ATP-induced dissociation of rabbit skeletal actomyosin subfragment 1. Characterization of an isomerization of the ternary acto-S1-ATP complex

The adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) induced dissociation of actomyosin subfragment 1 (S1) has been investigated by monitoring the light scattering changes that occur on dissociation. We have shown that ATP gamma S dissociates acto-S1 by a mechanism similar to that of ATP but at a rate 10 times slower. The maximum rate of dissociation is limited by an isomerization of the ternary actin-S1-nucleotide complex, which has a rate of 500 s-1 for ATP gamma S and an estimated rate of 5000 s-1 for ATP (20 degrees C, 0.1 M KCl, pH 7.0). The activation energy for the isomerization is the same for ATP and ATP gamma S, and both show a break in the Arrhenius plot at 5 degrees C. The reaction between acto-S1 and ATP was also followed by the fluorescence of a pyrene group covalently attached to Cys-374. We show that the fluorescence of the pyrene group reports the isomerization step and not actin dissociation. The characterization of this isomerization is discussed in relation to force-generating models of the actomyosin cross-bridge cycle.     

Energetics and mechanism of actomyosin adenosine triphosphatase.

Rate constants were determined for the reaction of actin with subfragment 1 (S1), S1-product complex, heavy meromyosin (HMM), and HMM-products complex for a range of temperatures, pH's, and ionic strengths. For actin concentrations up to 10 muM, the rate of reassociation of the product intermediate was equal to the rate of actomyosin subfragment 1 (acto-S1) or acto-HMM adenosine triphosphatase (ATPase). Therefore, under these conditions, the only important pathway for adenosine triphosphate hydrolysis is through the dissociation and recombination of S1 or HMM. The apparent rate constants for the association of S1 and S1-product with actin showed a similar large ionic strength dependence. The S1-product reaction had a large temperature dependence paralleling the rate of acto-S1 ATPase, while the reaction with S1 had a much smaller variation with temperature. The low value of the rate constant for the S1-product reaction and its relationship to the s1 areaction suggests that the apparent rate constant does not measure a simple second-order reaction. A plausible mechanism is a rapid equilibrium for the binding step, followed by a transition (product release) which increases the association constant. A refractory state could also reduce the apparent rate constant of recombination. An approximate assignment of equilibrium constants for the acto-S1 ATPase reaction was made based on the interpretation of the present evidence and equilibrium constnats for the S1 ATPase.     

Transient kinetic analysis of N-phenylmaleimide-reacted myosin subfragment-1

Alkylation of myosin's Cys-707 (SH1) and Cys-697 (SH2) has profound consequences for myosin's ability to interact with actin and hydrolyze MgATP. Pre-steady-state measurements of myosin-S1 alkylated at SH1 and SH2 by N-phenylmaleimide (NPM) in the presence of ATP were taken to identify the steps of the reaction that are altered. It was found that the rate constant most affected by this modification is the apparent rate of the ATP hydrolysis step. This rate constant is reduced 20000-fold, an effect comparable in magnitude to the effect of the same modification on the binding of MgATP to S1 or acto-S1 [Xie, L., and Schoenberg, M. (1998) Biochemistry 37, 8048]. In contrast, the rate constants of phosphate release and dissociation of acto-S1 by ATP were reduced <20-fold. For unmodified S1, the enhancement of fluorescence seen after addition of ATP had the same rate constant as the ATP hydrolysis step (S1.ATP if S1.ADP.Pi) measured by single-turnover experiments in a quench-flow experiment. This is consistent with results previously observed [Johnson, K. A., and Taylor, E. W. (1978) Biochemistry 17, 3432]. However, NPM-modified S1 exhibited virtually no fluorescence enhancement upon ATP binding. This provides further evidence that M.ATP is the predominant intermediate of NPM-S1-catalyzed ATP hydrolysis.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

Kinetic mechanism of 1-N6-etheno-2-aza-ATP hydrolysis by bovine ventricular myosin subfragment 1 and actomyosin subfragment 1. The nucleotide binding steps

The large change in fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) has been used to investigate the kinetic mechanism of etheno-aza nucleotide binding to bovine cardiac myosin subfragment 1 (myosin-S1) and actomyosin subfragment 1 (actomyosin-S1). The time course of nucleotide fluorescence enhancement observed during epsilon-aza-ATP hydrolysis is qualitatively similar to the time course of tryptophan fluorescence enhancement observed during ATP hydrolysis. In single turnover experiments, the nucleotide fluorescence rapidly increases to a maximum level, then decreases with a rate constant of 0.045 s-1 to a final level, which is about 30% of the maximal enhancement; a similar fluorescence enhancement is obtained by adding epsilon-aza-ADP to cardiac myosin-S1 or actomyosin-S1 under the same conditions (100 mM KCl, 10 mM 4-morpholinepropanesulfonic acid, 5 mM MgCl2, 0.1 mM dithiothreitol, pH 7.0, 15 degrees C). The kinetic data are consistent with a mechanism in which there are two sequential (acto)myosin-S1 nucleotide complexes with enhanced nucleotide fluorescence following epsilon-aza-ATP binding. The apparent second order rate constants of epsilon-aza-ATP binding to cardiac myosin subfragment 1 and actomyosin subfragment 1 are 2-12 times slower than those for ATP. Actin increases the rate of epsilon-aza-ADP dissociation from bovine cardiac myosin-S1 from 1.9 to 110 s-1 at 15 degrees C which can be compared to 0.3 and 65 s-1 for ADP dissociation under similar conditions. Although there are quantitative differences between the rate and equilibrium constants of epsilon-aza- and adenosine nucleotides to cardiac actomyosin-S1 and myosin-S1, the basic features of the nucleotide binding steps of the mechanism are unchanged.     

Demembranated muscle fibers catalyze a more rapid exchange between phosphate and adenosine triphosphate than actomyosin subfragment 1

The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.     

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.     

Probing actomyosin interactions with 2,4-dinitrophenol

Access to different intermediates that follow ATP cleavage in the catalytic cycle of skeletal muscle actomyosin is a major goal of studies that aim toward an understanding of chemomechanical coupling in muscle contraction. 2,4-Dinitrophenol (DNP, 10(-2) M) inhibits muscle contraction, even though it accelerates the ATPase activity of isolated myosin. Here we used myosin subfragment 1 (S1), acto-S1 and mammalian skinned fibers to investigate the action of DNP in the presence of actin. DNP increases acto-S1 affinity and at the same time reduces the maximum rate of turnover as [actin]-->infinity. In skinned fibers, isometric force is reduced to the same extent (K0.5 approximately equal to 6 mM). Although actin activates Pi release from S1 at all DNP concentrations tested, the combination of enhanced S1 activity and reduced acto-S1 activity leads to a reduction in the ratio of these two rates by a factor of 30 at the highest DNP concentration tested. This effect is seen at low as well as at high actin concentrations and is less pronounced with the analog meta-nitrophenol (MNP), which does not inhibit the acto-S1 ATPase. Arrhenius plots for acto-S1 are parallel and linear between 5 and 30 degrees C, indicating no abrupt shifts in rate-limiting step with either DNP or MNP. Analysis of the reduction in isometric force with increasing Pi concentrations suggests that DNP and MNP stabilize weakly bound cross-bridges (AM.ADP.Pi). In addition, MNP (10(-2) M) increases the apparent affinity for Pi.     

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)     

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.     

X-ray diffraction evidence for the lack of stereospecific protein interactions in highly activated actomyosin complex

The structure of actomyosin complex while hydrolyzing ATP was investigated by recording X-ray diffraction patterns from rabbit skeletal muscle fibers, in which exogenously introduced rabbit skeletal subfragment-1 (S1) was covalently cross-linked to the endogenous actin filaments in rigor by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Approximately two-thirds of the introduced S1 was cross-linked. The cross-linking procedure did not affect the profile of the S1-induced enhancement of the actin-based layer line reflections in rigor, indicating that the acto-S1 interactions remained highly stereospecific. In the presence of ATP, the MgATPase of the S1 was highly activated regardless of calcium levels, presumably because the availability of the stereospecific binding sites for both proteins was maximized by the cross-linking. However, the diffraction pattern in the presence of ATP was striking in that the intensity profile of the strong 1/5.9 nm(-1) layer lines was indistinguishable from that from bare actin filaments, despite the fact that the majority of the S1 was still associated with actin. The change of the intensity profiles upon addition of ATP was completely reversible. Model calculations showed that this result can be explained if the S1 is not only swinging around its pivoting point, but the pivoting point itself is also moving on the actin surface in a range of a few nanometers. The results suggest that the stereospecific binding sites, which have been considered important for actomyosin cycling, are paradoxically left unoccupied for most of the time in this highly activated actomyosin complex.     

Structural transition at actin's N-terminus in the actomyosin cross-bridge cycle

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of the weakly bound actomyosin states than in the formation of the strongly bound actomyosin states. It has been the goal of this project to determine whether the interaction of actin's N-terminus with myosin changes upon transition between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus and replacement of the C-terminal cysteine with alanine. The N-terminal cysteine was labeled stoichiometrically with pyrene maleimide, and the properties of the modified mutant actin were examined prior to spectroscopic measurements. Among these properties, actin polymerization, strong S1 binding, and the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast actin, while small changes were observed in the weak S1 binding and the in vitro motility of actin filaments. Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measured for the strongly (with or without ADP) and weakly (with ATP and ATPgammaS) bound acto-S1 states. The fluorescence increased in each case, but the increase was greater (by about 75%) in the presence of MgATP and MgATPgammaS than in the rigor state. This demonstrates a transition at the S1 contact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to structural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 states.     

Interaction of fluorescently labeled myosin subfragment 1 with nucleotides and actin

Isolated myosin heads (subfragment 1) were modified by covalent attachment of 5-(iodoacetamido)fluorescein or 5-(iodoacetamido)salicylic acid to the essential sulfhydryl group SH1. The extrinsic fluorescence of the modified proteins was sensitive to binding of nucleotides and F-actin. With the fluorescein derivative [subfragment 1 (S1) modified with 5-(iodoacetamido)fluorescein (IAF) at SH1 (S1-AF)], association with MgADP decreased the probe fluorescence by 30%, whereas binding to actin increased the emission by a factor of 2. In the ternary complex acto-S1-AF X MgADP, the effect of nucleotide on the intensity of the attached fluorescein canceled the effect of actin. The fluorescence state of this ternary complex was similar to that of S1-AF X MgADP. The emission of S1-AF was resolved into two components with lifetimes of 4.3 and 0.6 ns and relative contributions of 33% and 67%, respectively. Interaction of S1-AF with nucleotides and actin did not alter the lifetimes but significantly shifted their fractional contributions. Quenching studies showed that the short lifetime likely arose from the fluorescein moiety statically quenched by internal groups. Binding of MgADP to the salicylate derivative [S1 modified with 5-(iodoacetamido)salicylic acid at SH1 (S1-SAL)] induced a 25% enhancement of the probe fluorescence, whereas formation of acto-S1-SAL decreased the emission by 10% regardless of whether MgADP was bound to the protein. Both labeled S1 species bound MgADP with a similar affinity, comparable to that of unmodified S1 previously reported by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rate of MgADP binding to and dissociation from acto-S1

The rate of binding and dissociation of MgADP from its ternary complex with actin and S1 was measured by following the extent to which fixed concentrations of MgADP slow down MgATP-induced dissociation of acto-S1. The solution of the equations describing this process shows that at any MgADP concentration the apparent rate of acto-S1 dissociation should be proportional to a square root of the equilibrium constant for MgADP dissociation and to MgATP concentration. By measuring the apparent rate of acto-S1 dissociation as a function of MgATP concentration, the rate of MgADP binding and dissociation were determined as 5 X 10(6) M-1 X s-1 and 1400 s-1, respectively. These rates were unchanged by modification of SH1 thiol of S1 by a variety of fluorescence and spin-labels, but dissociation rate was drastically reduced when SH1 was labelled with 5-iodoacetamidofluorescein.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Inhibition of myofibrillar and actomyosin subfragment 1 adenosinetriphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenyl-5'-yl imidodiphosphate

Adenosine 5'-diphosphate (ADP), inorganic pyrophosphate (PPi), and adenyl-5'-yl imidodiphosphate (AMPPNP) act as competitive inhibitors of the ATPase of myofibrils and actomyosin subfragment 1 (acto-S1). At I = 0.2 M, pH 7, and 15 degrees C, the inhibition constants for rabbit myofibrils are 0.17, 3, and 5 mM, respectively; the values for frog myofibrils at 0 degrees C are very similar, being 0.22, 1.5, and 2.5 mM. The inhibition constant of AMPPNP is about 2 orders of magnitude larger than the reported dissociation constant for fibers [Marston, S. B., Rodger, C. D., & Tregear, R. T. (1976) J. Mol. Biol. 104, 263-276]. A possible reason for this difference is that AMPPNP binding results in the dissociation of one head of each myosin molecule. The inhibition constants for rabbit acto-S1 cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide measured under the same conditions were 0.12, 2.6, and 3.5 mM for ADP, PPi, and AMPPNP, respectively. The inhibition of cross-linked and native acto-S1 was compared at low ionic strength and was found to be similar. The value for ADP is very similar to reported values of the dissociation constant whereas the inhibition constants for AMPPNP and PPi are an order of magnitude weaker [Greene, L. E., & Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548].     

Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.     

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl₂, HMM hydrolyzed 3PP at a maximum rate of 0.016 s⁻¹ HMM⁻¹, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.     

ADP binding to myosin cross-bridges and its effect on the cross-bridge detachment rate constants

We have studied the binding of adenosine diphosphate (ADP) to attached cross-bridges in chemically skinned rabbit psoas muscle fibers and the effect of that binding on the cross-bridge detachment rate constants. Cross-bridges with ADP bound to the active site behave very similarly to cross-bridges without any nucleotide at the active site. First, fiber stiffness is the same as in rigor, which presumably implies that, as in rigor, all the cross-bridges are attached. Second, the cross-bridge detachment rate constants in the presence of ADP, measured from the rate of decay of the force induced by a small stretch, are, over a time scale of minutes, similar to those seen in rigor. Because ADP binding to the active site does not cause an increase in the cross-bridge detachment rate constants, whereas binding of nucleotide analogues such as adenyl-5'-yl imidodiphosphate (AMP-PNP) and pyrophosphate (PPi) do, it was possible, by using ADP as a competitive inhibitor of PPi or AMP-PNP, to measure the competitive inhibition constant and thereby the dissociation constant for ADP binding to attached cross-bridges. We found that adding 175 microM ADP to 4 mM PPi or 4 mM AMP-PNP produces as much of a decrease in the apparent cross-bridge detachment rate constants as reducing the analogue concentration from 4 to 1 mM. This suggests that ADP is binding to attached cross-bridges with a dissociation constant of approximately 60 microM. This value is quite similar to that reported for ADP binding to actomyosin subfragment-1 (acto-S1) in solution, which provides further support for the idea that nucleotides and nucleotide analogues seem to bind about as strongly to attached cross-bridges in fibers as to acto-S1 in solution (Johnson, R.E., and P. H. Adams. 1984. FEBS Letters. 174:11-14; Schoenberg, M., and E. Eisenberg. 1985. Biophysical Journal. 48:863-871; Biosca, J.A., L.E. Greene, and E. Eisenberg. 1986. Journal of Biological Chemistry. 261:9793-9800).