Binding of actin filaments to connectin

The binding of actin filaments to connectin, a muscle elastic protein, was investigated by means of turbidity and sedimentation measurements and electron microscopy. In the presence of less than 0.12 M KCl at pH 7.0, actin filaments bound to connectin. Long actin filaments formed bundles. Short actin filaments also aggregated into irregular bundles or a meshwork, and were frequently attached perpendicularly to long bundles. The binding of F-actin to connectin was saturated at an equal weight ratio (molar ratio, 50 : 1), as determined by a cosedimentation assay. Larger amounts of sonicated short actin filaments appeared to bind to connectin than intact F-actin. Myosin S1-decorated actin filaments did not bind to connectin. The addition of S1 to connectin-induced actin bundles resulted in partial disaggregation. Thus, connectin does not appear to interfere with actin-myosin interactions, since myosin S1 binds to actin more strongly than connectin.     

Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages.

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Adenosine triphosphatase activity and "thick filament" formation of chicken gizzard myosin in low salt media.

The ATPase activity of chicken gizzard myosin was studied by varying the KCl concentration in the reaction medium. The following was thus found: (a) A sharp depression of the activity occurred when the KCl concentration was reduced to less than 0.3 M, showing the minimum activity around 0.15 M KCl. (b) The activity depression was removed by addition of urea or bay papain-digestion, but not by addition of p-chloromercuribenzoate. (c) In the KCl concentration where the activity depression occurred, the ATPase reaction proceeded in two distinct phases; the activity was relatively high in the early phase of the reaction and declined into the later phase where the steady state reaction took place. (d) In the KCl concentrations higher than that particular concentration or in the presence of urea, the ATPase reaction proceeded in one phase. (e) The temperature dependence of the ATPase activity in the early phase was of an ordinary magnitude being approximately equal to that of the ATPase activity in 0.6 M KCl. In contrast, the temperature dependence of the activity in the later phase was unusually small. Gizzard myosin in various concentrations of KCl was also examined by measuring the turbidity and the light-scattering intensity, and by observation under an electron microscope. The following was thus found: (a) In the KCl concentration where the activity depression occurred, there was a stagnation in the turbidity decrease as the KCl concentration was gradually increased and also the formation of "thick filaments," each of which was approximately 0.6-0.9 micron in length and 20-30 nm in diameter with no central "bare zone." (b) Addition of ATP induced dissociation of the thick filaments, and the dissociation occurred during the early phase of the ATPaseeaction. (c) Moreover, the temperature dependence of the ATP-induced dissociation rate was approximately equal to that of the ATPase activity in the early phase. On the basis of the findings mentioned above, it is concluded that the activity depression results from the ATP-induced dissociation of myosin filaments. Moreover, since high concentrations of KCl or urea also caused dissociation of myosin filaments and yet did not produce the activity depression, it was strongly suggested that gizzard myosin in the ATP-dissociated form must be different from that in the urea- or KCl-dissociated form, probably in the physical state of some myosin aggregates which were not detectable by the physical methods we used. As a side-observation, gizzard myosin filaments formed in the presence of ADP were found to be unusually long (longer than 2 micron), and they looked very similar to the particular filaments of skeletal myosin that were reported, by Moos, to be formed in the absence of the C protein.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Purification and characterization of myosin from the clonal rat glial cell strain C-6.

A myosin was isolated from the clonal rat glial cell strain C-6 and compared with rat skeletal muscle myosin. After cell extracts were subjected to gel filtration chromatography in the presence of KI and magnesium pyrophosphate the C-6 myosin was rapidly purified by a procedure similar to that used for skeletal muscle myosin. The C-6 myosin resembles muscle myosin both physically and enzymatically. It contains heavy chains of 200,000 daltons and two classes of light chains of 17,000 and 19,000 daltons in approximately equal molar ratios. This myosin forms bipolar thick filaments in 0.1 M KCl and binds reversibly to skeletal muscle F-actin, the binding being inhibited by MgATP. Skeletal muscle F-actin stimulates the C-6 myosin adenosine triphosphatase 2- to 3-fold in the presence of KCl and Mg2+. The action activation of muscle myosin ATPase at low ionic strength is 10-fold greater than that of C-6 myosin. Ca2+ and EDTA stimulated the ATPase activities of both enzymes. When assayed in the presence of 0.6 M KCl and 1 mM EDTA the skeletal muscle myocin ATPase demonstrates substrate saturation while the C-6 myosin enzyme activity is stimulated by ATP concentrations above 2.5 mM.     

Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin

Effects of purealin isolated from a sea sponge, Psammaplysilla purea, on the enzymatic and physiochemical properties of chicken gizzard myosin were studied. At 0.15 M KCl, 40 microM purealin increased the Ca2+- and Mg2+-ATPase activity of dephosphorylated gizzard myosin to 2.5- and 3-fold, respectively, but decreased the K+-EDTA-ATPase activity of the myosin to 0.25-fold. In contrast, purealin had little effect on the ATPase activities of phosphorylated gizzard myosin. The ATP-induced decrease in light scattering of dephosphorylated gizzard myosin at 0.15 M KCl was lessened by 40 microM purealin. Electron microscopic observations indicated that thick filaments of dephosphorylated myosin were disassembled immediately by addition of 1 mM ATP at 0.15 M KCl, although they were preserved by purealin for a long time even after addition of ATP. Upon ultracentrifugation, dephosphorylated myosin sedimented as two components, the 10 S species and myosin filaments, in the solution containing 0.18 M KCl and 1 mM Mg X ATP in the presence of 60 microM purealin. These results suggest that purealin modulates the ATPase activities of dephosphorylated gizzard myosin by enhancing the stability of myosin filaments against the disassembling action of ATP.     

Diffusion of H-meromyosin in F-actin plus ATP solution at a very low electrolyte concentration

The translational diffusion coefficient (D) of H-meromyosin in actin (F-actin) and ATP solution was measured under conditions wherein the actin-activated ATPase activity is close to its maximal value at a very low electrolyte concentration. The results were compared with similar data obtained with 0.1 M KCl, where H-meromyosin and actin were almost completely dissociated. With 0.1 M KCl, it was found that there was no dependence of the D of H-meromyosin on actin concentration. On the other hand, at a very low electrolyte concentration, it was found that the D of H-meromyosin did depend on actin concentration; at a rather high actin concentration (and activation of ATPase), it was slightly larger than at low or zero actin concentrations. This behavior of D at a low electrolyte concentration is interpreted on the assumption that even in solution, H-meromyosin molecules can actively slide on actin filaments due to the ATPase activity.     

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.     

Effects of Ca2+ on the conformation and enzymatic activity of smooth muscle myosin

The influence of Ca2+ on the enzymatic and physical properties of smooth muscle myosin was studied. The actin-activated ATPase activity of phosphorylated gizzard myosin and heavy meromyosin is higher in the presence of Ca2+ than in its absence, but this effect is found only at lower MgCl2 concentrations. As the MgCl2 concentration is increased, Ca2+ sensitivity is decreased. The concentration of Ca2+ necessary to activate ATPase activity is higher than that required to saturate calmodulin. The similarity of the pCa dependence of ATPase activity and of Ca2+ binding to myosin and the competition by Mg2+ indicate that these effects involved the Ca2+-Mg2+ binding sites of gizzard myosin. For the actin dependence of ATPase activity of phosphorylated myosin at low concentrations of MgCl2, both Vmax and Ka are influenced by Ca2+. The formation of small polymers by phosphorylated myosin in the presence of Ca2+ could account for the alteration in the affinity for actin. For the actin dependence of phosphorylated heavy meromyosin at low MgCl2 concentrations, Ca2+ induces only an increase in Vmax. To detect alterations in physical properties, two techniques were used: viscosity and limited papain hydrolysis. For dephosphorylated myosin, 6 S or 10 S, Ca2+-dependent effects are not detected using either technique. However, for phosphorylated myosin the decrease in viscosity corresponding to the 6 S to 10 S transition is shifted to lower KCl concentrations by the presence of Ca2+. In addition, a Ca2+ dependence of proteolysis rates is observed with phosphorylated myosin but only at low ionic strength, i.e. under conditions where myosin assumes the folded conformation.     

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.     

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.     

Purification and characterization of myosin from bovine retina.

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.     

Subtilisin-cleaved actin: polymerization and interaction with myosin subfragment 1

Homogeneous preparations of actin cleaved into two fragments, the N-terminal 9- and C-terminal 36-kDa peptides, were achieved by proteolysis of G-actin with subtilisin at 23 degrees C at a 1:1000 (w/w) ratio of enzyme to actin. The subtilisin cleavage site was identified by sequence analysis to be between Met-47 and Gly-48. Although under nondenaturing conditions the two fragments remained associated to one another, the cleavage affected macromolecular interactions of actin. The rates of cleaved actin polymerization by MgCl2, KCl, and myosin subfragment 1 (S-1) were slower and the critical concentrations for this process were higher than in intact protein. Intact and cleaved actin formed morphologically indistinguishable filaments and copolymerized in the presence of MgCl2. The affinity of actin for S-1 was decreased by about 10-fold due to subtilisin cleavage, but the S-1 ATPase activity was activated to the same Vmax value by both intact and cleaved actins. DNase I inhibition measurements revealed lower affinity of cleaved actin for DNase I than that of intact protein. These results are discussed in terms of actin's structure.     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

Binding of connectin to myosin filaments

Binding of native connectin (2,100 kDa fragment of alpha-connectin) to myosin filaments was investigated using a sedimentation technique and densitometric estimations of the separated proteins. In the presence of 60 mM KCl and 5 mM phosphate buffer, pH 7.0, as much as 1.5 mol of connectin was bound to 1 mol of myosin, suggesting that some 150 connectin filaments bound to a single myosin filament of approximately 0.5 micron in length. This value was much more than the ratio found in muscle (12:1). It appeared that C protein did not affect the binding of connectin to myosin filaments.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

Purification and characterization of myosin from bovine retina

Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca²⁺ and a low activity in the presence of Mg²⁺. The Mg²⁺-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal mucle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.     

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.