Competitive and uncompetitive effects of 2,4-dinitrophenol on ATPase activities of rabbit skeletal actomyosin and myosin.

A kinetic study of the ATPase reactions catalyzed by myosin and actomyosin was carried out by varying the concentrations of ATP and 2,4-dinitrophenol (DNP). Mg-ATPase of myosin in the initial burst and that of actomyosin were both inhibited competitively by DNP. The dissociation contants for the DNP-myosin interaction (Ki) were estimated to be very similar, that is, 4.2 mM in the initial burst of ATP splitting, and 3.3 mM for the actomyosin ATPase. It is therefore suggested that DNP acts at the same site when it inhibits the burst splitting of ATP and the actomyosin ATPase. In contrast, Mg,-Ca-, and EDTA-ATPase activities of myosin in the steady state were all affected uncompetitively by DNP. Moreover, the Ki value for Mg-ATPase of myosin in the steady state was found to be 31 mM, which is much higher than those mentioned above for the initial burst and actomyosin ATPase. It is therefore suggested that the site at which DNP acts to inhibit the burst splitting of ATP is different from the site at which DNP acts to affect Mg-, Ca-, and EDTA-ATPases in the steady state.     

Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.     

Steady-state studies of the actin-activated adenosine triphosphatase activity of myosin.

Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

Dialdehyde derivatives of purine mononucleotides: substrate properties and affinity modification of myosin ATPase

It was demonstrated that the dialdehyde derivative of ATP is a good substrate for Ca-ATPase of heavy meromyosin (Km = (1.2-1.4) X 10(-4) M; V = VATP). At the same time, this compound can induce irreversible inhibition of the enzyme. Since oxo-ATP is rapidly hydrolyzed by myosin to form oxo-ADP, this inhibition is the result of the enzyme interaction with oxo-ADP. It was found that the kinetics of heavy meromyosin inhibition by oxo-ADP are typical of affinity modification; in this case ATP fully protects heavy meromyosin from the activity loss. Similar results on the irreversible inhibition of the ATPase activity under the action of oxo-ADP were obtained in the presence of myosin, heavy meromyosin, subfragment I and natural actomyosin and in the absence of bivalent cations, thus suggesting the modification of the active center of myosin ATPase.     

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

The effect of magnesium ions on the two-stage kinetics of superprecipitation and ATPase activity of natural actomyosin

The effect of magnesium ions on the two-stage kinetics of superprecipitation (SPP) and ATP activity of natural skeletal muscle actomyosin was studied. It was found that the changes in the ratios of two independent steps of SPP and ATPase activity are mainly induced by the Mg-ATP2- complex, but not by free Mg2+. These changes in the kinetics of SPP and ATPase are regarded as being due to the shift in the dynamic equilibrium between the two types of the actomyosin complexes in solution, each of which is characterized by different reaction mechanisms. The role of the Mg-ATP2(-)-induced alteration of at least two structural-and-functional states of actomyosin in muscle contractibility is discussed.     

The causes of the nonuniform thermostability of Mg-ATPase and of the contractility of muscle models

With longer periods of preliminary heat-treatment of actomyosin suspension the decrease in the rate of superprecipitation (SPP) is followed by that in the extent of SPP, and, finally, in the Mg-ATPase activity. A similar uncoupling of mechanical and enzymatic activities is observed when the ratio between the native and the inactivated myosin in reconstructed actomyosin varied. This uncoupling is supposed to result from the formation during heat-treatment of myosin bridges incapable of dissociating in the presence of Mg-ATP. The bridges affect largely the mechanical properties of actomyosin, and in a lesser degree, its enzymatic properties.     

Structure and function of the two heads of the myosin molecule. IV. Physiological functions of various reaction intermediates in myosin adenosinetriphosphatase, studied by the interaction between actomyosin and 8-bromoadenosine triphosphate.

The kinetic properties of the hydrolyses of 8-Br ATP and 8-SCH3 ATP by myosin [EC 3.6.1.3] and actomyosin were compared with those of ATP, and the following results were obtained. The Ca-NTPase activities of myosin using these two ATP analogs as substrates were smaller than that of ATPase, and the NTPase activities toward these analogs were strongly suppressed by EDTA. The Mg-NTPase activities toward these analogs were higher in a medium of high ionic strength than in a medium of low ionic strength, in contrast to the activity of Mg-ATPase. These analogs did not produce any initial burst of Pi liberation, activation of myosin NTPase by F-actin, or superprecipitation of actomyosin. The interactions between 8-Br ATP and HMM, acto-HMM, actomyosin, and myofibrils were studied in detail in the presence of Mg2+ in medium of low ionic strength. The Michaelis constant, Km, and the maximum rate, Vm, of 8-Br ATPase of HMM were 27 muM and 21 min-1, respectively. The fluorescence change of HMM induced by 8-Br ATP also followed the Michaelis-Menten equation, and the Michaelis constant, Kf1, was as low as 4 muM. Acto-HMM and acto-S-1 were fully dissociated by the addition of 8-Br ATP. The relation between the extent of dissociation of acto-HMM and the concentration of 8-Br ATP followed the Michaelis-Menten equation, and the apparent dissociation constant, Kd, was 22 muM. This Kd value is almost equal to the Km value of 8-Br ATPase of HMM described above. Myofibrillar contraction was not supported by 8-Br ATP. It was concluded that in the myosin NTPase reaction with 8-Br ATP as a substrate, M2NTP but not MNDPP is formed in route (1), while MNTP is formed in route (2). It was also concluded that the key intermediate for the actomyosin NTPase reaction is MNDPP, and that dissociation of acto-HMM is induced by the formation of M2NTP and MNTP in routes (1) and (2), respectively.     

Modulation of the substrate binding sites of platelet myosin by actin.

The studies reported in this paper were undertaken to compare the steady-state kinetics of ATPase of purified platelet actomyosin and myosin free of actin. Actomyosin exhibits highly sigmoid kinetics with at least two interacting ATP or UTP binding sites. These studies were done at O.6 m KCl where actin and myosin are generally supposed to be dissociated in the presence of these nucleotides (M. Gallaghar, T. C. Detwiler, and A. Stracher, 1976, in Cell Motility (Goldman, R., Pollard, T., and Rosenbaum, J., eds.), Vol. 3, Part A, pp. 475–485 Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.; A. Weber, 1969, J. Gen Physiol.53, 781–791). When the dissociation of platelet actomyosin was actually investigated by a sucrose density gradient technique under conditions which were similar to those of the steady-state kinetic experiments, only partial dissociation of actin from myosin was observed. This was especially true at low nucleotide concentrations where differences in the sigmoidicity of the saturation curves of actomyosin and actin-free myosin have been observed. These findings suggest that in platelet actomyosin, actin enhances the cooperativity of nucleotide binding sites of myosin by reducing the K_m for ATP or UTP. In contrast, the saturation curves of platelet myosin using either ATP or UTP as substrates are less sigmoidal and possess an intermediary plateau region, when analyzed by Hill and reciprocal plots, these data indicate both positive and negative cooperativity suggesting more than two substrate binding sites. Platelet myosin also hydrolyzed other nucleotides (the order of rates being ITP > UTP > UTP > ATP > CTP > GTP). The kinetics of ITP differed from that of ATP or UTP in that no plateau region was observed on the saturation curve. In addition, no cooperativity of ITP binding sites was seen at low substrate concentrations (up to 0.2 mm) but was instead observed at high ITP concentration. It is concluded that conformational changes in myosin induced by ITP may not be necessarily identical to those induced by ATP or UTP.     

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.     

Extraction of an actin-like protein from the plasmodium of a myxomycete and its interaction with myosin A from rabbit striated muscle

An actin-like protein was obtained from the plasmodia of a myxomycete, Physarum polycephalum. It forms a complex with muscle myosin A which behaves similarly to the actomyosin from rabbit striated muscle. On the addition of ATP the complex of this protein with myosin A shows a viscosity drop at high concentrations of KCl (～0.5 M). At low concentrations of KCl (～0.05 M) this complex superprecipitates from solutions containing 1 mM MgCl₂ and shows Mg-activated ATPase activity. That is, the actin-like protein converts the ATPase of myosin A to the actomyosin type.     

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.     

pH-dependent changes in the two-stage kinetics of superprecipitation reaction of natural actomyosin

The effect of pH on the two-stage kinetics of the superprecipitation (SPP) reaction of natural actomyosin was investigated. It was shown that the experimental dependencies appear as two intersecting bell-shaped curves reflecting the effects of pH on individual steps of the SPP reaction which are mediated by different molecular mechanisms. It was supposed that the both reaction mechanisms involve actomyosin complexes which have different structural states and differ also by the degree of dissociation in the presence of ATP. The shifts in the dynamic equilibrium between the two states of actomyosin may induce pH-modulations in the two-stage kinetics of SPP and, presumably, ATPase.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Association and dissociation of the thick and thin filaments within myofibrils in conditions of “contraction” and “relaxation”

1. The current assumption that the low ATPase activity of relaxed myofibrils is represented by the ATPase activity of myosin which has been set free during the dissociation of actomyosin was investigated. For this purpose, the ATPase activity of relaxed skeletal myofibrils of the rabbit and of the crab Maia squinado has been compared with the activity of contracted fibrils and of purified rabbit myosin in conditions of varying ionic strength, pH and concentrations of MgATP (i.e. MgATP²⁻ + MgHATP⁻) and Mg²⁺.

2. Contraction and relaxation of the fibrils was induced by changing the concentration of Ca²⁺ from about 5×10⁻⁵ to below 1×10⁻⁸ M.

3. In all conditions studied, the ATPase activity of relaxed fibrils was about 6–8 times less than that of the contracted fibrils, but it remained a typical actomyosin ATPase.

4. Quantitatively and qualitatively, this ATPase differs from the ATPase of myosin. For instance, its dependence on pH is the reverse of that of the myosin ATPase.

5. Calculation showed that the fibrils are dissociated by 90% in conditions of relaxation. Since the ATPase activity of myosin was merely some 2% of the actomyosin activity, the major part of the ATPase of fibrils, even at a dissociation of 90%, is bound to show the properties of the ATPase of actomyosin.

6. However, a dissociation of 90% cannot be distinguished from a dissociation of 100% by means of physical methods (viscosity, superprecipitation, resistance to stretch, etc.). This explains why physical methods indicate a “full” dissociation of actomyosin although, enzymatically, the ATPase is still of the actomyosin type.

7. The possible reasons are discussed for the discrepancy between the 100-fold increase in the ATP turnover and the 1000-fold increase in energy turnover of the living muscle during the transition from relaxed to active state. The most probable explanation seems to be an ATPase activity of myosin which is too high by a factor of ten as compared to the energy turnover of living muscle at the resting state. This high activity cannot be caused by a contamination of the myosin by Ca²⁺-insensitive actomyosin.     

The myosin cardiac loop participates functionally in the actomyosin interaction

The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Mediating the actomyosin interaction in myosin is an actin binding site distributed among several peptides on the myosin surface including surface loops contributing to affinity and actin regulation of myosin ATPase. A structured surface loop on beta-cardiac myosin, the cardiac or C-loop, was recently demonstrated to affect myosin ATPase and was indirectly implicated in the actomyosin interaction. The C-loop is a conserved feature of all myosin isoforms with crystal structures, suggesting that it is an essential part of the core energy transduction machinery. It is shown here that proteolytic digestion of the C-loop in beta-cardiac myosin eliminates actin-activated myosin ATPase and reduces actomyosin affinity in rigor more than 100-fold. Studies of C-loop function in smooth muscle myosin were also undertaken using site-directed mutagenesis. Mutagenesis of a single charged residue in the C-loop of smooth muscle myosin alters actomyosin affinity and doubles myosin in vitro motility and actin-activated ATPase velocities, thereby involving a charged region of the loop in the actomyosin interaction. It appears likely that the C-loop is an essential electrostatic binding site for actin involved in modulation of actomyosin affinity and regulation of actomyosin ATPase velocity.     

Comparison of kinetic properties of the ATPase reaction of arterial smooth muscle myosin with skeletal muscle myosin

Myosin was prepared from arterial smooth muscle, and a hybrid actomyosin was formed from arterial myosin and rabbit skeletal muscle F-actin. We performed kinetics on the ATPase reaction [EC 3.6.1.3] of arterial myosin and the hybrid actomyosin at high ionic strength, and compared the kinetic properties of arterial myosin ATPase with those of skeletal muscle myosin ATPase. No significant difference was found between these two myosins in the size of the initial Pi burst, the amount of bound nucleotides, and the rates of various elementary steps in the ATPase reaction. On the other hand, two important differences were observed between the hybrid actomyosin and skeletal muscle actomyosin: (i) The amounts of ATP necessary for complete dissociation of the hybrid and skeletal muscle actomyosins were 2 and 1 mol/mol of myosin, respectively. (ii) The rate of dissociation of the hybrid actomyosin induced by ATP was much lower than that of skeletal muscle actomyosin and also was lower than that of fluorescence enhancement.     

Heat resistance of MgATPase and contractility in muscle models

1. 1.|During the heating of a synthetic actomyosin suspension, the following sequence of events were observed. First, the rate of superprecipitation decreased; secondly the extent of superprecipitation decreased and finally the MgATPase activity was inhibited. At the same time the dissociating capability of actomyosin decreased in a solution of high ionic strength.

2. 2.|A similar lack of coincidence between the mechanical and the enzymatic activities of actomyosin was observed with an increasing proportion of inactivated myosin occurring in the reconstructed actomyosin complex.

3. 3.|The different heat resistance of contractility and MgATPase activity in muscle models may be caused by inactivated myosin bridges which form in the course of heat treatment so that the dissociating capacity of actomyosin in the presence of ATP is lost.

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.