The limited tryptic cleavage of chymotryptic S-1: an approach to the characterization of the actin site in myosin heads.

Limited tryptic proteolysis of S-1 (A₁+A₂) or S-1 (A₁) and S-1 (A₂) converts the heavy chain into 3 fragments of Mr = 27K-50K-20K. As a result the actin-stimulated ATPase activity of the fragmented heads is lost. When the digestion is performed using the complex F-actin-S-1, this ATPase activity is completely preserved and the heavy chain is split into only 2 fragments of Mr = 27K–70K. The specific protection by F-actin of the -COOH terminal region of the heavy chain at the joint 50K-20K against tryptic cleavage and loss of activity suggests that this part of the head can be involved in actin binding site and/or Mg²⁺ ATP hydrolysis by the acto-S-1 complex.     

Effect of mild heat treatment on the ATPase activity and proteolytic sensitivity of myosin subfragment-1

The K+-EDTA-activated ATPase activity of chymotryptic myosin subfragment-1 (S-1) decreased by 85-90% when S-1 was incubated over a 2-h period at 35 degrees C. Addition of F-actin, ATP, or ATP analogs, such as ADP or PPi, to S-1 before incubation at 35 degrees C prevented the loss of ATPase activity. The decrease in ATPase activity was also accompanied by changes in tryptic sensitivity. Instead of the normal peptide pattern--which is comprised of three heavy chain fragments (27K, 50K, and 20K)--only two fragments (27K and 20K) appeared on the sodium dodecyl sulfate-gel electrophoregram after limited tryptic digestion of thermally treated S-1. Addition of any ligand--e.g. ATP, ADP, pyrophosphate, or actin--which prevented the loss of ATPase activity during incubation at 35 degrees C also prevented the observed change in the tryptic peptide pattern of S-1. Tryptic digested S-1, whose heavy chain has been cleaved to 27K, 50K, and 20K fragments, also lost its ATPase activity upon mild heat treatment. The heat-treated trypsin-digested S-1 was subjected to a second tryptic digestion, which resulted in the disappearance of the 50K fragment, while the 50K fragment of tryptic S-1 not subjected to heat treatment was not susceptible to additional tryptic hydrolysis. The results indicate that the structural changes, that take place specifically in the 50K region of S-1 upon mild heat treatment, lead to both the loss of the ATPase activity and the changed tryptic sensitivity of S-1.     

Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.     

Antibody against 25,000 dalton tryptic fragment of subfragment-1 from chicken skeletal muscle myosin: functional implication of the 25,000 fragment region in subfragment-1

Antibody was prepared against the 25,000-dalton tryptic fragment of subfragment-1 from skeletal muscle myosin. The antibody was found to inhibit the Mg2+-ATPase activity and the initial P1-burst of the ATPase. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. The acto-S-1 ATPase activity was also inhibited by the antibody. These results suggest that there is a site in the 25K fragment region responsible for the transition of the myosin-ATP complex to another high energy complex.     

Isolation and characterization of the N-terminal 23-kilodalton fragment of myosin subfragment 1

The 23-kDa N-terminal tryptic fragment was isolated from the heavy chain of rabbit skeletal myosin subfragment 1 (S-1). The heavy-chain fragments were dissociated by guanidine hydrochloride following limited trypsinolysis, and the 23-kDa fragment was isolated by gel filtration and ion-exchange chromatography. Finally, the fragment was renatured by removing the denaturants. The CD spectrum of the renatured fragment shows the presence of ordered structure. The tryptophan fluorescence emission spectrum of the fragment is considerably shifted to the red upon adding guanidine hydrochloride which indicates that the tryptophans are located in relatively hydrophobic environments. The two 23-kDa tryptophans, unlike the rest of the S-1 tryptophans, are fully accessible to acrylamide as indicated by fluorescence quenching. The isolated 23-kDa fragment cosediments with F-actin in the ultracentrifuge and significantly increases the light scattering of actin in solution which indicates actin binding. The binding is rather tight (Kd = 0.1 microM) and ionic strength dependent (decreasing with increasing ionic strength). ATP, pyrophosphate, and ADP dissociate the 23-kDa-actin complex with decreasing effectiveness. The isolated 23-kDa fragment does not have ATPase activity; however, it inhibits the actin-activated ATPase activity of S-1 by competing presumably with S-1 for binding sites on actin. F-Actin binds to the 23-kDa fragment immobilized on the nitrocellulose membrane. The fragment was further cleaved, and one of the resulting peptides, containing the 130-204 stretch of residues, was found to bind actin on the nitrocellulose membrane, indicating that this region of the 23-kDa fragment participates in forming an actin binding site.     

Actomyosin interactions in the presence of ATP and the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.     

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.     

Mechanism of actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex.

We have investigated the steps in the actomyosin ATPase cycle that determine the maximum ATPase rate (Vmax) and the binding between myosin subfragment one (S-1) and actin which occurs when the ATPase activity is close to Vmax. We find that the forward rate constant of the initial ATP hydrolysis (initial Pi burst) is about 5 times faster than the maximum turnover rate of the actin S-1 ATPase. Thus, another step in the cycle must be considerably slower than the forward rate of the initial Pi burst. If this slower step occurs only when S-1 is complexed with actin, as originally predicted by the Lymn-Taylor model, the ATPase activity and the fraction of S-1 bound to actin in the steady state should increase almost in parallel as the actin concentration is increased. As measured by turbidity determined in the stopped-flow apparatus, the fraction of S-1 bound to actin, like the ATPase activity, shows a hyperbolic dependence on actin concentration, approaching 100% asymptotically. However, the actin concentration required so that 50% of the S-1 is bound to actin is about 4 times greater than the actin concentration required for half-maximal ATPase activity. Thus, as previously found at 0 degrees C, at 15 degrees C much of the S-1 is dissociated from actin when the ATPase is close to Vmax, showing that a slow first-order transition which follows the initial Pi burst (the transition from the refractory to the nonrefractory state) must be the slowest step in the ATPase cycle. Stopped-flow studies also reveal that the steady-state turbidity level is reached almost instantaneously after the S-1, actin, and ATP are mixed, regardless of the order of mixing. Thus, the binding between S-1 and actin which is observed in the steady state is due to a rapid equilibrium between S-1--ATP and acto--S-1--ATP which is shifted toward acto-S-1--ATP at high actin concentration. Furthermore, both S-1--ATP and S-1--ADP.Pi (the state occurring immediately after the initial Pi burst) appear to have the same binding constant to actin. Thus, at high actin concentration both S-1--ATP and S-1--ADP.Pi are in rapid equilibrium with their respective actin complexes. Although at very high actin concentration almost complete binding of S-1--ATP and S-1--ADP.Pi to actin occurs, there is no inhibition of the ATPase activity at high actin concentration. This strongly suggests that both the initial Pi burst and the slow rate-limiting transition which follows (the transition from the refractory to the nonrefractory state) occur at about the same rates whether the S-1 is bound to or dissociated from actin. We, therefore, conclude that S-1 does not have to dissociate from actin each time an ATP molecule is hydrolyzed.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Cross-linking of the 25- and 20-kilodalton fragments of skeletal myosin subfragment 1 by a bifunctional ATP analogue

The bifunctional photoreactive ATP analogue azidonitrobenzoyl-8-azido-ATP (ANB-8-N3-ATP) was synthesized. This ATP analogue carriers photoreactive azido groups at the eighth position of the adenine ring and at the 3' position of ribose. Photolysis of this analogue in the presence of skeletal muscle alpha-chymotryptic subfragment 1 (S-1) resulted in a new 120-kDa band, while photolysis in the presence of the tryptic S-1 produced a new 45-kDa band. The 45-kDa peptide was shown to be combined with the 25-kDa N-terminal and 20-kDa C-terminal fragments since it was labeled with a monoclonal antibody specific for the N-terminal 25-kDa segment of the S-1 heavy chain, and it was also found to retain the fluorescence of (iodoacetamido)fluorescein attached specifically to the SH-1 thiol of the C-terminal 20-kDa segment. These results indicate that the 25- and 20-kDa peptides are in close contact with the ATPase active site.     

Characterization of an actin flow artifact that occurs during the presteady state dissociation of actosubfragment-1

Models for the activation of the myosin subfragment-1 (S-1) ATPase activity by actin describe transitions that occur between kinetic intermediate states during steady state hydrolysis of ATP. These states consist of myosin-nucleotide complexes in rapid equilibrium binding with actin, but steady state measurements of actin binding during hydrolysis lead only to a weighted average of the individual binding constants involved. In the current work, in order to determine the individual binding constants involved in the activation process, we have investigated the presteady state kinetics of the dissociation of actomyosin by ATP. We find that an actin flow artifact appears to dominate the time course of dissociation, and characterization of this artifact reveals that its magnitude rises linearly (approximately) with the concentration of bound S-1. Attempts to subtract the actin flow artifact from the actoS-1 dissociation signal were not entirely successful due at least partially to the transient nature of the bound S-1 concentration in the first few milliseconds. However, further studies reveal that if the order of addition of actin, ATP, and S-1 are varied, the observed light scattering transients are essentially superimposable. One possible explanation of these data is that the binding constants for myosin-ATP and myosin-ADP-Pi to actin are equal. However, it is also possible that the flow artifact is so large that further analysis is precluded. In addition, we show that the actin flow artifact has little effect on the fluorescence measurements of the phosphate burst reported previously. Therefore, the prior interpretation of the fluorescence data remains unchanged.     

Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole

The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) reacted specifically with 1.9 lysyl residues/mol of the myosin subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+- and Ca2+-ATPase activities were almost completely inhibited, whereas the Mg2+-ATPase activity was increased to 180% of original activity. The actin-activated Mg2+-ATPase activity was decreased to 30% of original activity by this modification. However, affinity of S-1 for actin in the presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was quenched on addition of ATP, suggesting that ATP induced conformational changes around the NBD groups attached to S-1. Tryptic digestion of the modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa peptide of S-1, more precisely the 45-kDa peptide. These results confirm the recent reports that the 50-kDa peptide of S-1 is involved in the myosin ATPase reaction (K?rner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).     

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.     

Fluorescence studies on the nucleotide- and Ca2+-binding domains of molluscan myosin.

The effects of nucleotides and Ca2+ on the intrinsic tryptophan fluorescence of molluscan myosin and its proteolytic fragments were studied. By using these proteins from the scallop, Pecten maximus, the existence of two distinct tryptophan-containing domains was established, which respond independently to ATP and Ca2+-specific binding. The latter is located in the 'neck' region of the myosin, which constitutes the regulatory domain. Subfragment 1, lacking the regulatory domain, responded only to ATP binding. On the other hand a tryptic fragment comprising the regulatory domain responded only to Ca2+ binding. Subfragment 1, containing the regulatory domain, responded to both ATP and Ca2+, but its ATPase activity was Ca2+-insensitive. By contrast, the ATPase activity of HMM was Ca2+-sensitive. Increasing the ionic strength had a detrimental effect on Ca2+-sensitivity, and fluorescence studies on solubilized myosin were therefore of limited value. Myosin and its fragments from other molluscan species which were investigated produced similar changes to those of Pectan maximus.     

Characterization of the motor activity of mammalian myosin VIIA

Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.     

Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate.

A single cycle of adenosine 5'-triphosphate (ATP) hydrolysis by a complex of actin and myosin subfragment one (acto-S-1) was studied in a stopped-flow apparatus at low temperature and low ionic strength, using light scattering to monitor the interaction of S-1 with actin and fluorescence to detect the formation of fluorescent intermediates. Our results show that the addition of a stoichiometric concentration of ATP to the acto-S-1 causes a cycle consisting of first, a rapid dissociation of the S-1 from actin by ATP; second, a slower fluorescence change in the S-1 that may be related to the initial phosphate burst; and third, a much slower rate limiting recombination of the S-1 with actin. This latter step equals the acto-S-1 steady-state adenosine 5'-triphosphatase (ATPase) rate at both low and high actin concentrations, and like the steady-state ATPase levels off at a V max of 0.9s-1 at high actin concentration. Therefore, the release of adenosine 5'-diphosphate and inorganic phosphate is not the rate-limiting step in the acto-S-1 ATPase. Rather, a slow first-order step corresponding to the previously postulated transition from the refractory to the nonrefractory state precedes the rebinding of the S-1 to the actin during each cycle of ATP hydrolysis.     

Activation of skeletal S-1 ATPase activity by actin-tropomyosin-troponin. Effect of Ca++ on the fluorescence transient.

Regulation in striated muscles primarily involves the effect of changes in the free calcium concentration on the interaction of subfragment-1 (S-1) with the actin-tropomyosin-troponin complex (henceforth referred to as [acto]R). At low concentrations of free Ca++ the rate of ATP hydrolysis by (acto)R S-1 can be as much as 20-fold lower than that in the presence of high free Ca++, even though the binding of S-1 to (actin)R in the presence of ATP is virtually independent of the calcium concentration. This implies that the mechanism of regulation involves a kinetic transition between actin-bound states, rather than the result of changes in actin binding. In the current work, we have investigated the fluorescence transient that occurs with the binding and hydrolysis of ATP both at low and high free [Ca++]. The magnitude of this transition at low free [Ca++] is higher than at high free [Ca++]. At low free [Ca++], the rate of the fluorescence transient either stays constant or decreases slightly with increasing free actin concentrations, but at high free [Ca++] the rate increases slightly with increasing free actin concentration. The observed changes in rate are not great enough to be of regulatory importance. The results of the fluorescence transient experiments together with the binding studies performed at steady state also show that neither the binding of M.ATP or M.ADP.Pi to (actin)R is appreciably Ca++ sensitive. These data imply that an additional step (or steps) in the ATPase cycle, i.e., other than the burst transition, must be regulated by calcium.     

Incorporation of 6-carboxyfluorescein into myosin subfragment 1

We describe for the first time the introduction of a label into the "50K" domain of myosin subfragment 1 (S-1), and we investigate the properties of this fluorescent modification in relation to the ATPase and actin-binding activities, both residing in the myosin head. The labeling consists of a major incorporation of 6-carboxyfluorescein into the "50K" domain of S-1. Using different conditions for tryptic digestion that allowed a fragmentation of the "50K" domain with a loss of 5 kilodaltons (kDa) leading to a final product of 45 kDa, we have shown that the fluorescent dye remains in the 45-kDa final product. By studying cross-linking as a function of time, we have demonstrated that the "50K" domain and the 45-kDa fluorescent peptide are equally cross-linkable to actin. We have also investigated the K+EDTA-, Ca2+-, Mg2+-, and actin-activated ATPase activities of this modified S-1 and after purification observed no enzymatic changes.     

Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal alpha-actin. Fab fragments of these antibodies (S alpha N Fab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 degrees C. Binding experiments carried out in the presence of ATP at 5 degrees C revealed that the catalytic inhibition was related to the inhibition of S-1 binding to actin by Fab. At equimolar ratios of Fab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of Fab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.     

Cryoenzymic studies on actomyosin ATPase. Evidence that the degree of saturation of actin with myosin subfragment 1 affects the kinetics of the binding of ATP

The initial steps of actomyosin subfragment 1 (acto-S1) ATPase (dissociation and binding of ATP) were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The dissociation kinetics were followed by light scattering in a stopped-flow apparatus, and the binding of ATP was followed by the ATP chase method in a rapid-flow quench apparatus. The data from the chase experiments were fitted to E + ATP in equilibrium (K1) E.ATP----(k2) E*ATP, where E is acto-S1 or S1. The kinetics of the binding of ATP to acto-S1 were sensitive to the degree of saturation of the actin with S1. There was a sharp transition with actin nearly saturated with S1: when the S1 to actin ratio was low, the kinetics were fast (K1 greater than 300 microM, k2 greater than 40 s-1); when it was high, they were slow (K1 = 14 microM, k2 = 2 s-1). With S1 alone K1 = 12 microM and k2 = 0.07 S-1. With acto heavy meromyosin (acto-HMM) the binding kinetics were the same as with saturated acto-S1, regardless of the HMM to actin ratio. The dissociation kinetics were independent of the S1 to actin ratio. Saturation kinetics were obtained with Kd = 460 microM and kd = 75 S-1. The data for the saturated acto-S1 could be fitted to a reaction scheme, but for lack of structural information the abrupt dependence of the ATP binding kinetics upon the S1 to actin ratio is difficult to explain.(ABSTRACT TRUNCATED AT 250 WORDS)