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.     

Separation of subfragment-1 of H-meromyosin into two equimolar fractions with and without formation of the reactive enzyme-phosphate-ADP complex.

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of Pi liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1). The size of the initial burst of Pi liberation of acto-S-1 was 0.35 mole/mole of S-l at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/moleof S-1 at ATP concentrations above 1 mole/mole of S-1...     

Nucleotide-induced specific fluorescent labeling of the 23-kDa NH2-terminal tryptic peptide of myosin ATPase by the serine-reactive reagent 9-anthroylnitrile

The fluorescent reagent 9-anthroylnitrile (ANN) reacted preferentially with serine among various amino acids tested. When the myosin subfragment-1 (S-1) was incubated with ANN, the 9-anthroyl (AN) group was covalently incorporated into the S-1 heavy chain. The incorporation of the AN group was enhanced by the presence of ATP and ADP. In the presence of ATP, 0.98 mol of the AN group was maximally incorporated into S-1. The resulting S-1 derivative exhibited four absorption maxima in the range of 300-400 nm and fluoresced strongly with an emission maximum at 462 nm upon excitation at 390 nm. The spectral properties were similar to those of the AN-derivatives of serine and polyserine. When 0.98 mol of the AN group was incorporated into S-1, the K+- and Ca2+-ATPase activities decreased to 30%, while the Mg2+-ATPase activity increased to 220% of the original value. Tryptic digestion of the labeled S-1 revealed that the AN group was attached only to the NH2-terminal 23-kDa tryptic peptide of the S-1 heavy chain. Neither the 20-nor the 50-kDa peptide was labeled with ANN. The results suggest that a serine residue, which becomes more reactive in the presence of the nucleotide, is located in the 23-kDa tryptic peptide of S-1.     

Proteolytic fragmentation of myosin: location of SH-1 and SH-2 thiols.

The heavy chain fragmentation pattern of native myosin when digested by proteolytic enzymes is influenced by such conditions as the nature of the proteolytic agent, ionic strength and presence or absence of divalent cations. HMM and S-1 produced by digestion of 14CNEM-labelled myosin under various conditions were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Purified samples of these species were digested under controlled conditions by chymotrypsin and trypsin and a comparison of the observed heavy chain fragmentation patterns led to a sequential arrangement of the proteolytic fragments. The main features of this arrangement are the following: a 21K molecular weight tryptic peptide is found at the N-terminal side of myosin heavy chain. Adjacent to it is a 48K peptide, then a 19.5K peptide containing the two SH-1 and SH-2 thiols. These three peptides constitute the heavy chain of S-1. Adjacent to this S-1 heavy chain is a tryptic (and also chymotryptic) 40K peptide. The rest of the HMM heavy chain on the C-terminus is a sequence susceptible to both chymotrypsin and trypsin attack yielding an undefined number of small peptides.     

Effect of mild heat treatment on actin and nucleotide binding of myosin subfragment 1

Chymotryptic subfragment 1 (S-1) prepared from rabbit skeletal myosin has lost its ATPase activity upon incubation at 35 degrees C for 3 h. The loss in ATPase activity was accompanied by the perturbation of the structure of the 50K domain as indicated by a dramatic increase in the tryptic susceptibility of this domain without any change in the susceptibility of the other domains of S-1. The perturbation starts at the C-terminal region of the domain as suggested by the appearance of a 29K intermediate protein band in the tryptic peptide pattern of the heat-treated S-1. The heat-treated molecule essentially retained its actin and polyphosphate binding ability, and the actin binding was still sensitive to the presence of ATP or pyrophosphate. However, as opposed to native S-1, in heat-treated S-1 the addition of ATP does not induce an increase in tryptophan fluorescence, and, in the case of the treated species, the fluorescence of 1,N6-ethenoadenosine 5'-diphosphate added to the mixture is quenchable by acrylamide. This latter observation suggests that the binding of the adenine ring of the nucleotide has been altered following the heat treatment. The results indicate that the actin and polyphosphate binding sites of S-1 are distinct and that they are relatively independent of the adenine ring binding site.     

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.     

Reactions of 1-N6-ethenoadenosine nucleotides with myosin subfragment 1 and acto-subfragment 1 of skeletal and smooth muscle

The fluorescent nucleotides epsilon ADP and epsilon ATP were used to study the binding and hydrolysis mechanisms of subfragment 1 (S-1) and acto-subfragment 1 from striated and smooth muscle. The quenching of the enhanced fluorescence emission of bound nucleotide by acrylamide analyzed either by the Stern-Volmer method or by fluorescence lifetime measurements showed the presence of two bound nucleotide states for 1-N6-ethenoadenosine triphosphate (epsilon ATP), 1-N6-ethenoadenosine diphosphate (epsilon ADP), and epsilon ADP-vanadate complexes with S-1. The equilibrium constant relating the two bound nucleotide states was close to unity. Transient kinetic studies showed two first-order transitions with rate constants of approximately 500 and 100 s-1 for both epsilon ATP and epsilon ADP and striated muscle S-1 and 300 and 30 s-1, respectively, for smooth muscle S-1. The hydrolysis of [gamma-32P] epsilon ATP yielded a transient phase of small amplitude (less than 0.2 mol/site) with a rate constant of 5-10 s-1. Consequently, the hydrolysis of the substrate is a step in the mechanism which is distinct from the two conformational changes induced by the binding of epsilon ATP. An essentially symmetric reaction mechanism is proposed in which two structural changes accompany substrate binding and the reversal of these steps occurs in product release. epsilon ATP dissociates acto-S-1 as effectively as ATP. For smooth muscle acto-S-1, dissociation proceeds in two steps, each accompanied by enhancement of fluorescence emission. A symmetric reaction scheme is proposed for the acto-S-1 epsilon ATPase cycle. The very similar kinetic properties of the reactions of epsilon ATP and ATP with S-1 and acto-S-1 suggest that two ATP intermediate states also occur in the ATPase reaction mechanism.     

Inhibition of nuclear histone proteolysis by nucleotides]

Effects of nucleotides on the proteolysis of histones in nuclei incubated at 37 degrees C during 1, 3 and 20 h were studied. It has been shown that the H1 histone is removed first during proteolysis and then the H3 and H2B histones are digested. The H4 histone is not cleaved even after 20 h incubation. PMSF and ATP inhibit the H1 cleavage when its structure was not disturbed before ATP, CTP, ADP, NAD+, AMP and NADH inhibit the partial cleavage of the core histones H3 and H2B. ATP, CTP, AMP and NADH prevent the total digestion of H2B. ATP and, at lower extent, CTP prevent the H3 digestion. ATP, CTP, ADP and NAD+ inhibit the cleavage of the H2A histone in the experiments with 20 h incubation, when H4 is only resistant in the absence of nucleotides. The data obtained suggest an important role of ATP and other nucleotides in maintaining the structure of histones and chromatin.     

Binding of myosin to actin in myofibrils during ATP hydrolysis

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.     

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.     

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.     

Different binding properties and affinities for ATP and ADP among sulfonylurea receptor subtypes, SUR1, SUR2A, and SUR2B

ATP-sensitive potassium (K(ATP)) channels, composed of sulfonylurea receptor (SURx) and Kir6.x, play important roles by linking cellular metabolic state to membrane potential in various tissues. Pancreatic, cardiac, and vascular smooth muscle K(ATP) channels, which consist of different subtypes of SURx, differ in their responses to cellular metabolic state. To explore the possibility that different interactions of SURx with nucleotides cause differential regulation of K(ATP) channels, we analyzed the properties of nucleotide-binding folds (NBFs) of SUR1, SUR2A, and SUR2B. SURx in crude membrane fractions was incubated with 8-azido-[alpha-(32)P]ATP or 8-azido-[gamma-(32)P]ATP under various conditions and was photoaffinity-labeled. Then, SURx was digested mildly with trypsin, and partial tryptic fragments were immunoprecipitated with antibodies against NBF1 and NBF2. Some nucleotide-binding properties were different among SUR subtypes as follows. 1) Mg(2+) dependence of nucleotide binding of NBF2 of SUR1 was high, whereas those of SUR2A and SUR2B were low. 2) The affinities of NBF1 of SUR1 for ATP and ADP, especially for ATP, were significantly higher than those of SUR2A and SUR2B. 3) The affinities of NBF2 of SUR2B for ATP and ADP were significantly higher than those of SUR2A. This is the first biochemical study to analyze and compare the nucleotide-binding properties of NBFs of three SUR subtypes, and our results suggest that their different properties may explain, in part, the differential regulation of K(ATP) channel subtypes. The high nucleotide-binding affinities of SUR1 may explain the high ability of SUR1 to stimulate pancreatic K(ATP) channels. It is also suggested that the C-terminal 42 amino acids affect the physiological roles of SUR2A and SUR2B by changing the nucleotide-binding properties of their NBFs.     

Tryptic cleavage and substructure of bovine cardiac myosin subfragment 1

The method of limited tryptic proteolysis has been used to compare and contrast the substructure of bovine cardiac myosin subfragment 1 (S-1) to that of skeletal myosin S-1. While tryptic cleavage of cardiac S-1, like that of skeletal S-1, yields three fragments, the 25K, 50K, and 20K peptides, the digestion of cardiac S-1 proceeds at a 2-fold faster rate. The increased rate of cleavage is due entirely to an order of magnitude faster rate of cleavage at the 25K/50K junction of cardiac S-1 compared to that of skeletal, with approximately equal rates of cleavage at the 50K/20K junctions. Actin inhibits the tryptic attack at this latter junction, but its effect is an order of magnitude smaller for the cardiac than for the skeletal S-1. Furthermore, the tryptic susceptibility of the 50K/20K junction of cardiac S-1 in the acto-S-1 complex is increased in the presence of 2 mM MgADP. This effect is not due to partial dissociation of the cardiac acto-S-1 complex by MgADP. Our results indicate that in analogy to skeletal S-1, the cardiac myosin head is organized into three protease-resistant fragments connected by open linker peptides. However, the much faster rate of tryptic cleavage of the 25K/50K junction and also the greater accessibility of the 50K/20K junction in the cardiac acto-S-1 complex indicate substructural differences between cardiac and skeletal S-1.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.     

Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon

Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates. In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP. We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP. Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation. Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon. The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides. Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion. Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site.     

Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges

Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane regulation of the chromosomal replication activity of E. coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.     

Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.     

Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.