Binding of Nucleotides at the Active Site Modulates the Local and Global Conformation of Myosin in Muscle Fibres

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres to study the effect of the binding of nucleotides (ADP and AMP⋅PNP) to myosin. The thermal unfolding of muscle fibres showed three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMP⋅PNP markedly affected the transitions, implying the strong interaction between AMP⋅PNP and catalytic domain, and partial dissociation of heads from actin. ADP produced only small changes in transition temperatures. Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMP⋅PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The conformation of the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin differ from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state to actin might have different local conformations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nucleotides Induced Changes in Skeletal Muscle Myosin by DSC,TMDSC and EPR

Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum. In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region The monoton decreasing TMDSC heat capacities show that C _p of biological samples should not be temperature independent. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nucleotide Analogue Induces Global and Local Changes in Muscle Fibres

The effect of AMP.PNP on the thermal stability and dynamics of myosin head were investigated by using DSC and different spin label technique for chemically skinned muscle fibres prepared from rabbit. The thermal unfolding of the fibres in rigor, strong as well as weak-binding state showed a complex process characterizing at least three discrete domain regions with different stability (T _m =54, 58.4 and 62.3°C). The unfolding at 54°C refers to the catalytic domain of myosin, whereas transition at T _m =58.4°C represents the rod-like region. In the presence of AMP.PNP only the parameters of the last transition changed significantly (T _m =70.4°C) showing an increased interaction between actin and myosin heads being attached to actin. Measurements on MSL-fibres (labelled at Cys-707 of myosin) in the presence of AMP.PNP showed that about half of the cross-bridges dissociated from actin. This fraction had a dynamic disorder, the other population had the same spectral feature as in rigor. In contrast, on TCSL-fibres AMP.PNP increased the orientational disorder of myosin heads, a random population of spin labels was superimposed on the ADP-like spectrum showing conformational and motional changes in the internal structure of myosin heads. ST EPR measurements reported increased rotational mobility of spin labels in the presence of AMP.PNP. The DSC and EPR results suggest that in the presence of AMP.PNP the attached heads have the same global orientation as in rigor, but the internal structure undergoes a local conformational change. This revised version was published online in July 2006 with corrections to the Cover Date.     

Thermal analysis in biological and medical applications

In this paper, I will sum up our research activity from this field performed in the last about 25 years. I will focus on three main points: basic muscle research in the different intermediate states of ATP hydrolysis cycle during muscle contraction, R&D activities to develop and test different dairy products and TA application in some surgical and diagnostic problem. Our initial research concerned the investigation of thermal stability of main muscle proteins clarifies their basic unfolding characteristics. Later we extended the thermal stability investigation from protein solution to the myosin myofibrils and to the higher organization of muscle proteins, the muscle fibres, checking the effect of nucleotides. At that time, it became possible to stabilize the different intermediate states of ATP hydrolysis up to the time of DSC measurement, using different P_i analogues (V_i, AlF_x and BeF_x) and non-hydrolysable ATP analogue (AMP.PNP). This way the targets were AM.ADP.V_i (and with AlF_x or BeF_x) so-called weak binding state, AM.ADP the “strong” binding state as well as the “rigor” AM complex state. AM.AMP.PNP state was used to mimic the AM.ATP state. With our R&D cooperation, a cold spreadable butter was successfully developed. We were a partner in the development of Ca-enriched cheese, in its spreadable form too as well as in the development and testing of different dairy products using probiotic cultures. Our TA activity covers a wide range of medical applications, e.g. investigation of the different abnormalities of human leg skeletal muscle, different stages of degeneration of human vertebral disc, to judge the goodness/applicability of different suture technique on tracheal cartilage in primary airway reconstruction or the characterization of different self-expandable stents implantation in the oesophagus treatment. We have joined those groups who try to use DSC in the diagnosis of different diseases from blood plasma, e.g. in the case of breast cancer, melanoma, in psoriasis.     

Vanadate (Vi) and ADP induced domain motions in myosin head by DSC and EPR

Thermal stability and internal dynamics of myosin head in psoas muscle fibres of rabbit in the intermediate state AM.ADP.P_i - mimicked by AM.ADP.V_i - of the ATP hydrolysis cycle was studied by differential scanning calorimetry and spin label electron paramagnetic resonance spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding ADP and weakly binding AM.ADP.V_i state of myosin to actin. The transition at 54.0°C can be assigned to the 50 k actin-binding domain. The transition at highest temperature (67.3°C) represents the unfolding of actin and the contributions arising from the nucleotide-myosin head interaction. The transition at 58.4°C reflects the melting of the large rod part of myosin. Nucleotide binding (ADP, ATP plus orthovanadate) induced shifts of the melting temperatures and produced changes in the calorimetric enthalpies. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads in agreement with DSC findings. This revised version was published online in July 2006 with corrections to the Cover Date.     

DSC study on the motor protein myosin in fibre system

We have examined by DSC the complexes of myosin with actin in fibre system in the absence of nucleotides and the intermediate state of ATP hydrolysis by mimicking stable complex with myosin and ADP and beryllium fluoride in muscle fibres. Comparing the DSC results with other structural analogues of phosphate P_i leads the conclusion that the AM.ADP.BeF_x complex favours the AM.ADP.P_i complex in fibre system. The deconvolution of DSC scans resulted in four transitions, the first three transition temperatures were almost independent of the intermediate state of the muscle, the last transition temperature was shifted to higher temperature, depending on the actual intermediate states of ATP hydrolysis. In AM.ADP.V_i state the transition temperature at the second and third transitions (actin binding domain and myosin rod) varied only slightly, whereas the last one (the fourth transition) shifted markedly to higher temperature depending on the ternary complex, e.g. in case of ADP plus BeF_x it was 77.7 °C, the highest value in weakly binding state of myosin to actin. The sum of calorimetric enthalpies of the first and last curves was practically constant, but their fractions depended on the state of the muscle. In strongly binding state of myosin to actin (rigor, ADP state) the fraction of the first transition was much larger, than the last one, whereas in weakly binding state of myosin to actin, the fraction of the first transition decreased at the expense of the last one. It supports also the view that these transitions are parts of the same portion of the myosin molecule.     

Effect of Nucleotides on Thermal Transitions of Myosin

The internal dynamics and the thermal stability of myosin in rabbit psoas muscle fibres in different intermediate states of the ATP hydrolysis cycle were studied by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding and weakly binding state of myosin to actin. The transition at 58.4°C can be assigned to the nucleotide-binding domain. The transition at highest temperature represents the unfolding of the actin and the contributions arising from the actin-myosin interaction. The transition of 54°C reflects the interaction between the subunits of myosin. Nucleotide binding induced shifts of the melting temperatures and produced variations in the calorimetric enthalpy changes. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads. This revised version was published online in August 2006 with corrections to the Cover Date.     

DSC study of glycerol-extracted muscle fibers in intermediate states of ATP hydrolysis

Summary The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·V_i, ADP·AlF₄) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were T_m1=52.9±0.7°C, T_m2=57.9±0.7°C, T_m3=63.7±1.0°C and T_m4=67.8±0.7°C, but the last transition increased to higher temperature depending on the P_i analogue.     

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid‐State NMR Spectroscopy

DnaB helicases are bacterial, ATP‐driven enzymes that unwind double‐stranded DNA during DNA replication. Herein, we study the sequential binding of the “non‐hydrolysable” ATP analogue AMP‐PNP and of single‐stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid‐state NMR. Phosphorus cross‐polarization experiments monitor the binding of AMP‐PNP and DNA to the helicase. ¹³C chemical‐shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP‐PNP addition into a conformation apt for ssDNA binding, and AMP‐PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP‐PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid‐state NMR can provide for the characterization of protein–DNA interactions and the interplay with ATP or its analogues.     

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide- and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak- binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intermediate states of myosin head during atp hydrolysis cycle in psoas muscle fibres by EPR and DSC

Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle, myosin and actin. The process is driven by the energy liberated from the hydrolysis of ATP. In the presence of CaATP the energy released from hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion of myosin head while bound to actin. It is suggested that myosin heads attached to actin produce conformational changes during the hydrolysis process of ATP, which results in a strain in the head portion of myosin in an ATP-dependent manner. These structural changes lead to a large rotation of myosin neck region relieving the strain. Paramagnetic probes and EPR spectroscopy provide direct method in which the rotation and orientation of specifically labelled proteins can be followed during muscle activity. In order to find correlation between local and global structural changes in the intermediate states of the ATPase cycle, the spectroscopic measurements were combined with DSC measurements that report domain stability and interactions.     

Rapid determination of creatine, phosphocreatine, purine bases and nucleotides (ATP, ADP, AMP, GTP, GDP) in heart biopsies by gradient ion-pair reversed-phase liquid chromatography.

A simple binary solvent method has been developed for the simultaneous determination of creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, GTP, GDP, IMP, NAD, inosine, adenosine, hypoxanthine and xanthine. This allows separation of the most important nucleotides present in myocardial biopsies as, for example, in studies using 31P NMR spectroscopy. In NMR spectra ATP and PCr are the only visible high-energy phosphates, therefore the status of other nucleotides and bases cannot be determined. The nucleotides, AMP degradation products, PCr and Cr in pig and rat heart muscle were resolved with 35 mM K2HPO4, 6 mM tetrabutylammonium hydrogensulfate buffer, pH 6.0, and a binary acetonitrile gradient on medium-bore, 250 mm or 125 mm x 3.9-4.6 mm I.D. steel octadecyl-bonded (C18) columns at a flow-rate of 1.5 or 1.0 ml/min. This method, optimized for use with older high-performance liquid chromatography pumps (100 microliters displacement heads), resolves the major porcine and rat myocardial nucleotides and degradation products within 22 min. The amounts found in normoxic porcine muscle are: Cr 9.21 +/- 0.75; hypoxanthine 1.40 +/- 0.14; PCr 7.20 +/- 1.2; IMP 1.34 +/- 0.13; beta NAD 1.82 +/- 0.23; AMP 0.10 +/- 0.04; GDP 0.05 +/- 0.02; ADP 1.23 +/- 0.09; GTP 0.19 +/- 0.01; ATP 4.45 +/- 0.32 mumol/g wet weight. The method, incorporating adenosine tetraphosphate as an internal standard, allows the documentation of changes in both the high-energy phosphates and their degradation products in a single analysis of myocardial samples as small as 200 micrograms (wet weight).     

Study of the Simultaneous Binding of ADP and ATP on Coupling Factor CF-1 by a Modification of the Hummel and Dreyer Method

Abstract

A modification of the Hummel and Dreyer method⁽¹⁾, based on anion exchange separation is used here for the study of the simultaneous binding of ADP and ATP on spinach coupling factor CF₁.

This method gives the same results as gel filtration (dissociation constant and number of sites) when ADP alone is present.

The extent of binding of ADP and ATP is approximately the same when mixed in equimolecular ratio, but since endogenous ADP is irreversible bound, this nucleotide is predominant on CF₁.

The binding of one nucleotide is partly prevented by prealable mixing of CF₁ with the other nucleotide. This phenomenon occurs likely at the level of high affinity sites, where binding would not be entirely reversible, contrarily to low affinity sites.

This method is of potential application for other ligands separable by anion exchange chromatography and for other types of chromatography (reversed phase).     

Transient kinetic and isotopic tracer studies of the myosin adenosine triphosphatase reaction.

From transient kinetic studies of the Mg2+-dependent adenosine triphosphatase of myosin subfragment 1, prepared from rabbit skeletal muscle, a seven-step mechanism has been proposed. Features of this mechanism include two-step processes for ATP and ADP binding in which the binary complex isomerizes in addition to a rapid nucleotide association step. In the case of ATP a large negative standard free energy change is associated with the isomerization. An overall rate-limiting isomerization of the myosin-product complex prior to product release has been identified. Studies on the mechanism of cleavage of ATP bound to the active site indicate the process is readily reversible and can account for the observation that more than one oxygen of the product phosphate arises from water. This proposal has been substantiated by the finding that the oxygen atoms of the gamma-phosphoryl group of bound ATP also undergo extensive exchange with water.     

AMP.PNP affects the dynamical properties of monomer and polymerized actin

Actin is the component of several biological systems and it plays important role in different biological processes, especially in cell motility. The actin-based motility is accompanied with ATP-consume, and the irreversible ATP hydrolysis is coupled with the polymerization of monomer actin into filamentous form. When an actin monomer is incorporated into a filament, the ATPase is activated, and thereby the polymer formation is promoted. The polymer formation and the ATP hydrolysis is associated with internal motions and significant changes of the conformation in reaction partners. In this article, the ATP nucleotide in monomer actin was exchanged by its non-hydrolyzable analogue adenylyl-imidodiphosphate (AMP.PNP), and using two biophysical methods, electron paramagnetic resonance spectroscopy (EPR) and differential scanning calorimetry (DSC), we studied the local and global changes in globular and fibrous actin following the nucleotide exchange. The paramagnetic probe molecule—a maleimide spin label—was attached to Cys-374 site of monomer actin, and its rotational mobility was derived at different temperature. In DSC measurements the transition temperatures of samples with different bound nucleotides were compared. From the measurements we could conclude, that the nucleotide exchange induces changes in the internal rigidity of the actin systems, AMP.PNP-actins showed longer rotational correlation time and increased thermal transition temperature.     

NMR identification of transient complexes critical to adenylate kinase catalysis

A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.     

核苷磷酸盐对含双芘侧链荧光敏感器化合物的荧光猝灭与分子识别

合成了一种在水溶液中对核苷磷酸盐ATP,ADP,AMP阴离子具有识别能力的荧光化学敏感器分子---化合物1。通过检测化合物1在水溶液中芘激基缔合物荧光发射强度随核苷磷酸盐、腺嘌呤等的加入而引起的变化,求出不同核苷磷酸盐及腺嘌呤对芘激基缔合物荧光发射的猝灭常数,并进行了比较研究。利用荧光发射强度随不同核苷磷酸盐引入而发生的变化计算出化合物1对不同核苷磷酸盐进行配位的配位稳定常数。结果表明所合成的化合物1对ATP有着良好的识别力选择功能。此外,还利用分子力学的计算方法对化合物1及其与核苷磷酸盐形成配合物后的分子构象及几何尺寸作了计算,并结合实验结果进行了初步讨论。     

Quantitative analysis of cation binding to the adenosine nucleotides using the variable ionic strength method: validation of the Debye-Hückel-Onsager theory of electrophoresis in the absence of counterion binding

The free solution mobilities of the adenosine nucleotides 5'-adenosine triphosphate (ATP), 5'-adenosine diphosphate (ADP), 5'-adenosine monophosphate (AMP), and 3'-5'-cyclic AMP (cAMP) have been measured in diethylmalonate buffers containing a wide variety of monovalent cations. The mobilities of all nucleotides increase gradually with the increase in intrinsic conductivity of the cation in the BGE. However, at a given conductivity, the mobilities observed for ATP, ADP, and AMP in BGEs containing alkali metal ions and other cations are lower than these observed in BGEs containing tetraalkylammonium ions. Since the mobility of cAMP is independent of the cation in the BGE, the results suggest that the relatively low mobilities observed for ATP, ADP, and AMP in BGEs containing cations other than a tetraalkylammonium ion are due to cation binding, reducing the effective net charge of the nucleotide and thereby reducing the observed mobility. To measure the binding quantitatively, the mobilities of the nucleotides were measured as a function of ionic strength. The mobilities of ATP, ADP, and AMP decrease nonlinearly with the square root of ionic strength (I(1/2)) in BGEs containing an alkali metal ion or Tris(+). By contrast, the mobilities decrease linearly with I(1/2) in BGEs containing a nonbinding quaternary ammonium ion, as expected from Debye-Hückel-Onsager (DHO) theory. The mobility of cAMP, a nonbinding analyte, decreases linearly with I(1/2), regardless of the cation in the BGE. Hence, a nonlinear decrease of the mobility of an analyte with I(1/2) appears to be a hallmark of counterion binding. The curved mobility profiles observed for ATP, ADP, and AMP in BGEs containing an alkali metal ion or Tris(+) were analyzed by nonlinear curve fitting, using difference mobility profiles to correct for the effect of the physical properties of BGE on the observed mobilities. The calculated apparent dissociation constants range from 22 to 344 mM, depending on the particular cation-nucleotide pair. Similar values have been obtained by other investigators, using different methods. Interestingly, Tris(+) and Li(+) bind to the adenosine nucleotides with approximately equal affinities, suggesting that positively charged Tris(+) buffer ions can compete with alkali metal ions in Tris-buffered solutions.     

Borate-nucleotide complex formation depends on charge and phosphorylation state

Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 microM nucleotide and 500 microM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3) showed that borate complexation with nicotinamide nucleotides was significantly influenced by the charge on the nicotinamide group and the number of phosphate groups on the adenine ribose. Borate binding decreased in the order of NAD(+), NADH, NADP(+) and NADPH. To investigate the relationship between complex formation and phosphorylation, association constants (K(A)) of borate-adenine (AMP, ADP, ATP), -guanine (GMP, GDP, GTP), -cytidine (CMP, CDP, CTP) and -uridine (UMP, UDP, UTP) complexes were compared. The results showed that the number of nucleotide phosphate groups was inversely proportional to the relative abundance of the borate complexes, with the K(A) of borate-nucleotide complex decreasing in the order mono-, di- and tri-phosphates (AMP approximately GMP approximately CMP approximately UMP > ADP approximately GDP approximately CDP approximately UDP > GTP > ATP approximately CTP approximately UTP). At pH 7.4, using ammonium bicarbonate buffer, only borate-NAD(+) complex was observed. This indicates that the borate-NAD(+) complex may be the most physiologically relevant of those studied.     

The binding of Mn2+ and ADP to myosin.

The metal ion requirement of myosin-ADP binding was investigated by use of Mn2+. Mn2+ binds to two sets of noninteracting sites on myosin which are characterized by affinity constants of 10(6) and 10(3), M(-1) at 0.016 M KCl concentration. The maximum number of sites is 2 for the high affinity and 20-25 for the low affinity set. Binding of Mn2+ to the high affinity sites increases the affinity of ADP binding to myosin. F-actin inhibits ADP binding (Kiely, B., and Martonosi, A., Biochim. Biophys. Acta 172: 158-170 [1969]), but even at F-actin concentrations much higher than that required to saturate the actin binding sites of myosin or its proteolytic fragments, significant ADP binding remained. The actin insensitive portion of ADP binding was inhibited by 10(-4) M inorganic pyrophosphate or ATP. The results are discussed on the basis of a model in which actin and ADP bind to myosin at distinct but interacting sites.