The influence of substoichiometric concentrations of myosin subfragment 1 on the state of aggregation of actin under depolymerizing conditions

In 3 mM KCl, 2 mM Tris/HCl pH 7.5, 22 degrees C, 0.38 microM myosin subfragment 1 delays the depolymerization of F-actin (7.2 microM measured as monomer). The depolymerization proceeds rapidly for a few minutes and then slows down suddenly when the ratio between the monomers in the actin filaments and myosin subfragment 1 reaches the value of 11. At this time myosin subfragment 1 is substantially all bound to the actin polymers which form an irregular and discontinuous network of filaments running in doublets and in triplets, perhaps cross-linked by myosin subfragment 1. Depolymerization proceeds then for several hours, apparently ending up with the formation of the 1:1 actin-S1 heteropolymer. The ratio between the monomers in the actin filaments and myosin subfragment 1 at the end of the rapid depolymerization process is different for different protein preparations and may be as low as 5.5. In 2 mM Tris/HCl pH 7.5, 25 degrees C, 1 microM myosin subfragment 1 is able to induce the formation of undecorated actin filaments from 12 microM ATP--G-actin. These filaments probably originate by redistribution of myosin subfragment 1 between the newly formed 1/1 actin-S1 heteropolymer and G-actin in the medium, a process which allows the transient formation of undecorated actin filaments.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

Time-resolved electron microscopic analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly developed freezing system using liquid helium. Actin and S1 (100 microM each) formed a ropelike double-helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the ropelike double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the ropelike double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 35-200 ms are so called "weakly bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly bound S1 under a rapid association- dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly developed freezing system. This enabled us to directly compare the conformation of weakly and strongly bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly and strongly bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly and strongly bound S1 have different angles of attachment to actin filaments. As compared to strongly bound S1, weakly bound S1 showed a significantly broader distribution of attachment angles. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.     

Affinity chromatography of immobilized actin and myosin.

Actin and myosin were immobilized by coupling them to agarose matrices. Both immobilized G-actin and immobilized myosin retain most of the properties of the proteins in free solution and are reliable over long periods of time. Sepharose-F-actin, under the conditions used in this study, has proved unstable and variable in its properties. Sepharose-G-actin columns were used to bind heavy meromyosin and myosin subfragment 1 specifically and reversibly. The interaction involved is sensitive to variation in ionic strength, such that myosin itself is not retained by the columns at the high salt concentration required for its complete solubilization. Myosin, rendered soluble at low ionic strength by polyalanylation, will interact successfully with the immobilized actin. The latter can distinguish between active and inactive fractions of the proteolytic and polyalanyl myosin derivatives, and was used in the preparation of these molecules. The complexes formed between the myosin derivatives and Sepharose-G-actin can be dissociated by low concentrations of ATP, ADP and pyrophosphate in both the presence and the absence of Mg2+. The G-actin columns were used to evaluate the results of chemical modifications of myosin subfragments on their interactions with actin. F-Actin in free solution is bound specifically and reversibly to columns of insolubilized myosin. Thus, with elution by either ATP or pyrophosphate, actin has been purified in one step from extracts of acetone-dried muscle powder.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.     

The ghost muscle fiber with thin filaments reconstructed from nonmuscle actin--a model for studying the cytoskeleton using polarization microfluorimetry]

In muscle fibers which are free of myosin, tropomyosin and troponin thin filaments were reconstructed from muscle and non-muscle G-actin modified with 1,5-IAEDANS. Using polarized microfluorimetry it was shown that actin in such filaments maintained the ability to respond to conformational changes during actin interaction with subfragment of myosin (S1). The models of muscle fibers with reconstructed from non-muscle actin thin filaments are supposed to use for investigation of mechanisms of cell cytoskeleton functions with the help of polarized microfluorimetry.     

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 binding of myosin heads on heavy meromyosin and assembled myosin to actin in the presence of nucleotides. Measurements by the proteolytic rates method

The initial rates of tryptic digestion at the 50/20-kDa junction in myosin and myosin subfragment 1 were determined for the free proteins and their complexes with actin in the presence and absence of MgATP. The proteolytic reactions were carried out at 24 degrees C and under ionic strength conditions (mu) adjusted to 35, 60, and 130 mM. The percentages of myosin heads and myosin subfragment 1 bound to actin in the presence of MgATP were calculated from the rates of proteolysis for each set of digestion experiments. In all cases, the myosin heads in the synthetic filaments showed greater binding to actin than myosin subfragment 1. This binding difference was most prominent (3-fold) at mu = 130 mM. The binding of heavy meromyosin (HMM) to actin in the presence of MgADP was measured at 4 degrees C by ultracentrifugation and the proteolytic rates methods. Ultracentrifugation experiments determined the fraction of HMM molecules bound to actin in the presence of MgADP, whereas the proteolytic measurements yielded the information on the fraction of HMM heads bound to actin. Taken together, these measurements show that a significant fraction of HMM is bound to actin with only one head in the presence of MgADP under ionic conditions of 180 and 280 mM.     

Effect of nucleotide on the binding of N,N'-p-phenylenedimaleimide-modified S-1 to unregulated and regulated actin

In our previous study [Chalovich, J. M., Greene, L. E., & Eisenberg, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4909-4913], myosin subfragment 1 that was modified by having its two reactive thiol groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM) was found to resemble the myosin subfragment 1-adenosine 5'-triphosphate (S-1.ATP) complex in its interaction with actin. In the present study, we examined the effect of actin on adenosine 5'-diphosphate (ADP) trapped at the active site of pPDM.S-1. Our results indicate first that, in the presence of actin, ADP is no longer trapped at the active site but exchanges rapidly with free nucleotide. Different pPDM.S-1.nucleotide complexes were then formed by exchanging nucleotide into the active site of pPDM.S-1 in the presence of actin. The binding of pPDM.S-1.ATP or pPDM.S-1.PPi to actin is virtually identical with that of unmodified S-1 in the presence of ATP. Specifically, at mu = 18 mM, 25 degrees C, pPDM.S-1.ATP or pPDM.S-1.PPi binds to unregulated actin with the same affinity as does S-1.ATP, and this binding does not appear to be affected by troponin-tropomyosin. On the other hand, pPDM.S-1.ADP and pPDM.S-1 with no bound nucleotide both show a small, but significant, difference between their binding to actin and the binding of S-1.ATP; pPDM.S-1 and pPDM.S-1.ADP both bind about 2- to 3-fold more strongly to unregulated actin than does S-1.ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of regulation of cardiac actin-myosin subfragment 1 by troponin-tropomyosin

The effect of Ca2+ on the interaction of bovine cardiac myosin subfragment 1 (S-1) with actin regulated by cardiac troponin-tropomyosin was evaluated. The ratios of actin to troponin and to tropomyosin were adjusted to optimize the Ca2+-dependent regulation of the steady-state actin-activated magnesium adenosinetriphosphatase (MgATPase) rate of myosin S-1. At 25 degrees C, pH 6.9, 16 mM ionic strength, the extrapolated values for maximal adenosine 5'-triphosphate (ATP) turnover rate at saturating actin, Vmax, were 6.5 s-1 in the presence of Ca2+ and 0.24 s-1 in the absence of Ca2+. In contrast to this 27-fold regulation of ATP hydrolysis, there was negligible Ca2+-dependent regulation of cardiac myosin S-1 binding to actin. In the presence of ATP, the dissociation constant of regulated actin and cardiac myosin S-1 was 32 microM in the presence of Ca2+ and 40 microM in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. These dissociation constants are indistinguishable from the concentrations of actin needed to reach half-saturation of the myosin S-1 MgATPase rates, 37 microM actin in the presence of Ca2+ and 53 microM in its absence. Although there may be Ca2+-dependent regulation of cross-bridge binding in the intact heart, the present biochemical studies suggest that cardiac regulation critically involves other parts of the cross-bridge cycle, evidenced here by almost complete Ca2+-mediated control of the myosin S-1 MgATPase rate even when the myosin S-1 is actin-bound.     

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.     

Electron microscopic determination of the actin filament end at which cytochalasin B blocks monomer addition using the acrosomal actin bundle from horseshoe crab sperm

G-actin freed from exogenous ATP was added to the pieces of isolated acrosomal actin bundles from horseshoe crab sperm to form filaments as reported earlier (Tilney, L.G., Bonder, E.M., & DeRosier, D.J. (1981) J. Cell Biol. 90, 485-494). The growth of a filament was far more rapid at one end (the preferred end) than the other end. These ends were shown to correspond to the barbed and pointed ends, respectively, by decoration of the filaments with myosin subfragment 1. Cytochalasin B inhibited the monomer addition at the preferred end. This technique is useful in determining the ends to which actin filament end-binding proteins from nonmuscle cells bind, which are considered to regulate the actin polymerization in the cells.     

Interactions of myosin subfragment 1 isozymes with G-actin

The polymerization of G-actin by myosin subfragment 1 (S-1) isozymes, S-1(A1) and S-1(A2), and their proteolytically cleaved forms was studied by light-scattering, fluorescence, and analytical ultracentrifugation techniques. As reported previously, S-1(A1) polymerized G-actin rapidly while S-1(A2) could hardly promote the assembly reaction (Chaussepied & Kasprzak, 1989a; Chen and Reisler, 1990). This difference between the isozymes of S-1 was traced to the very poor, if any, ability of G-actin-S-1(A2) complexes to nucleate the assembly of actin filaments. The formation of G-actin-S-1(A2) complexes was verified in sedimentation velocity experiments and by fluorescence measurements using pyrene-labeled actin. The G-actin-S-1(A2) complexes supported the growth of actin filaments and accelerated the polymerization of actin in solutions seeded with MgCl2-, KCl-, and S-1(A1)-generated nuclei. The growth rates of actin-S-1(A2) filaments were markedly slower than those for actin-S-1(A1) filaments. Proteolytic cleavage of S-1 isozymes at the 50/20-kDa junction of the heavy chain greatly decreased their binding to G-actin and thus inhibited the polymerization of actin by S-1(A1). These results are discussed in the context of G-actin-S-1 interactions.     

Interaction of plasma gelsolin with G-actin and F-actin in the presence and absence of calcium ions

Plasma gelsolin formed a very tight 1:2 complex with G-actin in the presence of Ca2+, but no interaction between gelsolin and G-actin was detected in the presence of excess EGTA. However, the 1:2 complex dissociated into a 1:1 gelsolin:actin complex and monomeric actin when excess EGTA was added. Plasma gelsolin bound tightly to the barbed ends of actin filaments and also severed filaments in the presence of Ca2+ and bound weakly to the filament barbed end in the presence of EGTA. The 1:2 gelsolin-actin complex bound to the barbed ends of filaments but did not sever them. By blocking the barbed end of filaments with plasma gelsolin, we determined the critical concentration at the pointed end in 1 mM MgCl2 and 0.2 mM ATP to be 4 microM. The dissociation rate constant for ADP-G-actin from the pointed end was estimated to be about 0.4 s-1 and the association rate constant to be about 5 X 10(4) M-1 s-1. Finally, we obtained evidence that plasma gelsolin accelerates but does not bypass the nucleation step and, therefore, that the concentration of gelsolin does not directly determine the concentration of filaments polymerized in its presence. Thus, gelsolin-capped filaments may not provide an absolutely reliable method for determining the rate constant for the association of ATP-G-actin at the pointed ends of filaments, but a reasonable estimate would be 1 X 10(5) M-1 s-1 in 1 mM MgCl2 and 0.2 mM ATP.     

The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.     

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.     

The critical concentration of actin in the presence of ATP increases with the number concentration of filaments and approaches the critical concentration of actin.ADP

F-actin at steady state in the presence of ATP partially depolymerized to a new steady state upon mechanical fragmentation. The increase in critical concentration with the number concentration of filaments has been quantitatively studied. The data can be explained by a model in which the preferred pathway for actin association-dissociation reactions at steady state in the presence of ATP involves binding of G-actin . ATP to filaments, ATP hydrolysis, and dissociation of G-actin . ADP which is then slowly converted to G-actin . ATP. As a consequence of the slow exchange of nucleotide on G-actin, the respective amounts of G-actin . ATP and G-actin . ADP coexisting with F-actin at steady state depend on the filament number concentration. G-actin coexisting with F-actin at zero number concentration of filaments would then consist of G-actin . ATP only, while the critical concentration obtained at infinite number of filaments would be that for G-actin . ADP. Values of 0.35 and 8 microM, respectively, were found for these two extreme critical concentrations for skeletal muscle actin at 20 degrees C, pH 7.8, 0.1 mM CaCl2, 1 mM MgCl2, and 0.2 mM ATP. The same value of 8 microM was directly measured for the critical concentration of G-actin . ADP polymerized in the presence of ADP and absence of ATP, and it was unaffected by fragmentation. These results have important implications for experiments in which critical concentrations are compared under conditions that change the filament number concentrations.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

The binding of heat-treated myosin subfragment 1 to actin and substructure considerations

Heat treatment of myosin subfragment 1 at 35 degrees C caused about 95% inactivation of the catalytic function but did not block its binding to actin. Heat-treated subfragment 1 showed specific, strong, and close to stoichiometric binding to actin. MgATP but not MgADP dissociated these complexes. However, in contrast to intact subfragment 1, the heat-treated protein did not polymerize G-actin and was not protected from trypsin by the binding to actin. Tryptic degradation of the 50K fragment abolished, or reduced greatly, the binding of heat-treated subfragment 1 to actin in solution but not on nitrocellulose overlays. These results are discussed in the context of subfragment 1 substructure.     

Myosin subfragment-1 interacts with two G-actin molecules in the absence of ATP

The interaction between G-actin and myosin subfragment-1 (S1) has been monitored by pyrenyl-actin fluorescence and light scattering. In low ionic strength buffer and in the absence of ATP the polymerization of G-actin induced by myosin subfragment-1 is preceded by the formation of binary GS and ternary G2S complexes in which S1 interacts tightly in rapid equilibrium (K greater than 10(7) M-1) with one and two G-actin molecules, respectively. Pyrenyl fluorescence of G-actin is enhanced 4-fold in GS and 3-fold in G2S. At concentrations of G-actin and S1 in the micromolar range and above, G2S is the predominant species at G-actin/S1 ratios equal to or greater than 1. The isomer of myosin subfragment-1 carrying the A1 light chain, S1(A1), forms a tighter ternary complex than the isomer S1(A2). Actin-bound ATP is not hydrolyzed upon formation of GS and G2S. In the presence of one molar equivalent or more of myosin subfragment-1/mol of G-actin, in low ionic strength buffer containing no nucleotides, G-actin polymerizes faster in the presence of S1(A1) than in the presence of S1(A2). The interaction of S1 with G-actin is inhibited by the binding of ATP or ADP to S1, ATP having a higher affinity for S1 than ADP. The possible structural similarity of the G2S complex to the F-acto-S1 complex in the rigor state and the potential significance of a ternary (actin)2-myosin interaction for actomyosin-based motility are discussed.