Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue

Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.     

Troponin is a potential regulator for actomyosin interactions

Troponin extracted from rabbit skeletal muscle directly binds to an actin filament in a molar ratio of 1:1 even in the absence of tropomyosin. An actin filament decorated with troponin did not exhibit significant difference from pure actin filaments in the maximum rate of actomyosin ATP hydrolysis and the sliding velocity of the filament examined by means of an in vitro motility assay. However, the relative number of troponin-bound actin filaments moving in the absence of calcium ions decreased to half that in their presence. The amount of HMM bound to the filaments was less than 4% of actin monomers in the presence of TNs. In addition, actin filaments could not move when Tn molecules were bound in the molar ratio of about 1:1 although they sufficiently bind to myosin heads. These results indicate that troponin can transform an actin monomer within a filament into an Off-state without sterically blocking of the myosin-binding sites with tropomyosin molecules.     

Actomyosin II interaction modulates cell cortex stability

Myosin II controls the viscoelastic behavior of actin filaments, interacting with actin in an energy-dependent manner. Replacing adenosine triphosphate with adenosine diphosphate changes actomyosin sliding to cross-linking. Rheological measurements show a 3-4-fold increase of the elastic portion G' in actin filaments when myosin II is present at a molar ratio r(MA)=1:200. This observation is supported by the demonstration of inactive myosin heads along actin filaments using atomic force microscopy.     

1,3-Diethylurea-enhanced Mg-ATPase activity of skeletal muscle myosin with a converse effect on the sliding motility

We investigate the effects of urea and its derivatives on the ATPase activity and on the in vitro motility of chicken skeletal muscle actomyosin. Mg-ATPase rate of myosin subfragment-1 (S1) is increased by 4-fold by 0.3 M 1,3-diethylurea (DEU), but it is unaffected by urea, thiourea, and 1,3-dimethylurea at ≤ 1 M concentration. Thus, we further examine the effects of DEU in comparison to those of urea as reference. In in vitro motility assay, we find that in the presence of 0.3 M DEU, the sliding speeds of actin filaments driven by myosin and heavy meromyosin (HMM) are significantly decreased to 1/16 and 1/6.6, respectively, compared with the controls. However, the measurement of the actin-activated ATPase activity of HMM shows that the maximal rate, V_max, is almost unchanged with DEU. Thus, the myosin-driven sliding motility of actin filaments is significantly impeded in the presence of 0.3 M DEU, whereas the cyclic interaction of myosin with F-actin occurs during the ATP turnover, the rate of which is close to that without DEU. In contrast to DEU, 0.3 M urea exhibits only modest effects on both actin-activated ATPase and sliding motility of actomyosin. Thus, DEU has the effect of uncoupling the sliding motility of actomyosin from its ATP turnover.     

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.     

Cooperativity of thiol-modified myosin filaments. ATPase and motility assays of myosin function.

The effects of chemical modifications of myosin's reactive cysteines on actomyosin adenosine triphosphatase (ATPase) activities and sliding velocities in the in vitro motility assays were examined in this work. The three types of modifications studied were 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3- diazole labeling of SH2 (based on Ajtai and Burghart. 1989. Biochemistry. 28:2204-2210.), phenylmaleimide labeling of SH1, and phenylmaleimide labeling of myosin in myofibrils under rigor conditions. Each type of modified myosin inhibited the sliding of actin in motility assays. The sliding velocities of actin over copolymers of modified and unmodified myosins in the motility assay were slowest with rigor-modified myosin and most rapid with SH2-labeled myosin. The actin-activated ATPase activities of similarly copolymerized myosins were lowest with SH2-labeled myosin and highest with rigor-modified myosin. The actin-activated ATPase activities of myosin subfragment-1 obtained from these modified myosins decreased in the same linear manner with the fraction of modified heads. These results are interpreted using a model in which the sliding of actin filaments over myosin filaments decreases the probability of myosin activation by actin. The sliding velocity of actin over monomeric rigor-modified myosin exceeded that over the filamentous form, which suggests for this myosin that filament structure is important for the inhibition of actin sliding in motility assays. The fact that all cysteine modifications examined inhibited the actomyosin ATPase activities and sliding velocities of actin over myosin poses questions concerning the information about the activated crossbridge obtained from probes attached to SH1 or SH2 on myosin.     

Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay

In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.     

Myosin step size. Estimation from slow sliding movement of actin over low densities of heavy meromyosin

We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.     

Novel Mode of Cooperative Binding between Myosin and Mg-actin Filaments in the Presence of Low Concentrations of ATP

Cooperative interaction between myosin and actin filaments has been detected by a number of different methods, and has been suggested to have some role in force generation by the actomyosin motor. In this study, we observed the binding of myosin to actin filaments directly using fluorescence microscopy to analyze the mechanism of the cooperative interaction in more detail. For this purpose, we prepared fluorescently labeled heavy meromyosin (HMM) of rabbit skeletal muscle myosin and Dictyostelium myosin II. Both types of HMMs formed fluorescent clusters along actin filaments when added at substoichiometric amounts. Quantitative analysis of the fluorescence intensity of the HMM clusters revealed that there are two distinct types of cooperative binding. The stronger form was observed along Ca²⁺-actin filaments with substoichiometric amounts of bound phalloidin, in which the density of HMM molecules in the clusters was comparable to full decoration. The novel, weaker form was observed along Mg²⁺-actin filaments with and without stoichiometric amounts of phalloidin. HMM density in the clusters of the weaker form was several-fold lower than full decoration. The weak cooperative binding required sub-micromolar ATP, and did not occur in the absence of nucleotides or in the presence of ADP and ADP-Vi. The G680V mutant of Dictyostelium HMM, which over-occupies the ADP-Pi bound state in the presence of actin filaments and ATP, also formed clusters along Mg²⁺-actin filaments, suggesting that the weak cooperative binding of HMM to actin filaments occurs or initiates at an intermediate state of the actomyosin-ADP-Pi complex other than that attained by adding ADP-Vi.     

Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

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.     

Direct observation of molecular motility by light microscopy

We used video-fluorescence microscopy to directly observe the sliding movement of single fluorescently labeled actin filaments along myosin fixed on a glass surface. Single actin filaments labeled with phalloidin-tetramethyl-rhodamine, which stabilizes the filament structure of actin, could be seen very clearly and continuously for at least 60 min in 02-free solution, and the sensitivity was high enough to see very short actin filaments less than 40 nm long that contained less than eight dye molecules. The actin filaments were observed to move along double-headed and, similarly, single-headed myosin filaments on which the density of the heads varied widely in the presence of ATP, showing that the cooperative interaction between the two heads of the myosin molecule is not essential to produce the sliding movement. The velocity of actin filament independent of filament length (greater than 1 micron) was almost unchanged until the density of myosin heads along the thick filament was decreased from six heads/14.3 nm to 1 head/34 nm. This result suggests that five to ten heads are sufficient to support the maximum sliding velocity of actin filaments (5 micron/s) under unloaded conditions. In order for five to ten myosin heads to achieve the observed maximum velocity, the sliding distance of actin filaments during one ATP cycle must be more than 60 nm.     

The tail of myosin reduces actin filament velocity in the in vitro motility assay

It has been observed that heavy meromyosin (HMM) propels actin filaments to higher velocities than native myosin in the in vitro motility assay, yet the reason for this difference has remained unexplained. Since the major difference between these two proteins is the presence of the tail in native myosin, we tested the hypothesis that unknown interactions between actin and the tail (LMM) slow motility in native myosin. Chymotryptic HMM and LMM were mixed in a range of molar ratios (0-5 LMM/HMM) and compared to native rat skeletal myosin in the in vitro motility assay at 30 degrees C. Increasing proportions of LMM to HMM slowed actin filament velocities, becoming equivalent to native myosin at a ratio of 3 LMM/HMM. NH4+ -ATPase assays demonstrated that HMM concentrations on the surface were constant and independent of LMM concentration, arguing against a simple displacement mechanism. Relationships between velocity and the number of available heads suggested that the duty cycle of HMM was not altered by the presence of LMM. HMM prepared with a lower chymotrypsin concentration and with very short digestion times moved actin at the same high velocity. The difference between velocities of actin filament propelled by HMM and HMM/LMM decreased with increasing ionic strength, suggesting that ionic bonds between myosin tail and actin filaments may play a role in slowing filament velocity. These data suggest the high velocities of actin filaments over HMM result from the absence of drag generated by the myosin tail, and not from proteolytic nicking of the motor domain.     

Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.     

Calcium functionally uncouples the heads of myosin VI

This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.     

Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe³⁺-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.     

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.     

Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.     

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.     

Substructure of the myosin molecule: IV. Interactions of myosin and its subfragments with adenosine triphosphate and F-actin

The enzymic activity of several single-headed subfragments of myosin (HMM S-1 and single-headed HMM) has been compared to the double-headed derivative of myosin (HMM) both in the presence and absence of aetin. Under the assay conditions of our experiments, we find that HMM hydrolyses ATP at approximately twice the rate of any single-headed species. These results suggest a relatively independent functional role for each of the two heads of the myosin molecule.An attempt has been made to determine the stoichiometry of association between subfragments and actin, either in the absence of nucleotide or during the hydrolysis of ATP. It was originally thought that a comparison of the maximum turnover rate of HMM at infinite concentrations of actin with the maximum rate at infinite concentrations of enzyme (but with a fixed amount of actin) would yield the combining ratio of actin to HMM. However, the considerable variation of ATP turnover rates with the conditions of the experiment has made it impossible to reach any firm conclusions regarding stoichiometry. A more direct approach to the question of stoichiometry is possible in the absence of ATP. By reacting varying amounts of F-actin with a given concentration of subfragment and centrifuging the resulting complex, it is possible to determine the unbound concentration of subfragment in the supernatant. These data provide sufficient information to construct a Scatchard plot and show that twice as many moles of actin are bound by HMM as by HMM S-1. Furthermore, the association constant of actin for HMM is several orders of magnitude higher than that for the single-headed species.In connection with the question of why myosin has two “heads”, we have examined the ability of single-headed molecules to undergo the phenomenon of “superprecipitation”. We find that single-headed myosin (the preparation of which was discussed in the preceding paper) is able to superprecipitate in much the same manner as native myosin.We conclude from these studies that each head of the myosin molecule is able to function in a relatively independent fashion. These studies do not, of course, exclude the possibility of more subtle interactions between the heads of myosin which our techniques are not able to detect.