakened the affinity of S-1 for F-actin, the ratios of affinity of digested S-1 for F-actin to that of intact S-1 were all about 1/10 both in the absence and presence of nucleotides. This may be understood if we accept the assumption that two kinds of structure participate in the binding of S-1 with F-actin; one is independent of nucleotides and the other is dependent on them, with only the former being affected by the tryptic digestion of S-1.

Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions.

The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.

Reaction of two heads of gizzard myosin with ATP.

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of two heads of myosin with F-actin: binding of H-meromyosin with F-actin in the absence of nucleotide.

The bindings of S-1 and the two heads of HMM with pyrene-labeled F-actin were studied using the change in light-scattering intensity or that in the fluorescence intensity of the pyrenyl group. At low ionic strength (50 mM KCl), both S-1 and HMM became bound tightly with F-actin (Kd less than 0.1 microM) and both heads of HMM became bound to F-actin. The affinities of S-1 and HMM for F-actin decreased with increasing KCl concentration. In 1 M KCl, the Kd values of S-1 and HMM for F-actin were 11 and 0.58 microM, respectively. Thus, HMM was bound to F-actin 19 times more tightly than S-1. We compared the extent of binding of HMM to F-actin measured by a centrifugation method with that measured by the fluorescence change of pyrenyl-group, and found that even in 1 M KCl, HMM became bound to F-actin with a two-headed attachment. We measured the kinetics of binding and dissociation of acto-S-1 and acto-HMM from the time course of the change in light-scattering intensity after mixing S-1 or HMM with F-actin at 1 M KCl and that after mixing 1 M KCl with acto-S-1 or acto-HMM formed at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of myosin and its subfragment-1 with antibodies specific to the two heads of the myosin molecule.

It was shown by Miyanishi et al. [Miyanishi, T., Maita, T., Matsuda, G., and Tonomura, Y. (1982) J. Biochem. 91, 1845-1853] that the amino acid sequence around the reactive lysine residue is different between head B (Pi-burst head) and head A of the myosin molecule. Thus, we synthesized these two peptides, and prepared rabbit polyclonal antibodies against them. Each antibody bound strongly with both peptides. However, the binding of the antibodies with S-1 was inhibited by the peptide used for the antigen but unaffected by the non-antigen peptide, suggesting that only antibodies specific to each head can bind with S-1. Myosin was absorbed by either antibody A or B, which was immobilized on protein A in Staphylococcus aureus cells. However, half of S-1 was absorbed by each of the antibodies. The S-1 prepared showed about 0.5 mol of initial Pi-liberation per mol of S-1. The Pi-burst size of S-1 unbound to the immobilized anti-A antibody increased to almost 1 mol/mol S-1, while that of S-1 unbound to the anti-B antibody decreased to 0.15 mol/mol S-1. These results suggest the existence of two kinds of heads in the myosin molecule.

Structure of heads A and B of myosin studied by tryptic digestion of myosin subfragment-1.

We studied the difference in the structure of head B (P1-burst head) and head A of myosin by limited tryptic digestion of myosin subfragment-1 (S-1), and using antibodies (anti-A and anti-B) which bind specifically with each head. The antibodies were prepared using peptides with sequences identical to those around the reactive lysine residue of heads A and B. When myosin subfragment-1 (S-1) was cleaved limitedly by trypsin, S-1 heavy chain (100 kDa) was digested into fragments of 25, 50, and 20 kDa. Two fragments with molecular masses of 75 and 27 kDa were transiently produced in the initial phase of digestion. Anti-A and anti-B antibodies bound only with peptides that contained the reactive lysine residue [S-1 heavy chain (100 kDa), 75-, 27-, and 25-kDa peptides], thus showing specific binding with antigen peptide. However, the 27-kDa fragment bound more strongly with anti-B antibody than with anti-A antibody. When S-1 was separated into fractions rich in S-1A and S-1B using insoluble anti-A or anti-B antibody, each antibody bound more strongly with the S-1 heavy chain (100 kDa) of its corresponding fraction by Western immunoblotting. These results suggest that the antibodies react specifically with peptides even after SDS-PAGE and membrane-blotting, and that the structure of the 25 kDa 50 kDa junction differs between heads A and B of myosin.

Acceleration of the ATPase activity of glycerol-treated muscle fibers by repeated stretch-release cycles.

Glycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KC1, 2 mM MgC1(2), and 10 micron CaC1(2) at pH 7.8 and 0 degrees C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 s at a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (alpha seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that: 1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1--2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, alpha, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (alpha=0). 2. The ATPase activity of oscillated muscle fibers increased with increase in the value of alpha, reached a maximal level, then decreased gradually with further increase of alpha to a value slightly larger than that of static fibers. At 0 degrees C, the value of alpha for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20 degrees C, the alpha value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers. 3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, alpha, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of alpha of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0 degrees C and 20 degrees C during one cycle of the changes in the rate of ADP liberation.

Standard free energy changes for formation of various intermediates in the reaction of H-meromyosin ATPase.

Two reaction intermediates of H-meromyosin (HMM) ATPase [EC 3.6.1.3], E2AT32P, and (see article), were formed by mixing excess HMM with AT32P. Then a large excess of unlabelled ATP was added, and the amount of AT32P liberated from E2AT32P was measured as the difference between the total amount of AT32P in the reaction mixture and the amount of AT32P bound to HMM, obtained by filtering the mixture after adding charcoal to adsorb nucleotides (charcoal-filtration method). The amount of free AT32P was also measured as the amount of glucose-6-32P formed within 15 sec after adding large excesses of hexokinase [EC 2.7.1.1] and glucose to the reaction mixture. The rate constant, k-2, for the step E2ATP yields E plus ATP was calculated at various KCl concentrations from the time-course of liberation of AT32P. The intermediate, (see article), was formed by mixing HMM with AT32P in a molar ratio of 1:2, and the rate constant, k-6, for the step (see article) was also determined by the same procedures used for k-2. In 0.5 M KCl and 2 mM MgCl2 at pH 7.8 and 0 degrees, k-2 and k-6 were 0.002 sec-1 and 0.1 sec-1 or more, respectively. From the rate constants determined in this work and the rate and equilibrium constants which we reported previously, the standard free energy changes (kcal/mole) for formation of various reaction intermediates in the reaction of HMM ATPase in 0.5 M KCl and 2 mM MgCl2 at pH 7.8 and 0 degrees were calculated to be as follows: (see article).

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide.

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.

Elementary steps of the actin-activated ATPase reaction of cardiac muscle myosin subfragment-1.

The rates of the elementary steps of the actomyosin ATPase reaction were measured using the myosin subfragment-1 of porcine left ventricular muscle. The results could be explained only by the two-route mechanism for actomyosin ATPase (Inoue, Shigekawa, & Tonomura (1973) J. Biochem. 74, 923-934), in which ATP is hydrolyzed via routes with or without accompanying dissociation of actomyosin. The dependence on the F-actin concentration of the rate of the acto-S-1 ATPase reaction in the steady state was measured in 5 mM KCl at 20 degrees C. The maximal rate, Vmax, and the dissociation constant for F-actin of the ATPase, Kd, were 3.0 s-1 and 2.2 mg/ml, respectively. The Kd value was almost the same as that determined from the extent of binding of S-1 with F-actin during the ATPase reaction. The rate of recombination of the S-1-phosphate-ADP complex, S-1ADPP, with F-actin, vr, was lower than that of the ATPase reaction in the steady state. Thus, ATP is mainly hydrolyzed without accompanying dissociation of acto-S-1 into S-1ADPP and F-actin. In the cardiac acto-S-1 ATPase reaction, the rate of the ATPase reaction in the steady state and that of recombination of S-1ADPP with F-actin were about 1/5 those of the skeletal acto-S-1 ATPase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

ATPase characteristics of myosin from nematode Caenorhabditis elegans purified by an improved method. Formation of myosin-phosphate-ADP complex and ATP-induced fluorescence enhancement.

Myosin was purified rapidly from the nematode Caenorhabditis elegans by an improved method. Crude actomyosin was extracted from the worms at low ionic strength. Paramyosin was removed by repeating the precipitation of myosin filaments in the presence of Mg2+ and the dissolution of them in 0.6 M NaCl. Actin was removed by ultracentrifugation in the presence of Mg-ATP and finally by column chromatography on DEAE-cellulose. This method gave a good yield of myosin (20-30 mg from 50 g wet weight of worms), and its EDTA(K+)-ATPase activity was about 3-fold higher than that of myosin prepared by the method of Harris and Epstein (1979). ATP hydrolysis by nematode myosin showed an initial Pi-burst due to formation of the myosin-phosphate-ADP complex. Tryptophan fluorescence of myosin was enhanced about 8% by ATP. The relationship between the structure and function of myosin is discussed based on the above results and the amino acid sequences of myosins from rabbit skeletal muscle and Caenorhabditis elegans.

Oxygen exchange reaction during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments.

The oxygen exchange during ATP hydrolysis by glycerinated muscle fibers, myofibrils, and synthetic actomyosin filaments was studied from the distribution of the [18O]Pi species produced by the hydrolysis of [gamma-18O]ATP. The products were mixtures of two species, one with a low extent of oxygen exchange and the other with a high extent. The low and high extents of oxygen exchange in these two Pi species were the same as those of the acto-S-1 ATPase reaction through the routes with and without the dissociation of actomyosin, respectively (Yasui, M., Ohe, M., Kajita, A., Arata, T., & Inoue, A. [1988] J. Biochem. 104, 550-559). During isometric contraction of glycerinated muscle fibers at 20 degrees C, the fraction of ATP hydrolysis with low extent of oxygen exchange was 0.83 and 0.70, respectively, in 0 and 120 mM KCl. In myofibrils, the fraction of ATP hydrolysis with a low extent of oxygen exchange was 0.72-0.88 in 0-120 mM KCl at 20 degrees C. Therefore, in glycerinated muscle fibers and myofibrils ATP seems to be mainly hydrolyzed through a route without the dissociation of actomyosin, especially at low ionic strength and at room temperature when the tension development is high. ATP hydrolysis through this route may be coupled with muscle contraction.

Oxygen exchange during the acto-subfragment-1 ATPase reaction: evidence for the two-route mechanism of the actomyosin ATPase reaction.

The oxygen exchange occurring during the acto-S-1 ATPase reaction was analyzed based on the distribution of 18O-labeled species of P1 using [gamma-18O]ATP as a substrate. Evidence was found for the two-route mechanism in which ATP is hydrolyzed via the dissociation of acto-S-1 into F-actin and the S-1-phosphate-ADP complex, S-1PADP, and their recombination, and also hydrolyzed without the dissociation of acto-S-1 (Inoue, A., Shigekawa, M., & Tonomura, Y. (1973) J. Biochem. 74, 923-934; Inoue, A., Ikebe, M., & Tonomura, Y. (1980) J. Biochem. 88, 1663-1677). When ATP was mainly hydrolyzed without the dissociation of acto-S-1, the extent of oxygen exchange was low. When ATP was hydrolyzed by both routes, the distribution of product P1 with 3, 2, 1, and 0 18O atoms showed a mixture resulting from low and high oxygen exchange. The rate of ATPase without the dissociation of acto-S-1 can be estimated from the rate of the overall reaction (v), the rate of recombination of S-1PADP with F-actin (vr), and the extent of dissociation of acto-S-1 (a). The distribution of the P1 species measured was almost equal to that calculated from the ratio of ATP hydrolysis via the two pathways as avr and v-avr, respectively. This result indicates that the rates of the dissociation of acto-S-1PADP into S-1PADP and F-actin and their recombination are much lower than the rate of decomposition of the acto-S-1PADP complex into acto-S-1 + ADP + Pi.

X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP.

To clarify the structure of the ATP hydrolysis intermediates (ADP.Pi bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeFx = 3,4 and AlF4) and vanadate (VO4(Vi)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (approximately 50% decrease in intensity) was observed in the presence of BeFx among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca(2+)-activated fibers, while the first actin layer line at approximately 1/36.7 nm-1 retained a rigor-like profile with an intensity weakened by approximately 50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (approximately 1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeFx. These results indicate that a substantial fraction (approximately 40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeFx bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603-608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.