The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin.

The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.

Recent advances into vanadyl, vanadate and decavanadate interactions with actin.

Although the number of papers about "vanadium" has doubled in the last decade, the studies about "vanadium and actin" are scarce. In the present review, the effects of vanadyl, vanadate and decavanadate on actin structure and function are compared. Decavanadate (51)V NMR signals, at -516 ppm, broadened and decreased in intensity upon actin titration, whereas no effects were observed for vanadate monomers, at -560 ppm. Decavanadate is the only species inducing actin cysteine oxidation and vanadyl formation, both processes being prevented by the natural ligand of the protein, ATP. Vanadyl titration with monomeric actin (G-actin), analysed by EPR spectroscopy, reveals a 1:1 binding stoichiometry and a K(d) of 7.5 µM(-1). Both decavanadate and vanadyl inhibited G-actin polymerization into actin filaments (F-actin), with a IC(50) of 68 and 300 µM, respectively, as analysed by light scattering assays, whereas no effects were detected for vanadate up to 2 mM. However, only vanadyl (up to 200 µM) induces 100% of G-actin intrinsic fluorescence quenching, whereas decavanadate shows an opposite effect, which suggests the presence of vanadyl high affinity actin binding sites. Decavanadate increases (2.6-fold) the actin hydrophobic surface, evaluated using the ANSA probe, whereas vanadyl decreases it (15%). Both vanadium species increased the ε-ATP exchange rate (k = 6.5 × 10(-3) s(-1) and 4.47 × 10(-3) s(-1) for decavanadate and vanadyl, respectively). Finally, (1)H NMR spectra of G-actin treated with 0.1 mM decavanadate clearly indicate that major alterations occur in protein structure, which are much less visible in the presence of ATP, confirming the preventive effect of the nucleotide on the decavanadate interaction with the protein. Putting it all together, it is suggested that actin, which is involved in many cellular processes, might be a potential target not only for decavanadate but above all for vanadyl. By affecting actin structure and function, vanadium can regulate many cellular processes of great physiological significance.

Severing of F-actin by yeast cofilin is pH-independent.

Cofilin plays an important role in actin turnover in cells by severing actin filaments and accelerating their depolymerization. The role of pH in the severing by cofilin was examined using fluorescence microscopy. To facilitate the imaging of actin filaments and to avoid the use of rhodamine phalloidin, which competes with cofilin, alpha-actin was labeled with tetramethylrhodamine cadaverine (TRC) at Gln41. The TRC-labeling inhibited actin treadmilling strongly, as measured by epsilonATP release. Cofilin binding, detected via an increase in light scattering, and the subsequent conformational change in filament structure, as detected by TRC fluorescence decay, occurred 2-3 times faster at pH 6.8 than at pH 8.0. In contrast, actin filaments severing by cofilin was pH-independent. The pH-independent severing by cofilin was confirmed using actin labeled at Cys374 with Oregon Green 488 maleimide. The depolymerization of actin by cofilin was faster at high pH.

Regional differences in extracellular purine degradation in the prostatic and epididymal portions of the rat vas deferens.

1. The aim of the present study was to compare ecto-nucleotidase activities in rat bisected vas deferens using 1,N6-etheno(epsilon)-nucleotides (epsilon-ATP and epsilon-AMP) as substrates. Degradation was estimated by measuring the disappearance of the substrate and the appearance of its metabolites using HPLC with fluorescence detection. Incubation of tissue preparations (prostatic or epididymal portions) with 300 nmol/L epsilon-ATP at 37 degrees C caused a partial disappearance of epsilon-ATP and appearance of its metabolites (epsilon-ADP, epsilon-AMP and epsilon-adenosine). Incubation at 25 degrees C reduced epsilon-ATP degradation more in the prostatic than in the epididymal portion. 2. Incubation of tissue preparations with epsilon-AMP at 37 degrees C resulted in the disappearance of epsilon-AMP and the appearance of epsilon-adenosine, which was more pronounced in the epididymal than in the prostatic portion. Incubation at 25 degrees C reduced epsilon-AMP degradation more in the epididymal than in the prostatic portion. 3. Decreasing pH from 7.4 to 6.5 enhanced epsilon-AMP degradation only in the prostatic portion, whereas increasing pH from 7.4 to 8.5 enhanced epsilon-AMP degradation in both portions, but more markedly in the epididymal portion. The alkaline phosphatase inhibitors levamisole (10 mmol/L) and beta-glycerophosphate (10 mmol/L) reduced epsilon-AMP degradation only in the epididymal portion. 4. In conclusion, the results of the present study are compatible with the presence, in the bisected rat vas deferens, of an ecto-nucleotidase system that is involved in the degradation of extracellular purines, which may differ between the epididymal and prostatic portions, with the epididymal portion presenting a different and higher capacity to form adenosine.

Interdependence of profilin, cation, and nucleotide binding to vertebrate non-muscle actin.

The interaction of profilin and non-muscle beta,gamma-actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Profilin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides primarily due to an increased nucleotide dissociation rate constant, but the nucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile intermediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold. Profilin binds NF-actin with high affinity, K(D) = 0.013 microM, and slows the observed denaturation rate of NF-actin. Addition of ATP to NF-actin weakens the affinity for profilin and addition of Mg(2+) to ATP-actin further weakens the affinity for profilin. The high-affinity Mg(2+) of actin regulates binding of both nucleotide and profilin to actin and is important for actin interdomain coupling. The data suggest that profilin binding to actin weakens nucleotide binding to actin by disrupting Mg(2+) coordination in the actin central cleft.

Characterization of nucleotide transport into rat brain synaptic vesicles.

ATP transport to synaptic vesicles from rat brain has been studied using the fluorescent substrate analogue 1,N6-ethenoadenosine 5'-triphosphate (epsilon-ATP). The increase in intravesicular concentration was time dependent for the first 30 min, epsilon-ATP being the most abundant nucleotide. The complexity of the saturation curve indicates the existence of kinetic and allosteric cooperativity in the nucleotide transport, which exhibits various affinity states with K0.5 values of 0.39 +/- 0.06 and 3.8 +/- 0.1 mM with epsilon-ATP as substrate. The Vmax values obtained were 13.5 +/- 1.4 pmol x min(-1) x mg of protein(-1) for the first curve and 28.3 +/- 1.6 pmol x min(-1) x mg of protein(-1) considering both components. This kinetic behavior can be explained on the basis of a mnemonic model. The nonhydrolyzable adenine nucleotide analogues adenosine 5'-O-3-(thiotriphosphate), adenosine 5'-O-2-(thiodiphosphate), and adenosine 5'-(beta,gamma-imino)triphosphate and the diadenosine polyphosphates P1,P3-di(adenosine)triphosphate, P1,P4-di(adenosine)tetraphosphate, and P1,P5-di(adenosine)pentaphosphate inhibited the nucleotide transport. The mitochondrial ATP/ADP exchange inhibitor atractyloside, N-ethylmaleimide, and polysulfonic aromatic compounds such as Evans blue and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid also inhibit epsilon-ATP vesicular transport.

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.

Studies of chromaffin granule functioning by flow cytometry: transport of fluorescent epsilon-ATP and granular size increase induced by ATP.

Flow cytometry techniques, usually employed to characterize cellular populations, are reported here to be a valuable tool to approach the study of subcellular organelle functioning. Chromaffin granules rendered fluorescent by using an antibody against their membrane protein, synaptophysin, are detectable by flow cytometry. Moreover, these storage granules are able to transport the fluorescent ATP analogue, epsilon-ATP (1,N6-ethenoadenosine 5'-triphosphate), and the resulting granular fluorescence increase can also be followed by this technique. The saturation studies show a non-hyperbolic kinetic behaviour, with a two step curve. The K0.5 values were 0.26 and 2.5 mM and Hill numbers 1 and 6 respectively. In addition, an unexpected granular size increase, which was dependent on the epsilon-ATP concentration, occurred together with the fluorescence increase. Other nucleotide triphosphate substrates of V-ATPase, such as ATP or GTP, but not the non-hydrolyzable analogue ATP gamma S (adenosine 5'-O-(3-thiotriphosphate), mimic this effect, which exhibited sigmoidal saturation curves with K0.5 values of 1.8 and 3.1 mM for ATP and epsilon-ATP respectively. The V-ATPase inhibitors, suramin, EGTA or EDTA significantly reduced the granular size increase in the presence of ATP. Extragranular addition of noradrenaline has no effect by itself on the granular size, but significantly reduced the granular size increase induced by ATP. This effect was reversed by the amine transport inhibitor reserpine. The granular size increase induced by ATP was more effective in the presence of Cl- than Br- or I-. Moreover, no increase occurred in the presence of F- or acetate. The Cl- channel blockers were poorly effective, and only 2-(phenylamino)-benzoic acid (DPC) exhibited an effect on the ATP-induced granular size increase.

Structure determination and characterization of Saccharomyces cerevisiae profilin.

The structure of profilin from the budding yeast Saccharomyces cerevisiae has been determined by X-ray crystallography at 2.3 A resolution. The overall fold of yeast profilin is similar to the fold observed for other profilin structures. The interactions of yeast and human platelet profilins with rabbit skeletal muscle actin were characterized by titration microcalorimetry, fluorescence titrations, and nucleotide exchange kinetics. The affinity of yeast profilin for rabbit actin (2.9 microM) is approximately 30-fold weaker than the affinity of human platelet profilin for rabbit actin (0.1 microM), and the relative contributions of entropic and enthalpic terms to the overall free energy of binding are different for the two profilins. The titration of pyrene-labeled rabbit skeletal actin with human profilin yielded a Kd of 2.8 microM, similar to the Kd of 2.0 microM for the interaction between yeast profilin and pyrene-labeled yeast actin. The binding data are discussed in the context of the known crystal structures of profilin and actin, and the residues present at the actin-profilin interface. The affinity of yeast profilin for poly-L-proline was determined from fluorescence measurements and is similar to the reported affinity of Acanthamoeba profilin for poly-L-proline. Yeast profilin was shown to catalyze adenine nucleotide exchange from yeast actin almost 2 orders of magnitude less efficiently than human profilin and rabbit skeletal muscle actin. The in vivo and in vitro properties of yeast profilin mutants with altered poly-L-proline and actin binding sites are discussed in the context of the crystal structure.

The role of profilin in actin polymerization and nucleotide exchange.

Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.

The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP.

The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells. Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP. Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation.

Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation.

Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.

Effects of subtilisin cleavage of monomeric actin on its nucleotide binding.

The kinetics of ATP exchange on subtilisin-cleaved G-actin was investigated by measuring the fluorescence of 1,N6-ethenoadenosine 5'-triphosphate. The apparent dissociation rate of ATP (k-ATP) was 2.8-fold larger than that of intact G-actin in the presence of 300 microM free Ca2+. Analysis of the dependence of k-ATP on free Ca2+ showed that the dissociation rate constant of tightly bound Ca2+ was not significantly changed by subtilisin cleavage. On the other hand, an equilibrium binding study using 8-amino-2-[(2-amino-5-methylphenoxy)-methyl]-6-methoxyquinoline N,N,N',N'-tetraacetic acid (Quin 2) showed that the affinity of tightly bound Ca2+ for G-actin was reduced by about 13-fold after subtilisin treatment. Consequently, the stabilization by Ca2+ of ATP was weak in cleaved G-actin. Furthermore, the kinetic analysis of ATP exchange revealed that the binding equilibrium between ATP and divalent cation-free cleaved G-actin was much slower than that in the case of intact G-actin.

Presence of epsilon-adenosine tetraphosphate in chromaffin granules after transport of epsilon-ATP.

Adenosine 5'-tetraphosphate (Ap4) is a natural constituent of chromaffin granules with concentration values of 2.2 +/- 0.1 nmol/mg of protein and a ratio 245 +/- 40 times lower with respect to ATP (n = 4). The granular transport of epsilon-ATP resulted in a time- and concentration-dependent production of epsilon-adenosine tetraphosphate (epsilon-Ap4) at the intragranular level. The epsilon-Ap4 formation followed a hyperbolic saturation kinetic at low epsilon-ATP concentrations with K(m) value of 0.4 microM epsilon-ATP intragranular (1.15 pmol/mg of granular protein). Intragranular concentrations of epsilon-ATP higher than 500 pmol/mg of protein (approximately to 175 microM intragranular) resulted in a non-saturable production of epsilon-Ap4.

Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics.

We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai et al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from approximately 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm2 dmol-1) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg(2+)-ATP and Ca(2+)-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M-1 s-1. The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M-1 s-1, which makes the "orientation factor" 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg(2+)-ATP-actin, 4.4 x 10(-9) M for Mg(2+)-epsilon ATP-actin, and 1.2 x 10(-10) M for Ca(2+)-ATP-actin.

Myosin subfragment 1 activates ATP hydrolysis on Mg(2+)-G-actin.

The interaction of myosin subfragment 1 isoenzyme A2 (S1A2) with Mg(2+)-G-actin was studied. Polarization titrations of 1,5-IAEDANS-Mg(2+)-G-actin and of epsilon ATP-Mg(2+)-G-actin with S1A2 provided evidence that, similar to Ca(2+)-G-actin, the proteins form a tight binary complex. Significant amounts of oligomeric forms of actin in the presence and absence of S1 were not detected. The effect of S1A2 on the rates of nucleotide and metal dissociation and hydrolysis from Mg(2+)-actin was measured. The hydrolysis rate for [gamma-32P]ATP-actin in the G-acto-S1A2 complex (k- = 0.016 s-1) was faster than the rate of 32P liberation from the complex (k- = 0.004 s-1), obtained by measuring the liberation of [32P]orthophosphate from [alpha-32P]ATP-actin in the presence of a large excess of alkaline phosphatase. This indicates that most of actin's ATP was hydrolyzed before it was released to solution and that the dissociating nucleotide was ADP, for which the dissociation rate is higher than that for ATP. In agreement with this mechanism, S1A2 accelerated the dissociation of epsilon ATP but inhibited the dissociation of epsilon ADP from the complex. The activation of actin's ATPase is specific for Mg(2+)-G-actin and does not occur in Ca(2+)-G-actin. The effect of deoxyribonuclease I on the rates of nucleotide dissociation and hydrolysis was examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Quenching of fluorescent nucleotides bound to myosin: a probe of the active-site conformation.

The conformation of the active ATPase site of myosin subfragment 1 (S1) and actomyosin in myofibrils was probed by measuring the solvent accessibility of the bound ethenonucleotides epsilon ADP and epsilon ATP (during steady-state hydrolysis). Solvent accessibility was determined by measuring the quenching of fluorescence produced by the solvent-phase quencher acrylamide, 25-400 mM. The fraction of the nucleotides that were specifically bound to the active site was determined following sedimentation in the presence and absence of 5 mM ADP. In agreement with previous investigations, both epsilon ATP and epsilon ADP were almost completely protected from the quencher when bound to the active site of myosin. The solvent accessibility of both epsilon ADP and epsilon ATP varied with both temperature and ionic strength. The nucleotides became more accessible at higher temperatures and higher ionic strength. At 1 M KCl the quenching curve was biphasic, indicating that the nucleotide pocket of myosin can exist in both a closed form that allows little quenching and a more open form that allows considerable quenching. However, the transition between forms was not strongly coupled to the state of the nucleotide, with a similar protection observed for both epsilon ADP and for epsilon ATP during steady-state cycling. epsilon ADP bound to acto-S1 or to actomyosin in myofibrils displayed the same degree of protection as seen with S1 alone. A similar result is obtained during steady-state hydrolysis. Thus nucleotides in the myosin pocket do not become more accessible to the solvent when myosin binds to actin in either rigor-ADP or active complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Domain motion in actin observed by fluorescence resonance energy transfer.

Actin is composed of two well-separated globular domains which are further subdivided into two subdomains [Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., & Holmes, K. C. (1990) Nature 347, 37-44]. Subdomains 1 and 2 constitute the small domain, and subdomains 3 and 4 comprise the large domain. In order to test a hinge bending domain motion in actin such as observed in many kinases, fluorescence resonance energy transfer between two probes attached to each of the two domains was measured by steady-state and time-resolved fluorometers. The adenine base is bound in a hydrophobic pocket between subdomains 3 and 4, and Tyr-69 is located at subdomain 2. In the present study, the adenine moiety was labeled with a fluorescence donor, epsilon ATP, and tyrosine-69 was labeled with the energy acceptor, dansyl chloride. Assuming the random orientation factor k2 = 2/3, the distance between epsilon-adenine moiety and dansyl chloride attached to Tyr-69 in G-actin was determined to be 2.46 nm from steady-state fluorescence measurements. The addition of DNase I did not appreciably change the distance (less than 0.1 nm). The distance decreased to 2.27 nm during polymerization by the addition of phalloidin under physiological salt conditions. On the other hand, time-resolved fluorescence energy transfer measurements have been used to investigate a distribution of distances for a donor-acceptor pair. In G-actin, the mean distance between probes was 2.79 nm with a full width at half-maximum of 3.91 nm, indicating a large number of conformational substates in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of the actomyosin interfaces.

Recombinant DNA methods were used to obtain soluble, undenatured fragments of the heavy chain of myosin subfragment 1 (S-1). These fragments were of preselected lengths and could include protease-sensitive segments that are destroyed when other preparation methods are used. Actin binding by each of the three contiguous segments (residues 1-248, 249-524, and 518-722, essentially spanning the entire S-1 heavy chain) was demonstrated. ATP binding, comparable to that of native S-1, was obtained only with a segment consisting of residues 1-524. Competition among the various fragments for actin was also studied. The data are discussed in relation to the recently reported resolved structure of S-1 [Rayment, I., Rypnieski, R. W., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993) Science 261, 50-58].

Myosin subfragment 1 inhibits dissociation of nucleotide and calcium from G-actin.

The dissociation rates of 1,N6-ethenoadenosine 5'-triphosphate (epsilon ATP) and of Ca2+ from G-actin and its complex with myosin subfragment 1 (S1) were measured by recording a large decrease in the fluorescence intensity of the dissociating nucleotide. Under the experimental conditions employed, the binary G-acto-S1A2 complex does not polymerize (Chaussepied, P., and Kasprzak, A. A. (1989) Nature 342, 950-953). The released nucleotide was hydrolyzed either by alkaline phosphatase or by apyrase; to trap Ca2+, EDTA was used. From the anisotropy of N-iodoacetyl-N'-(5-sulfo-1- naphthyl)ethylenediamine (1,5-IAEDANS)-actin, it was established that during the dissociation of epsilon ATP, the G-acto-S1 complex remained stable and the equilibrium of the system was unaltered. The reactions followed first order kinetics. The dissociation rate constant, kd for epsilon ATP decreased from 5.5 x 10(-4) s-1 for free G-actin to 1 x 10(-4) s-1 for G-acto-S1A2; for Ca2+, kd was also similarly reduced from 2.8 x 10(-2) s-1 to 4 x 10(-3) s-1. Two proteolytically derived actin variants were also examined. For free subtilisin-cleaved actin, kd for epsilon ATP was elevated 2-fold but was almost unchanged for Ca2+. In the complex of the cleaved G-actin with S1A2, kd for both epsilon ATP and for Ca2+ were reduced. The removal of the last 3 amino acids from actin produced a derivative whose behavior in binding to S1, as well as in the kinetics of epsilon ATP and Ca2+ dissociation, was undistinguishable from the unmodified protein.