The role of the bound nucleotide in the polymerization of actin.

Three mucleotides, ATP, ADP, and an unsplit-table analog of ATP (adenylyl imidodiphosphate (AMPPNP)), were bound to monomeric actin, and their effects on the rate and extent of the actin polymerization were studied. The kinetics of polymerization, assayed by the change in OD232, followed a simple exponential curve. The rates of polymerization were equal for bound ATP and AMPPNP; both of which were three to five times faster than the rate for ADP. The concentration of actin monomers in apparent equilibrium with the polymer, G(180 degrees longitude), was determined. Values of G(180 degrees longitude) in 100 mM KCl were found for different nucleotides to be: G-ATP(180 degrees longitude) = 0.7 mu-M, G-AMPPNP(180 degrees longitude) = 0.8 MU-M, and G-ADP(180 degrees longitude) = 3.4 mu-M. The equilibrium constant of the polymerization is given by K = [G(180 degrees longitude)]-minus 1 when no nucleotide is split. The polymerization of actin-ATP is more complex due to the splitting of the nucleotide and our data require that this polymerization involves more than one step. The kinetic parameters for the polymerization of actin-ATP can be explained by a simple scheme in which the nucleotide dephosphorylation occurs in a step following the polymerization step. The conclusions are: (1) the binding of ATP to actin monomer promotes polymerization slightly more than the binding of ADP, (2) actin bound ATP provides less than 4 kJ/mol of free energy to promote polymerization, and (3) the dephosphorylation of the nucleotide is not coupled to polymerization.     

Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state

The correlation between the time courses of actin polymerization under continuous sonication and the associated ATP hydrolysis has been studied. ATP hydrolysis was not mechanistically coupled to polymerization, i.e. not necessary for polymerization, but occurred on F-actin in a subsequent monomolecular reaction. Under sonication, polymerization was complete in 10 s while hydrolysis of ATP on the polymer required 200 s. A value of 0.023 s-1 was found for the first order rate constant of ATP hydrolysis on the polymer at 25 degrees C, pH 7.8, in the presence of 0.2 mM ATP, 0.1 mM CaCl2, and 1 mM MgCl2, independent of the F-actin concentration. The conversion of ATP X F-actin to ADP X F-actin was accompanied by an increase in fluorescence of a pyrenyl probe covalently attached to actin, consistent with a 2-fold greater fluorescence for ADP X F-actin than for ATP X F-actin, with a rate constant of 0.022 s-1. In contrast, the fluorescence of F-actin labeled with 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole did not change significantly when ATP or ADP was bound. The direct consequence of the uncoupling between polymerization and ATP hydrolysis is the formation of an ATP cap at the ends of the filaments, which maintains the stability of the polymer, while most of the filament contains bound ADP. The heterogeneity of the filament with respect to ATP and ADP results in a nonlinear relationship between the rate of elongation and the concentration of G-actin with a discontinuity at the critical concentration, where the rate of growth is zero. In this respect, F-actin in ATP behaves similarly to microtubules in GTP.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

Evidence that F-actin can hydrolyze ATP independent of monomer-polymer end interactions

The rate of ATP hydrolysis in solutions of F-actin at steady state in 50 mM KC1, 0.1 mM CaC12 was inhibited by AMP and ADP. The inhibition was competitive with ATP (Km of about 600 microM) with Ki values of 9 microM for AMP and 44 microM for ADP. ATP hydrolysis was inhibited greater than 95% by 1 mM AMP. AMP had no effect on the time course of actin polymerization, ATP hydrolysis during polymerization, or the critical actin concentration. Simultaneous measurements of G-actin/F-actin subunit exchange and nucleotide exchange showed that nucleotide exchange occurred much more rapidly than subunit exchange; during the experiment over 50% of the F-actin-bound nucleotide was replaced when less than 1% of the F-actin subunits had exchanged. When AMP was present it was incorporated into the polymer, preventing incorporation of ADP from ATP in solution. F-actin with bound Mg2+ was much less sensitive to AMP than F-actin with bound Ca2+. These data provide evidence for an ATP hydrolysis cycle associated with direct exchange of F-actin-bound ADP for ATP free in solution independent of monomer-polymer end interactions. This exchange and hydrolysis of nucleotide may be enhanced when Ca2+ is bound to the F-actin protomers.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

Binding of 1,N6-ethanoadenosine triphosphate to actin

G-actin is known to bind one molecule of ATP. Its polymerization to F-actin is accompanied by the splitting off of the terminal phosphate of the bound nucleotide. We have found that the fluorescent 1,N⁶-ethanoadenosine triphosphate (?ATP) can substitute for ATP in G-actin and that G-actin containing bound ?ATP possesses essentially full polymerizability. The binding of this ATP analog has been studied by following the inactivation of the ?ATP·G-actin complex. The binding constant (4?5.7 × 10⁶ M^?1) obtained in the absence of EDTA is about 50% of that for ATP, while the binding constant obtained in the presence of EDTA (0.9?3.0 × 10⁵ M^?1) is comparable to those for ATP and ADP. These findings suggest that ?ATP can be used as a structural probe for actin. The fluorescence lifetime of ?ATP bound to G·actin is 36 nsec. The rotational relaxation time of ?ATP·G-actin is near 60 nsec. at 20°C.     

Structure of the actin-myosin complex produced by crosslinking in the presence of ATP

The structure of the actin-myosin complex during ATP hydrolysis was studied by covalently crosslinking myosin subfragment 1 (S1) to F-actin in the presence of nucleotides (especially ATP) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The fluorescence energy transfer was measured between N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine and 6-(iodoacetamide)fluorescein bound to the SH1 thiol of S1 and the Cys374 thiol of actin. The covalent acto-S1, produced by crosslinking in the absence of nucleotide or in the presence of ADP, showed transfer efficiency of 0.50 to 0.52 and intersite distance of 4.5 to 4.7 nm, which were equal to those obtained with non-crosslinked acto-S1 in the absence of nucleotide. However, the covalent acto-S1, produced by crosslinking in the presence of either 5'-adenylyl imidodiphosphate (AMPPNP) at high ionic strength or ATP, showed a significant decrease in the efficiency to 0.26 to 0.34 and hence an increase in the distance to 5.2 to 5.5 nm. These results suggest that AM-ATP and/or AM-ADP-P (formed during ATP hydrolysis) and AM-AMPPNP have a very different conformation from AM and AM-ADP (in which A is actin and M is myosin).     

Effects of chaetoglobosin J on the G-F transformation of actin

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.     

Actin-facilitated assembly of smooth muscle myosin induces formation of actomyosin fibrils

To identify regulatory mechanisms potentially involved in formation of actomyosin structures in smooth muscle cells, the influence of F-actin on smooth muscle myosin assembly was examined. In physiologically relevant buffers, AMPPNP binding to myosin caused transition to the soluble 10S myosin conformation due to trapping of nucleotide at the active sites. The resulting 10S myosin-AMPPNP complex was highly stable and thick filament assembly was suppressed. However, upon addition to F-actin, myosin readily assembled to form thick filaments. Furthermore, myosin assembly caused rearrangement of actin filament networks into actomyosin fibers composed of coaligned F-actin and myosin thick filaments. Severin-induced fragmentation of actin in actomyosin fibers resulted in immediate disassembly of myosin thick filaments, demonstrating that actin filaments were indispensable for mediating myosin assembly in the presence of AMPPNP. Actomyosin fibers also formed after addition of F-actin to nonphosphorylated 10S myosin monomers containing the products of ATP hydrolysis trapped at the active site. The resulting fibers were rapidly disassembled after addition of millimolar MgATP and consequent transition of myosin to the soluble 10S state. However, reassembly of myosin filaments in the presence of MgATP and F-actin could be induced by phosphorylation of myosin P-light chains, causing regeneration of actomyosin fiber bundles. The results indicate that actomyosin fibers can be spontaneously formed by F-actin-mediated assembly of smooth muscle myosin. Moreover, induction of actomyosin fibers by myosin light chain phosphorylation in the presence of actin filament networks provides a plausible hypothesis for contractile fiber assembly in situ.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

High microfilament concentration results in barbed-end ADP caps.

Current theory and experiments describing actin polymerization suggest that site-specific cleavage of bound nucleotide following F-actin filament formation causes the barbed ends of microfilaments to be capped first with ATP subunits, then with ADP bound to inorganic phosphate (ADP.Pi) at steady-state. The barbed ends of depolymerizing filaments consist of ADP subunits. The decrease in stability of the barbed-end cap accompanying the transition from ADP.Pi to ADP allows nucleotide hydrolysis and subsequent loss of Pi to regulate F-actin filament dynamics. We describe a novel computational model of nucleotide capping that simulates both the spatial and temporal properties of actin polymerization. This model has been used to test the effects of high filament concentration on the behavior of the ATP hydrolysis cycle observed during polymerization. The model predicts that under conditions of high microfilament concentration an ADP cap can appear during steady-state at the barbed ends of filaments. We show that the presence of the cap can be accounted for by a kinetic model and predict the relationship between the nucleotide concentration ratio [ATP]/[ADP], the F-actin filament concentration, and the steady-state distribution of barbed-end ADP cap lengths. The possible consequences of this previously unreported phenomenon as a regulator of cytoskeletal behavior are discussed.     

Diphasic transformations of F-actin. Effects of urea and MgCl2 on F-actin.

Asakura, Taniguchi and Oosawa [1]proposed that muscle actin polymer under sonic vibration is in a different state from that of the ordinary double stranded helical structure (F-actin), characterised by partially interrupted structures of F-actin, a state of "f-actin". In order to confirm different states for actin polymers [1, 2], physicochemical studies were made by measurements of viscosity, flow birefringence, electric birefringence, fluorescence, electron microscopy, quasielastic light scattering and ATP splitting. The following results were obtained. (1) F-actin polymers can undergo two processes of depolymerization upon treatment with urea and various salts as judged by measurements of flow birefringence and viscosity: one is a rapid process in a solution containing K+ or Ca2+ and urea; the other is a slow process following a brief rapid one in a solution containing Mg2+ and urea. (2) In the presence of Mg2+ and a suitable concentration of urea, F-actin (FMU-actin) appeared to exhibit different properties than ordinary F-actin; it had lower viscosity and lower flow birefringence and it had on the whole a more flexible polymer structure, also judging from experiments of quasielastic light scattering, electric birefringence. The different structure was confirmed directly be electron microscopic observation. The aromatic side chains of FMU-actin were also more mobile than those of F-actin judging from fluorescence measurements. The transformation between F-actin and FMU-actin was reversible. (3) The state of FMU-actin polymers was also characterized by ATP splitting; FMU-actin split about one mole of ATP into ADP and inorganic phosphate per mole of actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer is required for ATP splitting. F-actin in solutions containing K+ or Ca2+ and urea did not split ATP. FMU-actin activated on Mg-ATP-ase of myosin at nearly the same rate as that of F-actin. (4) We have postulated a flexible filament model (f-actin). The relationships between the structure of f-actin and its functional role for force generation during contraction are discussed.     

Crystalline actin tubes. I. Is the conformation of the lanthanide-induced actin tube monomer more like F-actin than G-actin?

Actin, isolated from rabbit skeletal muscle, forms highly-ordered aggregates when it binds six moles of the lanthanide ion, Gd3+. In the presence of 0.1 M KCl, these aggregates are referred to as actin tubes. The monomer contained in the repeating subunit of these tubes possess a number of functional characteristics which include: (i) binding to myosin or subfragment-1 of myosin; (ii) rapid conversion into filamentous Gd-actin which can activate myosin ATPase activity; (iii) a slow rate of exchange of the bound nucleotide; (iv) a slow rate of exchange of the metal cation; (v) a resistance to digestion by proteolytic enzymes. Additionally, the monomer of the Gd-actin tube structures appears to stoichiometrically bind ATP and exhibit a lower minimum protein concentration for tube formation than is needed for the formation of F-actin. The properties listed above suggest that the actin monomer, which comprises the Gd-actin tubes, bears little resemblance to either the G-actin monomer or the recently-described actin monomer conformation that exists under conditions that favour polymerization. The data suggest that the actin molecules which comprise the Gd-actin tube structures contain sites which bind myosin, nucleotide and metal cations and that these sites are similar to the sites on F-actin.     

Molecular movements promoted by metal nucleotides in the heavy-chain regions of myosin heads from skeletal muscle

Molecular movements generated in the heavy-chain regions (27-50-20(X 10(3)) Mr) of myosin S1 on interaction with nucleotides ATP, AMPPNP, ADP and PPi were investigated by limited proteolysis of several enzyme-metal nucleotide complexes in the absence and presence of reversibly bound and crosslinked F-actin. The rate and extent of the nucleotide-promoted conversion of the NH2-terminal 27 X 10(3) Mr and 50 X 10(3) Mr segments into products of 22 X 10(3) Mr and 45 X 10(3) Mr, respectively, were estimated to determine the amplitude of the molecular movements. The 22 X 10(3) Mr peptide was identified by amino acid sequence studies as being derived from cleavage of the peptide bond between Arg and Ile (at position 23 to 24). The 45 X 10(3) Mr peptide, previously shown to represent the NH2-terminal part of the 50 X 10(3) Mr region, would be connected to the adjacent C-terminal 20 X 10(3) Mr region by a pre-existing loop segment of about 5 X 10(3) Mr; the proteolytic sensitivity of the latter region is increased particularly by nucleotide binding. The tryptic reaction proved to be a sensitive indicator of the conformational state of the liganded heavy chain as the rate of peptide bond cleavage in the two regions is dependent on the nature of the bound ligand; it decreases in the order: ATP greater than AMPPNP greater than ADP greater than PPi. It depends also on the nature of the metal present, Mg2+ and Ca2+ being much more effective than K+. Binding of F-actin to the S1-MgAMPPNP complex affords significant protection against breakdown of 27 X 10(3) Mr and 50 X 10(3) Mr peptides, but with concomitant hydrolysis of the 50 X 10(3) Mr-20 X 10(3) Mr junction. Additionally, interaction of MgATP with HMM modulates the tryptic fission of the S1-S2 region. The overall data provide a molecular support for the two-state model of the myosin head and emphasize the involvement of the 50 X 10(3) Mr unit in the mechanism of coupling between the actin and nucleotide binding sites.     

Cooperativity in F-actin filaments on binding of myosin subfragments, demonstrated by fluorescence of 1, N6- ethenoadenosine diphosphate.

The fluorescence lifetime of 1,N⁶-ethenoadenosine diphosphate (?-ADP) is 33 ns when bound to F-actin at 4 °C. When heavy meromyosin or myosin subfragment-1 binds to the F-actin filament, the lifetime of ?-ADP drops, reaching 29 ns when every actin monomer is bound to a myosin head. The change in lifetime is a consequence of cooperative conformational changes among the actin monomers. The results of these experiments support the contention that there are differences in the ways in which the two heads of the myosin molecule interact with the actin filament.     

Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.     

Rotational dynamics of actin-bound intermediates in the myosin ATPase cycle.

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to detect the microsecond rotational motions of spin-labeled myosin subfragment one (MSL-S1) bound to actin in the presence of the ATP analogues AMPPNP (5'-adenylylimido diphosphate) and ATP gamma S [adenosine 5'-O-(3-thiotriphosphate)], which are believed to trap myosin in strongly and weakly bound intermediate states of the actomyosin ATPase cycle, respectively. Sedimentation binding measurements were used to determine the fraction of myosin heads bound to actin under ST-EPR conditions and the fraction of heads containing bound nucleotide. ST-EPR spectra were then corrected to obtain the spectrum corresponding to the ternary complex (actin.MSL-S1.nucleotide). The ST-EPR spectrum of MSL-S1.AMPPNP bound to actin is identical to that obtained in the absence of nucleotide (rigor complex), indicating no rotational motion of MSL-S1 relative to actin on the microsecond time scale. However, MSL-S1-ATP gamma S bound to actin is rotationally mobile, with an effective rotational correlation time (tau r) of 17 +/- 2 microseconds. This motion is similar to that observed previously for actin-bound MSL-S1 during the steady-state hydrolysis of ATP [Berger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8753-8757]. We conclude that, in solution, the weakly bound actin-attached states of the myosin ATPase cycle undergo microsecond rotational motions, while the strongly bound intermediates do not, and that these motions are likely to be involved in the molecular mechanism of muscle contraction.     

Impact of profilin on actin-bound nucleotide exchange and actin polymerization dynamics

We have investigated the effects of profilin on nucleotide binding to actin and on steady state actin polymerization. The rate constants for the dissociation of ATP and ADP from monomeric Mg-actin at physiological conditions are 0.003 and 0.009 s-1, respectively. Profilin increases these dissociation rate constants to 0.08 s-1 for MgATP-actin and 1.4 s-1 for MgADP-actin. Thus, profilin can increase the rate of exchange of actin-bound ADP for ATP by 140-fold. The affinity of profilin for monomeric actin is found to be similar for MgATP-actin and MgADP-actin. Continuous sonication was used to allow study of solutions having sustained high filament end concentrations. During sonication at steady state, F-actin depolymerizes toward the critical concentration of ADP-actin [Pantaloni, D., et al. (1984)J. Biol. Chem. 259, 6274-6283], our analysis indicates that under these conditions a significant number of filaments contain terminal ADP-actin subunits. Addition of profilin to this system increases the polymer concentration and increases the steady state ATPase activity during sonication. These data are explained by the fast exchange of ATP for ADP on the profilin-ADP-actin complex, resulting in rapid ATP-actin regeneration. An important function of profilin may be to provide the growing ends of filaments with ATP-actin during periods when the monomer cycling rate exceeds the intrinsic nucleotide exchange rate of monomeric actin.     

Polymerization of ADP-actin and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer

Polymerization under sonication has been developed as a new method to study the rapid polymerization of actin with a large number of elongating sites. The theory proposed assumes that filaments under sonication are maintained at a constant length by the constant input of energy. The data obtained for the reversible polymerization of ADP-actin under sonication have been successfully analyzed according to the proposed model and, therefore, validate the model. The results obtained for the polymerization of ATP-actin under sonication demonstrate the involvement of ATP hydrolysis in the polymerization process. At high actin concentration, polymerization was fast enough, as compared to ATP hydrolysis on the F-actin, to obtain completion of the reversible polymerization of ATP-actin before significant hydrolysis of ATP occurred. A critical concentration of 3 microM was determined as the ratio of the dissociation and association rate constants for the interaction of ATP-actin with the ATP filament ends in 1 mM MgCl2, 0.2 mM ATP. The plot of the rate of elongation of filaments versus actin monomer concentration exhibited an upward deviation at high actin concentration that is consistent with this result. The fact that F-actin at steady state is more stable than the ATP-F-actin polymer at equilibrium suggests that the interaction between ADP-actin and ATP-actin subunits at the end of the ATP-capped filament is much stronger than the interaction between two ATP-actin subunits.