A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Effect of crosslinking by glutaraldehyde on interaction of F-actin with heavy meromyosin.

Crosslinking of F-actin by a bifunctional reagent glutaraldehyde resulted in a marked decrease of viscosity and length of F-actin filaments. The extent and rate of superprecipitation of actomyosin reconstituted from the modified actin were lower than those of unmodified actin-myosin complex, but activation of heavy meromyosin ATPase by the crosslinked actin was higher than by unmodified one. Heavy meromyosin ATPase activated by the crosslinked actin was distinctly less dependent on KCl concentration than that activated by unmodified actin. Turbidity of the modified acto-heavy meromyosin in the presence of ATP exceeded the sum of turbidities of actin and heavy meromyosin, whereas in the case of unmodified acto-heavy meromyosin the turbidity was comparable to that for noninteracting system. The difference in activation of heavy meromyosin. ATPase by the cross-linked and unmodified actin, clearly seen at room temperature, significantly diminished when temperature was lowered to 0 degrees C.     

Kinetics of steady state ATPase activity and rigor complex formation of acto-heavy meromyosin

1. Actin and heavy meromyosin, initially mixed in a Mg-ATP solution, began to form the rigor complex slowly after ATP in the solution had been completely hydrolyzed.

2. This was because the heavy meromyosin-product complex formed via ATP hydrolysis was almost completely dissociated from actin even in the absence of ATP and as soon as this heavy meromyosin-product complex was decomposed, the heavy meromyosin combined with actin forming the rigor complex.

3. Linear plots were obtained when the reciprocal of the excess rate of the actin-accelerated rigor complex formation was plotted against the reciprocal of the added actin concentration as similar with those made on the steady acto-heavy meromyosin ATPase.

4. The V of the rigor complex formation process was about 1/5 of that of the steady acto-heavy meromyosin ATPase activity, showing that the actomyosin ATPase activity could not be explained merely by the actin-accelerated decomposition of the heavy meromyosin-product complex.

5. The same analyses were carried out on myosin subfragment 1.

6. Our results could be explained by considering the two non-identical active sites of myosin, and we propose the following scheme for the actomyosin ATPase.

7. Actin accelerates the rate-limiting bond hydrolysis in the ATPase occurring at one active site of myosin, as well as the rate-limiting decomposition of the heavy meromyosin-product complex formed at another site.     

The mechanism of ATP hydrolysis by smooth muscle myosin and subfragments using steady state titration and 18O exchange.

The mechanisms of increases in the ATPase rates of smooth muscle acto-myosin, acto-heavy meromyosin (HMM) and acto-subfragment 1 (S1) were investigated using steady state titration and 18O exchange. Phosphorylation increased the phosphate release rates both from acto-myosin and acto-HMM. Steady state titration at high enzyme concentrations and 18O exchange at substoichiometric ATP concentrations showed that gizzard myosin was kinetically homogeneous, whereas HMM and S1 prepared by various published methods were heterogeneous. At high ATP concentrations, a small population of HMM and S1 hydrolyzed ATP with a low amount of oxygen exchange.     

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.     

Fluorometric method for estimating the kinetic parameters of beta-naphthyl triphosphate and ATP hydrolysis by acto-heavy meromyosin

We have established a method to estimate the values of various kinetic parameters of acto-heavy meromyosin (acto-HMM) ATPase, using a fluorescent ATP analog, beta-naphthyl triphosphate (beta-NapP3); from the fluorescence intensity change accompanying beta-NapP3 hydrolysis, the various kinetic parameters of beta-NapP3 hydrolysis, including its product inhibition, were obtained. beta-NapPd3 hydrolysis is inhibited competitively by ATP, resulting in different time courses of fluorescence intensity change in the presence and absence of ATP. From this difference, the values of kinetic parameters of ATP hydrolysis, including its product inhibition, can be estimated. By extending this method to the acto-HMM system, seventeen parameters in a reaction scheme for the concurrent hydrolysis of ATP and beta-NapP3, including association constants between F-actin and substrate-free or substrate-bound HMM, were obtained. The kinetic-parameters estimated for ATP hjydrolsis were in good agreement with those in the literature.     

The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin.

Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase.     

Nonlinear dependence on actin of acto-heavy meromyosin 18O exchange and ATPase rate at low actin concentrations

Hydrolysis of highly enriched [gamma-18O]ATP in unlabeled water by acto-heavy meromyosin at low actin concentration was found to be heterogeneous in that significant amounts of both the species containing 0 or 3 18O-labeled oxygens/phosphate were formed. Detailed quantitative comparison with theoretical distributions over a wide range of actin concentrations, however, indicated that the pathway which catalyzed ATP hydrolysis with a low extent of exchange only made a significant contribution at a low actin concentration and did not represent a major fraction of the total hydrolysis seen at higher actin concentrations. This low exchange component was also detected in the dependence on actin of the steady state ATPase. At low actin the steady state ATPase rate increased more rapidly as a function of actin concentration than predicted by the Km and Vmax for actin activation observed at moderate to high actin levels. This extra ATP hydrolysis at low actin correlates with that predicted for the low exchange pathway both with respect to the fraction of the ATP hydrolyzed and to its dependence on the actin concentration.     

Cross-linking of actin filaments by heavy meromyosin.

The structure of acto-heavy meromyosin has been examined by electron microscopy. When heavy meromyosin is mixed with actin at ~ 2 mg/ml a gel is formed. At lower actin concentrations more ordered assemblies are formed in which the actin filaments are in “rafts” about 300 Å apart cross-linked by heavy meromyosin. These results indicate that in solution the two heads of a heavy meromyosin molecule can bind to different actin filaments.     

Motion of actin filaments in the presence of myosin heads and ATP.

We measured, by fluorescence correlation spectroscopy, the motion of actin filaments in solution during hydrolysis of ATP by acto-heavy meromyosin (acto-HMM). The method relies on the fact that the intensity of fluorescence fluctuates as fluorescently labeled actin filaments enter and leave a small sample volume. The rapidity of these number fluctuations is characterized by the autocorrelation function, which decays to 0 in time that is related to the average velocity of translation of filaments. The time of decay of the autocorrelation function of bare actin filaments in solution was 10.59 +/- 0.85 s. Strongly bound (rigor) heads slowed down the diffusion. Direct observation of filaments under an optical microscope showed that addition of HMM did not change the average length or flexibility of actin filaments, suggesting that the decrease in diffusion was not due to a HMM-induced change in the shape of filaments. Rather, slowing down of translational motion was caused by an increase in the volume of the diffusing complex. Surprisingly, the addition of ATP to acto-HMM accelerated the motion of actin filaments. The acceleration was the greatest at the low molar ratios of HMM:actin. Direct observation of filaments under an optical microscope showed that in the presence of ATP the average length of filaments did not change and that the filaments became stiffer, suggesting that acceleration of diffusion was not due to an ATP-induced increase in flexibility of filaments. These results show that some of the energy of splitting of ATP is impaired to actin filaments and suggest that 0.06 +/- 0.02 of HMM interferes with the diffusion of actin filaments during hydrolysis of ATP.     

The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.     

Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.     

A kinetic study of the energy storing enzyme-product complex in the hydrolysis of ATP by heavy meromyosin

The hydrolysis of ATP by heavy meromyosin was studied by means of the measurement of the development of enthalpy. The results were compared with the rate of change in the intensity of the ultraviolet difference spectrum of heavy meromyosin. It is shown that as far as the enthalpy change is concerned: (1) most of the excess energy associated with ATP does not directly dissipate into the solution during the rapid hydrolysis of ATP in the initial stage of the reaction but is stored in stable form in an enzyme-product complex, (2) the ultraviolet difference spectrum of heavy meromyosin is due specifically to the reaction via the enzyme-product complex, suggesting that a local conformation of heavy meromyosin is changed because of the excess energy stored in the complex, and (3) the complex dominantly exists during the steady splitting of ATP.     

Inhibition of actin-activated myosin Mg(2+)-ATPase in smooth muscle by ruthenium red.

Ruthenium red was found to inhibit actin-activated myosin Mg(2+)-ATPase in smooth muscle and to bind to myosin heavy chain, but not to F-actin. The inhibition by Ruthenium red of actin-activated Mg(2+)-ATPase was of the competitive type with respect to actin (Ki 4.4 microM) and of the non-competitive type with respect to ATP (Ki 6.6 microM). However, Ruthenium red scarcely dissociated the acto-heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F-actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F-actin, but for activation of the Mg(2+)-ATPase.     

Dark-field light microscopic study of the flexibility of F-actin complexes

The flexibility of F-actin complexed with saturating amounts of myosin subfragments has been measured by the use of a dark-field light microscope and a high-sensitivity television camera. When dilute solutions of F-actin complexes were observed in the microscope, single filaments in flexural thermal motion were visible to the eye. Images of the fluctuating filaments were recorded on videotapes using the high-sensitivity camera, and these records were used for the analysis of fluctuation to calculate flexibility in the framework of statistical mechanics of thermal fluctuation in semi-flexible rods. The analysis was carried out by two different methods. In method A, we selected many filaments (the entire length appeared near focus occasionally in the limited period of 10 to 100 seconds), measured the mean square end-to-end distance 〈R²〉 of each filament during the period and also its contour length L, and calculated a parameter λ representing flexibility by the equation given by Landau & Lifshitz (1958): $\text{〈R}{}^2\text{〉 =}\text{[2λL ? 1 +}\text{exp}\text{(?2λL)]}\text{2λ}{}^2$. Then, we obtained a value for λ = 0.040 ± 0.010 μm^?1 for the acto-heavy meromyosin filament at 24.0 °C ± 1.0 deg. C, and λ = 0.027 ± 0.005 μm^?1 for the acto-tropomyosin-heavy meromyosin filament at the same temperature.In method B, still photographs were taken of the video screen to collect a great number of filaments or parts of filaments which appeared just in focus over their length, and the contour length L of each filament and the angle θ(L) between the tangents at its two ends were measured, on the basis of the assumption that the whole length of each filament was in a plane perpendicular to the direction of view. The data were treated statistically and the results were approximated with 〈cosθ(L)〉 = exp(?λL), which holds for an ensemble of filaments with flexibility λ but in two-dimensional thermal motion (Landau & Lifshitz, 1958). The λ-values obtained by this method for acto-heavy meromyosin and acto-tropomyosin-heavy meromyosin filaments were both in good agreements with those obtained by method A, confirming the reliability of our measurement.F-actin complexed with a saturating amount of myosin subfragment-1 was examined by method B, and its flexibility was shown to be little different from that of acto-heavy meromyosin filaments.     

Enzymatic mechanochemistry. I. The interaction of heavy meromyosin with “immobilized adenosine triphosphate”

ATP was covalently bound to an agarose gel. The insolubilized ATP was found to be capable of specifically binding heavy meromyosin. The adsorbed heavy meromyosin could be eluted by ATP in solution. Both binding and elution by ATP of heavy meromyosin were not much effected by Ca²⁺, Mg²⁺ or EDTA.While the water-soluble polyalanine-myosin was also found to be adsorbed, myosin in 0.5 M KCl did not seem to be adsorbed by agarose-ATP.Both Mg²⁺ and Ca²⁺ appear to activate the splitting of bound ATP by heavy meromyosin to practically the same extent.We prepared water-soluble derivatives of ATP in which ATP underwent the same chemical modification required for its coupling to agarose but in which the agarose component was absent. Their splitting by heavy meromyosin was also activated by Mg²⁺ though to a lesser extent but actin did not influence this reaction.Possible relations between our findings and the various stages of the reaction between myosin and ATP, as well as the potential use of columns filled with insolubilized NTPs for the separation and purification of myosin and of its subfragments, are discussed.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Heterogeneity in the actin activation of myosin

The soluble proteolytic fragments of myosin, heavy meromyosin and subfragment 1, were prepared with varying amounts of the proteases chymotrypsin and papain, respectively. The actin-activated ATP hydrolysis were examined with oxygen-18-labeled ATP. Each preparation of heavy meromyosin and subfragments 1 displayed two pathways of ATP hydrolysis, called respectively the high and low oxygen exchange mechanisms. The contributions of the two mechanisms were found to be sensitive to the potassium chloride concentration. With a fixed concentration of actin (300 microM), the contribution of the low-exchange mechanism decreased from a maximum of 90% of the ATP hydrolysis at 10 and 20 mM KCl to 12% at 180 mM KCl. The results suggested that the two mechanisms were competing reactions catalyzed by a single species of myosin.     

The mechanism of inhibition of the actin-activated myosin MgATPase by calponin.

Calponin inhibits the actin-activated ATPase of smooth muscle myosin and thus has been proposed as a thin filament-based regulatory component in smooth muscle. To obtain information on the mechanism of inhibition by calponin we have used chemical modification of actin and cross-linking of actin and subfragment 1. Modification of Lys 61 of actin had no effect on the inhibition by calponin of acto-heavy meromyosin ATPase, i.e. different from tropomyosin-troponin. In addition, modification of the acidic N-terminal region of actin did not impair the ability of calponin to bind to F-actin. Finally, calponin was effective in inhibiting ATPase activity of cross-linked acto-subfragment 1. Therefore the mechanism of inhibition by calponin is distinct from troponin-tropomyosin and caldesmon in that it does not involve either the N-terminal acidic region of actin nor the area around Lys 61 and does not fit a simple steric blocking model.     

The rates of formation and dissociation of actin-myosin complexes. Effects of solvent, temperature, nucleotide binding and head-head interactions.

The rates of formation and dissociation of actin-subfragment 1 and actin-heavy mero-myosin complexes were measured by using light-scatter and the change in fluorescence of N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine (IAEDANS)-labelled acting as probes. Association rate measurements were made at low protein concentration, where the transients approximated to single exponentials with rate constants proportional to the concentration of reactant in excess. Dissociation rate measurements were made by displacing IAEDANS-actin from myosin with excess native actin and by a salt jump. The second-order rate constant of association for actin-subfragment 1 was 3 x 10(6) M-1 . s-1 in 60 mM-KCl at 13 degree C. It was decreased 10-fold in 500 mM-KCl and in 50% (v/v) glycol. It was decreased 6-fold when MgADP or Mg[beta gamma-imido]ATP bound to myosin. The dissociation rate constant was 0.012 s-1 in 60 mM-KCl at 13 degree C. It was increased 4-fold by 500 mM-KCl, 25-fold by 50% glycol, 8-fold by MgADP binding and 170-fold by Mg[beta gamma-imido]ATP binding. Ea for association was 70 kJ . mol-1 and for dissociation 35 kJ . mol-1. Heavy meromyosin associated at twice the rate observed for subfragment 1 and dissociated at less than one-twentieth of the rate for subfragment 1 (60 mM-KCl, 25 degree C), but when Mg[beta gamma-imido]ATP bound actin-heavy meromyosin dissociated at one-half the rate for subfragment 1. There were significant correlations between increase in the dissociation rate constant, decrease in binding constant and increase in magnitude of conformational change. The association rate constant did not correlate with any property of the actin-myosin complex.