Nucleotide Exchange, Structure, and Mechanical ... - Pollard Lab
Val. 267, NO.
THEJOURNALOF RIOLOCICALCHEMISTRY
62 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Issue of October 5, PP. 20339-20345,1992 Printed in U.S A .
Nucleotide Exchange, Structure, and Mechanical Properties of Filaments Assembled from ATP-actin and ADP-actin* (Received for publication, April 6, 1992)
Thomas D. Pollard$, Ilya Goldberg, and William H. Schwarz$ From the Departmentof Cell Biology and Anatomy, The Johns HopkinsMedical School, Baltimore, Maryland 21205 and the 21218 §Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland
The adenine nucleotide bound in the central cleft of the actin molecule stabilizes the protein, but in spite of extensive research (for review see Korn et al., 1987; Pollard, 1990; and Carlier, 1991) the underlying function of the nucleotide and its hydrolysis in the assembly of actin filaments in live cells has remained an enigma for years (for review see Cooper, 1991). It is well established that when actin with bound ATP (referred to here as ATP-actin)polymerizes, the ATP on each molecule is hydrolyzed (Strauband Feuer, 1950) afterits incorporation into a filament (Pollard and Weeds, 1984) in an irreversible reaction (Carlier et al., 1988) to ADP and inorganic phosphate (Pi). The hydrolysis reaction may occur randomly on the polymerized subunits (Pollard and Weeds, 1984) or may be favored at the interface between a central region consisting of ADP-actin and newly added ATP-actin subunits near the ends of the filament (Carlier et al., 1987). The Pi remains bound to the filament for some time (Carlier and Pantaloni, 1986),making rapidly growing filamentsa heterogeneous mixture of subunits with ATP, ADP-P,, or ADP. Eventually the Pi dissociates leaving filaments composed largely of ADP-actin with a few ATP- and ADP-Piactin subunits near the ends. * This work was supported by National Institutes of Health Research Grant GM-26338. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondenceshould be addressed Dept. of Cell Biology and Anatomy, The Johns Hopkins Medical School, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-5664; Fax: 410.9554129.
The nucleotide composition of monomers has an easily measured impact on the elongation of actin filaments (for review see Korn et al., 1987). At the barbed end, we find by direct electron microscopic measurements that ATP-actin binds with a diffusion-limited rate constantof lo7M" s-l and dissociates slowly, about 1 s" (Pollard, 1986; Drenckhahn and Pollard, 1986). (Similar values of these and other rate constants have been measured by other methods; for review see Korn et al., 1987.) In contrast, ADP-actin binds about 20% as fast and dissociates about 5 times faster than ATPactin. ADP-P-actin has a critical concentration similar to ATP-actin (Rickard and Sheterline, 1988; Wanger and Wegner, 1987; Carlier and Pantaloni, 1988), but there arenot yet any direct measurements of the elongation rate constants. The effect of nucleotide on growth at thepointed end is less clear. Both ATP- andADP-actin associate at rates lower than expected for a diffusion-limited reaction (Pollard, 1986; Drenckhahn and Pollard, 1986). Both dissociate slowly, especially ADP-actin. The ratios of the association and dissociation rate constantsgive the critical actin monomer concentrations for elongation. In ATP with physiological concentrations of divalent cations, the critical concentration at the pointed end is substantially higher than at the barbed end. This should lead to a slow flux of subunitsthrough the filaments from the barbed end to the pointed end driven by ATP hydrolysis. These properties suggest that nucleotide hydrolysis might be used by cells to power subunit flux through actin filaments and possibly also to prepare the filaments for rapid disassembly at a time different from the time of assembly. In live cells actin subunits flux rapidly through filaments at the leading edge (Wang, 1985; Forscher and Smith,1988), and many actin filaments turn over rapidly (Amato and Taylor, 1986; Theriot and Mitchison, 1991). Neither the rapid flux nor turnover appears to be compatible with the relatively slow dynamics of purified actin filaments i n vitro, so we do not yet understand how the polymer dynamics in vivo are related to nucleotide hydrolysis (for reviewsee Cooper, 1991). Excluded volume effects (Drenckhahn and Pollard, 1986) or accessory proteins could speed up subunit exchange in the cell. Recent observations of Janmey et al. (1990), showing a possible effect of nucleotide hydrolysis on actin filament structure, mechanical properties, and nucleotide exchange, suggested a particularlyexciting new hypothesis regarding the function of the nucleotide. Janmey et al. presented evidence that filaments assembled from ADP-actin differed from filaments assembled from ATP-actin in several ways eventhough both consist of ADP-actin subunits. Their ADP-actin filaments were irregular in contourand slightly larger in diameter thanATP-actin filaments. Nevertheless, solutions of the ADP-filaments had a much higher dynamic elastic modulus than ATP-actin filaments, a difference that the authors at-
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Actin monomers with bound ATP, ADP, or fluorescent analogues of these nucleotides exchange the nucleotide on a second time scale, whereas filaments assembled from each of these species exchange their nucleotide with the solution at least 1,000 times slower than monomers. Filaments assembled from either ATP-actin or ADP-actin are indistinguishable by electron microscopy after negative staining. The dynamic elasticity and viscosity of filaments assembled from ATP-actin or ADP-actin or mixtures of these two species are the same over a wide range of frequencies. These observations do not support a recent suggestion (Janmey, P. A., Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., and Hartwig, J. H. (1990) Nature 347, 9599) that ATP hydrolysis within actinfilaments stiffens the polymer and alters both their structure and affinity for nucleotides. The difference in observations between these two studies may be related to time-dependent changes in ADP-actin prepared in slightlydifferent ways.
20340
Nucleotides
Actin
1983) and a two-step mechanism EXPERIMENTALPROCEDURES
Protein Preparation-Actin was purified from rabbit skeletal muscle and gel filtered in Buffer G on Sephadex G-150 (MacLean-Fletcher and Pollard, 1980). Buffer G consists of 2 mM Tris-C1, pH 8.0, at 25 "c,0.2 mM ATP, 0.5 mM dithiothreitol, 0.1 mM CaC12, 1 mM sodium azide. Mg-ADP-actin was made by the method of Pollard (1986) using soluble hexokinase or, for the nucleotide exchange experiments, by a modification using hexokinase-agarose beads as follows. One milliliter of actin at a concentration of 30-50 yM was mixed gently with 50 pl of Dowex 1 (Bio-Rad AG-X2 washed in 2 mM Tris, p H 8.0) for 5 min at 4 "C. The Dowex binds free nucleotides. The Dowex was removed by centrifugation at 14,000 X g for 30 s, and 852 p1 of nucleotide-free actin monomers from the supernatant was added t o a microcentrifuge containing 100 pl of packed hexokinase-agarose beads (Sigma H-2005; washed with 2 mM Tris-C1, pH 8.0, 0.5 mM dithiothreitol; final concentration 2 units/ml), 5 or 10 pl of 100 mM ADP (final concentration 0.5 or 1 mM), 8 p1 of 10 mM MgCl, (final concentration 80 PM), 20 p1 of 10 mM EGTA' (final concentration 200 yM), and 10 pl of 100 mM glucose (final concentration 1 mM). The sample was mixed gently for 4 h at 0-4 "C. After pelleting the hexokinase-agarose beads, the Mg-ADP-actin in the supernatantwas stored on ice and used the same day. Etheno-ADP (Molecular Probes, Inc.) was exchanged onto the actin in the same way using a final concentration of etheno-ADP of 0.5 mM. Etheno-ADP-actin is not stable for more than a few hours at 0 "C based on a progressive loss in theamplitude of the signal when ATP is exchanged for the ethenoADP. Etheno-ATP was exchanged onto the actin in the same way with the omission of the hexokinase beads using a finalconcentration of 0.5 mM etheno-ATP. Actin-Nucleotide Exchange Experiments-The binding or dissociation of etheno-ATP or etheno-ADP was measured at 25 "C by fluorescence (Waechterand Engel, 1975) with a Perkin-Elmer-Cetus Instruments 640-A spectrofluorometerusing an excitation wavelength of 365 nm and emission wavelength of 410 nm. The fluorescence of the etheno-nucleotides is higher when bound to actin than when free. The relationship of the fluorescence intensitytothe absolute extent of a reaction was established as follows. First, we measured the fluorescence change at the end of each reaction as a function of the actin monomer concentration (as in Fig. 1). The fluorescence intensity was always directly proportional to the actin monomerconcentration, Second, from the initial fluorescence in experiments such as Fig. 2 0 with mixtures of filaments and monomers, we established that thefluorescence of etheno-ADP is the same bound to monomeric and polymeric actin. (A similar comparison is not possible for etheno-ATP, since it is hydrolyzed during polymeri-
eN
+ A + eN*A
N+AeNA
where e N is etheno-ATP or etheno-ADP, A is actin, eN* is the high fluorescence form of the nucleotide analog bound to actin, and N is ATP or ADP. The simulations were initiated by adding the second nucleotide to anequilibrium mixture of actin and thefirst nucleotide. The association rate constantswere assumed to be 10 PM-' s" (Nowak et al., 1988). We searched by trial and error for the single set of dissociation rate constants, one for each of the four nucleotides, that allowed the kinetic simulations to fit simultaneously the full-time courses of the fluorescence changes in all four types experiments and all actin concentrations in Fig. 1. Initially the ratios of these dissociation rate constants were constrained by the ratios of the corresponding Kd values as determined above but had to be adjusted slightly during the simulations to achieve a good fit to all of the data. The optimal rate constants are given in the legend of Fig. 1, and simulations are illustrated in Fig. 1 as continuous curves. Actin Polymerization-Some of the actin was modified with pyrenyl-iodoacetamide(MolecularProbes, Inc.) and used to measure actin polymerization (Pollard, 1984). The critical concentration for polymerization was determined from the dependence of the steadystate fluorescence or the initial rate of nucleated actin polymerization on the concentration of actin (Pollard, 1984). Having establishedthat actin filaments do not exchange bound nucleotide at an appreciable rate, we also used the extent of fluorescent nucleotide exchange as a function of the concentration of actin as a new method to measure the critical concentration of monomers in the presence of filaments. Electron Microscopy-Actin filaments on glow-discharged carbon films were negatively stained with 1% uranyl acetate (Cooper and Pollard, 1982). Four grids each of ATP-actin andADP-actin filaments were coded and randomized before one author took micrographs at 46,000 X of one area of eight grid squares with well spread and evenly stained filaments. Coded prints were scored for straight filaments by two blinded observers. Rheological Measurements-The dynamic viscosity and elasticity were measured in a cone and plate rheometer with small amplitude oscillations as described by Sato et al. (1985). ADP-actin was made by the method of Pollard (1986). Samples were degassed to avoid bubbles during the long incubations. Actin stocks were warmed to 25 "C and diluted with ADP or ATP buffer and concentrated salts to give final concentrations of 34 p M actin, 50 mM KCl, 1 mMMgC12, 1 mM EGTA, 0.2 mM nucleotide, 10 mM imidazole, pH 7.0. Samples were immediately transferred to the rheometer and incubated at 25 "C The abbreviation used is: EGTA, [ethylenebis(oxyethyleneni- for 8-12 h to attain an equilibrium configuration before measurements were made of the dynamic viscosity and elasticity. tri1o)jtetraacetic acid.
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tributed to the greaterflexibility of the ADP-actin filaments, zation.) Third, we titrated 4 PM etheno-ATP-actin or etheno-ADPwhich caused them to be more entangled than the stiff rod- actin with ATP. From the titration data we calculated the fluoreslikeATP-actin filaments. TheADP-actinfilaments ex- cence of the etheno-nucleotides when free and when bound to actin. changed their bound ADP faster than the ADPof filaments For etheno-ATP binding to actin increased the fluorescence 16 fold; for etheno-ADP binding increased the fluorescence 18-fold. From the assembled from ATP-actin. The addition of ATP stiffened dependence of the equilibrium fluorescence on the concentration of the ADP-actin filaments andgave them properties more like ATP at each point in the titration, we calculated the relative values ATP-actinfilaments. It was postulated that hydrolysis of of the dissociation constants for the etheno-nucleotides and ATP ATP within an actin filament "traps . . . the monomers . . . (similar to themethod of Waechter and Engel, 1975). The ratios were conformationally and stores elastic energy" that might be Kd ATP/Kd etheno-ATP = 0.3 and Kd ATP/Kd etheno-ADP = 0.015. "available for releaseby actin-binding proteins that transduceFrom the nucleotide concentrations and thesteady-state fluorescence at the end of the experiments in (Fig. 1C) we calculated the ratio of force or sever actin filaments." Kd ADP/Kd etheno-ATP to be 1.5. These relative equilibrium conSince this is the most interesting suggestion in years restants and thenucleotide concentrations were used to scale all of the garding the function of the bound nucleotide of actin, we fluorescence data. thought that it is important to confirm the key observations For exchange experiments with polymerized actin, we first deterof Janmey et al. (1990). In this paper we report new data on mined the critical concentration for the actin species being tested nucleotideexchange, filamentstructure,andmechanical (see below) and then prepared the samples for exchange experiments properties of actin assembled from ADP-actin or ATP-actin. by mixing concentrated monomer and polymer stocks to give the We prepared the ADP-actinquickly in the cold by a modifi- criticalconcentration of monomer and variableconcentrations of cation of current methods. Using this ADP-actin and ATP- polymer. This avoided prolonged incubations that otherwise would been required to allow either monomers or polymers to attain actin, we found noevidence for rapidexchange of any nucleo- have steady state after dilution into the polymerizing buffer. tide between polymerized actin and the solution and were Kinetic Simulation Methods-Rate constants for the exchange of unable to detect significant differences in theoverall structure nucleotides on actin monomers were determined by kinetic simulation or mechanical properties of filaments assembled from ATP- using an Apple Macintosh version (HOPKINSIM, Daniel Wachsactin or ADP-actin. stock, Johns Hopkins Medical School) of KINSIM (Barshop et al.,
Nucleotides
Actin
20341
Although the exchange of actin-bound nucleotide is relatively slow in calcium-containing solutions (Gershman etal., Preparation of Actin-Nucleotide Complexes-Actin with 1989), ATP, ADP, etheno-ATP,and etheno-ADPall exbound Mg2+ and ADP or etheno-ADP prepared by exchange in a low ionic strength buffer containing M e , EGTA, glu- change on a second time scale in buffers with M e and EGTA cose, and hexokinase had a critical concentration10-20 times (Fig. 1).Under these conditions, M$+ is bound to the high et al., 1986) in the higher than the corresponding ATP-actins (Table I). The affinity divalent cation site (Gershman cleft of the actin molecule (Kabsch et al., 1990). The time hexokinase acts asa scavenger for any ATP dissociated from course is essentially the same when the Mg-EGTA buffer has the actin or synthesized from ADP by contaminating adenylate kinase (Pollard, 1986; Gershman et al., 1989). Soluble either a low ionic strength favoring depolymerization of the hexokinase was left in the ADP-actin preparations used for actin or high enough concentrations ofKC1 and M e to rheological studies to remove any ATP generated during the strongly favor polymerization of the actin molecules. Simulation of the time course of these exchange reactions equilibration and measurements. allowed us toselect dissociation rate constantsfor each of the To measure the exchange of ADP bound to the actin for ATP from the buffer, it was necessary to remove the hexoki- four nucleotides (Fig. 1 legend) that together account the nase from the actin. This was accomplished by a new method kinetic data from four different types of nucleotide exchange using hexokinase immobilized on agarose beads. The hexoki- experiments (Fig. 1).We used the two-step mechanism exnase activity that could be achieved in the exchange reaction plained under“Experimental Procedures.” The simulation us to model the reactions without simpliusing the commercial preparation of hexokinase-agarose was approach allowed fying assumptions about irreversibility used in earlier analytabout 10 times lower than theconcentration of soluble hexoical solutions (Waechter and Engel, 1975). Although the nukinase known to be effective in producing ATP-free actin in cleotide exchange rate constants are very sensitive to experiprevious studies (Pollard, 1986). To reduce the need for high mental conditions (Frieden and Patane, 1988), our complete hexokinase activity, free ATP was removed from the starting set of values for the rate constants is in general agreement sample of actin before adding ADP andthe immobilized with literature values obtained for subsets of these constants enzyme. In steady-state criticalconcentrationexperiments under somewhat differentconditions(Kuehl and Gergely, the resulting Mg-ADP-actin has the same critical concentra1969; Waechter and Engel, 1975; Nowak et al., 1988). tion that made with soluble hexokinase (Table I). Thus the Nucleotide Exchange by PolymerizedActin-Samples of two preparations of Mg-ADP-actin have the same ratio of polymerized actin assembled from monomers with bound dissociation and association rate constants for polymer elonATP, ADP, etheno-ATP, or etheno-ADP exchanged a small gation. It is formally possible that theabsolute values of these fraction of their nucleotide on a second time scale, but the rate constants may differ for the two preparations. Since the rate of exchange by most of the actin in these samples was critical concentration is very sensitive to low concentrations too slow to detect in 20 min (Fig. 2). This confirms for all of ATP-actin in ADP-actin (Ohm and Wegner, 1991), these nucleotides previous observations onfilaments assembled results are strong evidence that the hexokinase-agarose effi- from ATP-actin (Martonosi et al., 1960). ciently removes all of the ATP from the ADP-actin preparaThe time course for the exchangeable fraction of nucleotide tion like soluble hexokinase (Pollard, 1984). in each of these polymerized actin samples was similar to Nucleotide Exchange on Actin Monomers-We first carried samples of monomer without filaments, and theamplitude of out a comprehensive set of nucleotide exchange experiments the signal was equal to or slightly less than that predicted by with actin monomers (Fig. 1) to establisha quantitative the critical concentration of monomers measured independmechanism that would allowus to interpretexperiments with ently by steady-state or kinetic methods. Beyond this signal more complex mixtures containing both monomers and fila- arising from exchange of nucleotide by the monomers, there ments. Others have investigated nucleotide exchange by actin was no additional reproducible signal attributable to nucleomonomers (see for example Kuehl and Gergely, 1969; tide exchange by the polymeric actin over the time course of Waechter and Engel, 1975; Frieden and Patane, 1988; Nowak our experiments. In the experiments with ATP-actin plus et al., 1988), but not for all of the relevant nucleotides and etheno-ATP, we detected no exchange of nucleotide by polynot in a buffer than approximates physiological ionic condi- mer concentrations up to 50 times the critical concentration tions. of monomers, where exchange, even at a low rate, would have been obvious (Fig. 2 A ) . The signal to noise ratio is even better TABLE I with etheno-ATP-actin reacted with ATP. In these experiCritical concentrationsfor actin polymerization ments, polymer concentrations 60 times higher than the critConditions: 50 mM KCI, 1 mM MgCl,, 1 mM EGTA, 0.2 mM ical concentration did not exchange over a period of 20 min nucleotide, 0.5 mM dithiothreitol, 10 mM imidazole (pH 7.0). The underlined values were obtained with samples of actin prepared with (Fig. 2B). In the experiments with ADP-actin filaments the hexokinase-agarose beads. The other ADP-actin samples were pre- critical concentration is much higher, so the highest polymer pared with soluble hexokinase. The samples used for elongation rate concentration tested was six times the monomer concentraand steady-state polymer concentration measurements contained 5% tion. Nevertheless, no nucleotide exchange of the polymerized pyrene-labeled actin. actin was detected over times greater than 10 half-times for exchange of nucleotide on the monomers in the same sample Method ATP-actin ADP-actin (Fig. 2C). Similarresults were obtained with filaments of PM PM PM etheno-ADP-actin where the signal to noise ratio was even Elongation rate 0.04 1.35 better (Fig. 2 0 ) . 0.90 0.05 Given the sensitivity of these assays, the half-time for the 0.05 exchange of nucleotide on the subunits of actin filaments is Steady-state polymer 0.10 1.20 concentration 0.05 more than 1,000 times greater than the half-times for the 1.15 1.35 exchange of the same nucleotide bound to actin monomers. Nucleotide exchange 1.0 This conclusion holds true for filaments assembled from ATP0.9 actin, ADP-actin, etheno-ATP-actin, or etheno-ADP actin. RESULTS
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Ethe::iy-
20342
Actin Nucleotides 1 .o
4
TA+eT==eTA+T
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0.6
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1000
500
0
1500
2000
3
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DA + eT==eTA + D ,
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50
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Seconds FIG. 1. Time course of nucleotide exchange by actin monomers. Experimental data are given as open symbols and dashed line. Theoretical kinetic curves shown as continuous lines were calculated using KINSIM and the following rate constants: k , = 10 pL"' s-' for all nucleotide binding reactions; k- = 0.0045 s" for ATP, 0.015 s-' for etheno-ATP, 0.023 s-l for ADP, and 0.09 s" for etheno-ADP. Conditions: 2 mM Tris-C1, pH 7.5; 500 pM dithiothreitol; 200 pM EGTA, 80 p~ MgCI,. All samples contained nucleotides and etheno-adeninenucleotides as indicated and low concentrations of CaCl, that varied with the actin concentrations in the range of 3-10 p ~ Panel . A , exchange of ATP bound to actin for etheno-ATP. ATP-actin concentrations were 0.2 p M monomers plus 1 F M filaments (0) and 0.5 p~ monomers (0).The final ATP concentrations were 0.8 and 2 p ~ The . etheno-ATP concentration was 200 p ~ Panel . B , exchange of etheno-ATP bound to actin for ATP. The etheno-ATP-actinmonomer concentrations were 1 p~ (0), 2 /IM( O ) ,and 3 p~ (A). The final etheno-ATP concentrations were 17, 34, and 51 p ~ The . ATP concentration was 1,000 PM. Panel C , exchange of ADP hound to actin for etheno-ATP. ADP-actin monomer concentrations were 1 F M (O),2 pM (El),and 3 FM (A). The final ADP concentrations were 31,62, and 93 p ~ The . etheno-ATPconcentration was 200 p ~ Under . these conditions there was a significant concentration of ADP-actin in equilibrium with etheno-ATP-actin and the free nucleotides at the end of the reaction. Punel D, exchange of etheno-ADP bound to actin for ATP. The etheno-ADP-actin monomer concentrations were 1 p~ (O),2 p~ (El), and 3 p~ (A). The final etheno-ADP concentrations were 17,34, and51 p M . The ATP concentration was 1,000 pM.
Electron Microscopy of Actin Filaments Assembled from DISCUSSION ATP- or ADP-Monomers-Although there is alwayssome In a physiological buffer containing M e , KCl, and EGTA, heterogeneity in the appearance of actin filaments prepared a single set of rate constants can account for the timecourses for electron microscopy by negative staining, we could not detect any systematic differences in the gross appearance of of a wide range of nucleotide exchange experiments (Fig. I), values are reliable. All are actin filaments assembled from ATP-actin (Fig. 3, A-C) or so we are confident that these ADP-actin (Fig. 3, D-F). In particular, all of the grids con- based on a reasonable assumption that the association rate s-', a value citedby Nowak et al. (1988) tained filaments with smooth, gently curving contours. Most constants are 10 p"' from unpublishedwork. ofthe grids alsocontained someirregular filaments,but Four adeninenucleotides exchanged on a second time scale blinded observers could not distinguish two populations of of the from actin monomers, but we detected very little exchange on grids made with ATP-actin or ADP-actin. The quality stain varied from one grid square to the next but was not actin filaments over much longerperiods of time even in related to the nucleotide. One of four grids with ADP-actin experiments with high concentrations of filaments (Fig. 2). had considerable lateral aggregation of the filaments, but this Conservatively, we estimate that the nucleotide bound to actin was not characteristic of the other ADP grids. These obser- filaments polymerized from either ATP- or ADP-actin exvations rule out gross differences in filaments of ATP-actin changes 1,000 times slower than ATP or ADP on actin monand ADP-actin but do not preclude subtle differences in the omers. This exchange rate for subunits within filamentsis an packing or shape of the subunits that might be detected by upper limit, since some nucleotide exchanges into polymers high resolution reconstructions. Mechanical Properties of Actin Filaments Assembled from indirectly because of nucleotide exchange on monomers that off the endsof filaments. Theslow rate ATP- or ADP-Monomers-We detected no reproducible dif- then exchange on and of nucleotide exchange by polymerized actinisconsistent ferences in the viscous or elastic modulii of actin filaments assembled from ATP-actin or ADP-actin over a wide range with the atomic model of the actin filament (Holmes et al., long pitch helix appear of frequencies (Fig. 4). Filaments assembled from mixturesof 1990) wheresubunit contacts along the well ATP- and ADP-actin (made by converting most but not all to block the exitof the nucleotide from its binding site as as constraining the type of interdomain motions that open of the buffer ATP to ADP) had similar mechanical properties.
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2
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Actin Nucleotides TA+eT==eTA+T
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. these conditions there was a significant concentration of ADP-actin in equilibrium with etheno-ATPwere 31, 62, 93, and 124 p ~ Under actin and thefree nucleotides at the endof the reaction, accounting for the incomplete exchange of ADP. Panel D,exchange of etheno-ADP bound to actin for ATP. All samples contained 1.0 p~ etheno-ADP-actin monomers and 1,000 p~ ATP. The etheno-ADP-actin filament 2 p M (O), and 3 pM (A). The final etheno-ADP concentrationswere 17, 34, 51, and 68 p ~ . concentrations were 0 (bold line), 1 p~ (0),
the nucleotide binding site in hexokinase. Our conclusions are infull agreement with the long accepted belief that ADP is tightly bound to the subunits in actin filaments (Martonosiet al., 1960) but differ from two previous studies of nucleotide exchange by actin filaments using etheno-ATP fluorescence. Wang and Taylor (1981) followed the exchange of etheno-ATP for ATP and the reverse reaction with 5 p~ actin in a polymerization buffer with 2 mM MgC12 & 100 mMKC1. They observed two phases of fluorescence change. Initially, fluorescence changed rapidly with an amplitude of about 20% (-1 p M actin) and half-time of 5-10 min ( k 0.001 s-l). They interpreted thisfirst phase as therapid exchange of nucleotide on monomers and addition of some of these monomers to theends of filaments. They did not measure the critical concentration, but the amplitude of their fast phase was about 10 times larger than expected from later determinations under similar conditions (Table I).We cannot explain this difference from our experiments. The rate of the second phase of the fluorescence change was variable. In 100 mMKC1 with 50 pM MgCI2 the rate was near zero. In 2 mM MgC12 the maximum rate was about 0.1 nM s" and was attributed to the flux of subunits throughfilaments. Our calculation of their subunit exchange rate (0.1 nM subunits/ s/2 nM filamentends = 0.05 subunits/filament end/s) is consistent with the rate of subunit flux (about 0.1 s-l) calculated from the elongation rate constants (Pollard, 1986), so it is not necessary to postulate rapid exchange of nucleotide by subunits within the polymers. These conclusions are consistent with ours. Janmey et al. (1990) reported that solutions of ADP-actin filaments exchange bound nucleotide more rapidly than ATPactin filaments. They capped the barbed end of the filaments
-
to eliminate the slow flux of subunits observed by Wang and Taylor (1981). From the the partial time courses of nucleotide exchange in polymeric actin reported by Janmey et al. we estimate first-order rate constantsof 0.005 s-l for ADP-actin and 0.001 s" for ATP-actin.Their signal came from the binding of etheno-ATP, butthese values should approximate the dissociation rate constantsfor ADP and ATP. Both values are about &fold lower than our dissociation rate constants for ADP and ATP from monomeric actin, a difference that is expected because of the Ca2+in their buffer which slows the rate of nucleotide dissociation (Frieden and Patane, 1988). We suggest that their signal arose in large part from the critical concentration of monomers in their filament samples, as in our experiments in Fig. 2. The reason is that Janmeyet al. calibrated their fluorescence signal by assuming that 100% of the nucleotide in samples of filaments exchanged in 20 h. They calculated the fluorescence increment per molecule of actin-bound nucleotide from the totalactin concentrationand the fluorescence change at that time. Twenty hours is probably inadequate for complete equilibration of nucleotide with filaments capped on their rapidly exchanging ends by gelsolin. Consequently, they may have underestimated the magnitude of the fluorescence change. This explains why their experiments appear to show extensive exchange of nucleotide on polymerized actin on a minute time scale. The presence of gelsolin may also account for the relatively small difference in the amplitudes of exchange that they observed for ATPactin andADP-actin. Without gelsolin the fluorescence signal arising from the monomers present ina filament sample is 10 times higher for ADP-actin than ATP-actin (Fig. 2) because of the large difference in their critical concentrations at the barbed end. However, with the barbed ends capped by gelsolin,
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Seconds
FIG. 2. Time course of nucleotide exchange by mixtures containing a critical concentration of actin monomers and a range of concentrations of actin filaments. Note that therewas no exchange by the filaments above that expected from the critical concentrations of actin monomers. Panel A , exchange of ATP bound to actin for etheno-ATP. All samples contained 0.2 p~ ATP-actin monomers and 200 f i etheno-ATP. ~ ATP-actin filament concentrations were 1p M (0) and 3 pM (A).The final ATP concentrations were 7.2 and 20.4 p ~ Panel . B , exchange of etheno-ATP bound to actin for ATP. All samples contained 0.1 @M etheno-ATP-actin monomers and 1,000 PM ATP. The etheno-ATP-actin filament concentrations were 0 (bold line), 1 pM (O),2 pM (U), and 3 pM (A). The final etheno-ATP concentrations were 17, 19, 36, and 53 p M . Panel C , exchange of ADP bound to actin for etheno-ATP. All samples contained 1.0 p~ ADP-actin monomers and 200 p~ etheno-ATP. ADP-actin filament concentrations were 0 (bold line), 1 p~ (0), 2 p M (O), and 3 p M (A). The final ADP concentrations
20344
Nucleotides
Actin
0
ATP. q' ATP. G
0
ADP-q' ADP.G
8
E
100
0 1 FIG.4. Comparison of the rheological properties of ATPand ADP-actin filaments. Actin filaments at were analyzedby oscillation in a cone-plate rheometer at 25 "C over a wide range of frequencies. Conditions:34 PM ATP-actin (circles);34 p~ ADP-actin (squares). The filled symbols represent the elastic modulus ( G ' ) is dynes/cm2, and the open symbob represent the dynamic viscosity in poise.
the critical concentrationsare determined by the pointed ends where the ATP-actin and ADP-actin differ only by a factor of 2-3 (Coue and Korn, 1985; Pollard, 1986). We used nucleotide exchange by monomers and competitive nucleotide bindingexperiments (see "Experimental Procedures") to calibrate the absolute fluorescence increment for each nucleotide exchange pair. In our experiments with mixtures of filaments and thecritical concentrationof monomer, only the critical concentration of actin monomers exchanged rapidly. Even without capping the barbed ends, no nucleotide exchange pair gave a substantial fluorescence signal from filaments during the 20-min duration of our experiments. Because the nucleotide exchange rate by polymerized actin is so slow, a new experimental design will be required to detect any subtle differences in nucleotide exchange rates of filaments assembled from ADP- and ATP-actin. We found no difference in the electron microscopic appearance (Fig. 3) or mechanical properties (Fig. 4) of actin filaments assembled from ADP- or ATP-actin. Our observations do not exclude small differences in the structureof ADP- and ATP-actin filaments that may exist at the atomic level, but both the microscopy and rheology differ from the report of Janmey et al. (1990). First, our ADP-actin filaments have smooth, not irregular, profiles just like ATP-actin filaments.
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FIG. 3. Electron micrographs of negatively stained actin filaments. A-C, filaments assembled from ATP-actin. D-F, filaments assembled from ADP-actin. Magnifications:A, R, D,and E = 113,000 X; C and F = 173,000 X. Bars = 0.1 um.
20345
Actin Nucleotides
through the filaments and to preparefilaments for rapid disassembly at a time subsequentto theirassembly. Acknowledgments-WethankDr. Pascal Goldschmidt-Clermont for suggesting the use of the hexokinase-agarose beads for making ADP-actin andDr.PaulJanmeyfor exchanging ideas, data, and proteins in our ongoing effort to reconcile our experimental results. REFERENCES Amato, P. A., and Taylor, D. L. (1986) J. Cell Bid. 102,1074-1084 Barshop, B. A,, Wrenn, R. F., and Frieden,C. (1983) Anal. Biochem. 1 3 0 , 134145 Carlier, M.-F. (1991) J. Bid. Chem. 2 6 6 , 1-4 Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 26,7789-7792 Carlier, M.-F., and Pantaloni, D.(1988) J. Biol. Chem. 263,817-825 Carlier, M.-F., Pantaloni, D., and Korn, E.D. (1987) J. Bid. Chem. 262,30523059 Carlier, M.-F., Pantaloni, D., Evans, J. A., Lambooy, P. K., Korn, E. D., and Webb, M. R. (1988) FEBS Lett. 236,211-214 Cooper, J. A. (1991) Annu. Reu. Physiol. 5 3 , 585-605 Cooper, J. A., and Pollard, T. D. (1982) Methods Enzymol. 85,182-210 Coue, M., and Korn, E. D. (1985) J. Biol. Chem. 260,15033-15041 Drenckhahn, D., and Pollard, T. D. (1986) J. Bid. Chem. 261,12754-12758 Drewes, G., and Faulstich, H. (1991) J. Bid. Chem. 2 6 6 , 5508-5513 Forscher, P., and Smith,S. J. (1988) J. Cell Biol. 107,1505-1516 Frieden, C., and Patane, K. (1988) Biochemistry 2 7 , 3812-3820 Gershman, L., Selden, L., and Estes,J. (1986) Biochem. Biophys. Res. Commun. 135,607-614 Gershman. L.. Selden., L... Kinosian. H. J.. and Estes., J . (1989) Biochim. Bioohvs. . . ” Acta 996,109-115 Holmes, K., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 3 4 7 , 4449 Janmey, P.A., Hvidt, S., Peetermans, J., Lamb, J., Ferry, J. D., and Stossel, T. P. (1988) Biochemistry 27,8218-8227 Janmey, P. A,, Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., andHartwig, J. H. (1990) Nature 347,95-99 Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347,37-44 Korn, E. D., Carlier, M.-F., and Pantaloni, J. (1987) Science 238,638-644 Kuehl, W. M., and Gergely, J. (1969) J. Biol. Chem. 244,4720-4729 MacLean-Fletcher, S., and Pollard, T. D. (1980) Biochem. Biophys. Res. Commun. 9 6 , 18-27 Martonosi, A,, Gouvea, M. A,, and Gergely, J. (1960)J. Bid. Chem. 236,17001711 Nowak, E., Strezelecka-Golaszwska,H., and Goody, R. S. (1988) Biochemistry 2 7 , 1785-1792 O’Donoghue, S. I., Miki, M., and dosRemedios, C.G. (1992) Arch. Biochem. Biophys. 2 9 3 , 110-116 Ohm, T., and Wegner, A. (1991) Biochemistry 3 0 , 1193-1197 Pollard, T. D. (1984) J. Cell Biol. 9 9 , 769-777 Pollard, T. D. (1986) J. Cell Biol. 103,2747-2754 Pollard, T. D. (1990) Curr. Opin. Cell Biol. 2 , 33-40 Pollard, T.D., and Weeds, A. G. (1984) FEBS Lett. 170,94-98 Rickard, J. E., and Sheterline, P. (1988) J. Mol. Bid. 201,675-681 Sato. M..Leimbach. G.. Schwarz. W. H.. and Pollard. T. D. (1985) J. Bid. , , Chem. 260,8585-8592 Straub, F. B., and Feuer, G. (1950) Biochim. Biophys. Acta 4,455-470 Theriot, J. A., and Mitchison, T. J. (1991) Nature 3 5 2 , 126-131 Waechter, F., and Engel, J. (1975) Eur. J. Biochem. 57,453-459 Wang, Y:L. (1985) J. CellBiol. 1 0 1 , 597-602 Wang, Y.-L., and Taylor, D. L. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,55035507 Wanger, M., and Wegner, A. (1987) Biochim. Biophys. Acta 9 1 4 , 105-113 ,
I
,
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Second, our ADP-actin filaments and ATP-actin filaments had similar mechanical properties, whereas the ADP-actin filaments of Janmey et al. were about 10 times more rigid than their ATP-actin filaments. Furthermore, the absolute values of the elastic modulii of filaments prepared from fresh actin are much lower in our laboratory than Janmey’s, an unresolved, long-standing difference (see Sato et al., 1985; Janmey et al., 1988). The experimental design differed in two ways: ( a ) methods to prepare the actin, and ( b ) the inclusion of gelsolin in the rheological experiments of Janmey et al. to control the length of the filaments. Differences in the actin rather than the gelsolin seem more likely to account for the difference in rigidity. The differences in the observations between the two laboratories have not yet been resolved but may lie in the preparation of the ADP-actin. Our current methods take advantage of rapid exchange of nucleotide by Mg-actin (Gershman et al., 1989) to make ADP-actin quickly in the cold in an effort to avoid an inevitable, time-dependent (k = 2 X loT5s-l at 0 “C) conformational change that can be detected in ADP-actinby sulfhydryl titration of Cys-10 (Drewes and Faulstich, 1991). We never freeze purified actin preparations. Janmey et al. used a slower, early method from our laboratory (Pollard, 1984) to prepareADP-actin from frozen stocks. Ca-ATPactin is incubated with hexokinase and glucose at room temperature for several hours. These ADP-actin preparationsare not completely stable (Pollard, 1984), perhaps because of the conformational change. It was necessary to incubate their ADP-actin for 2 h at room temperature before the differences with ATP-actinbecame apparent. TheCys-10 conformational change should have occurred on part of the ADP-actin prepared by this method and may account for the substantial differences in the properties of the ADP-actin in the two laboratories. It is particularly interesting that the structural differences in ADP-actin filamentsobserved by Janmey et al. (1990) were reversed rapidly by ATP just like the Cys-10 conformational change (Drewes and Faulstich, 1991). O’Donoghue et al. (1992) also observed that ADP-actin prepared by our 1984 method has a higher viscosity than ATP-actin. Given that ADP- and ATP-actinfilaments are the same by our criteria, new evidence will be necessary to confirm the hypothesis that nucleotide hydrolysis within actin filaments is used to modulate their mechanical properties. Rather, we expect that nucleotide hydrolysis is used to drive subunit flux
~
THEJOURNALOF RIOLOCICALCHEMISTRY
62 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Issue of October 5, PP. 20339-20345,1992 Printed in U.S A .
Nucleotide Exchange, Structure, and Mechanical Properties of Filaments Assembled from ATP-actin and ADP-actin* (Received for publication, April 6, 1992)
Thomas D. Pollard$, Ilya Goldberg, and William H. Schwarz$ From the Departmentof Cell Biology and Anatomy, The Johns HopkinsMedical School, Baltimore, Maryland 21205 and the 21218 §Department of Chemical Engineering, The Johns Hopkins University, Baltimore, Maryland
The adenine nucleotide bound in the central cleft of the actin molecule stabilizes the protein, but in spite of extensive research (for review see Korn et al., 1987; Pollard, 1990; and Carlier, 1991) the underlying function of the nucleotide and its hydrolysis in the assembly of actin filaments in live cells has remained an enigma for years (for review see Cooper, 1991). It is well established that when actin with bound ATP (referred to here as ATP-actin)polymerizes, the ATP on each molecule is hydrolyzed (Strauband Feuer, 1950) afterits incorporation into a filament (Pollard and Weeds, 1984) in an irreversible reaction (Carlier et al., 1988) to ADP and inorganic phosphate (Pi). The hydrolysis reaction may occur randomly on the polymerized subunits (Pollard and Weeds, 1984) or may be favored at the interface between a central region consisting of ADP-actin and newly added ATP-actin subunits near the ends of the filament (Carlier et al., 1987). The Pi remains bound to the filament for some time (Carlier and Pantaloni, 1986),making rapidly growing filamentsa heterogeneous mixture of subunits with ATP, ADP-P,, or ADP. Eventually the Pi dissociates leaving filaments composed largely of ADP-actin with a few ATP- and ADP-Piactin subunits near the ends. * This work was supported by National Institutes of Health Research Grant GM-26338. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondenceshould be addressed Dept. of Cell Biology and Anatomy, The Johns Hopkins Medical School, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-5664; Fax: 410.9554129.
The nucleotide composition of monomers has an easily measured impact on the elongation of actin filaments (for review see Korn et al., 1987). At the barbed end, we find by direct electron microscopic measurements that ATP-actin binds with a diffusion-limited rate constantof lo7M" s-l and dissociates slowly, about 1 s" (Pollard, 1986; Drenckhahn and Pollard, 1986). (Similar values of these and other rate constants have been measured by other methods; for review see Korn et al., 1987.) In contrast, ADP-actin binds about 20% as fast and dissociates about 5 times faster than ATPactin. ADP-P-actin has a critical concentration similar to ATP-actin (Rickard and Sheterline, 1988; Wanger and Wegner, 1987; Carlier and Pantaloni, 1988), but there arenot yet any direct measurements of the elongation rate constants. The effect of nucleotide on growth at thepointed end is less clear. Both ATP- andADP-actin associate at rates lower than expected for a diffusion-limited reaction (Pollard, 1986; Drenckhahn and Pollard, 1986). Both dissociate slowly, especially ADP-actin. The ratios of the association and dissociation rate constantsgive the critical actin monomer concentrations for elongation. In ATP with physiological concentrations of divalent cations, the critical concentration at the pointed end is substantially higher than at the barbed end. This should lead to a slow flux of subunitsthrough the filaments from the barbed end to the pointed end driven by ATP hydrolysis. These properties suggest that nucleotide hydrolysis might be used by cells to power subunit flux through actin filaments and possibly also to prepare the filaments for rapid disassembly at a time different from the time of assembly. In live cells actin subunits flux rapidly through filaments at the leading edge (Wang, 1985; Forscher and Smith,1988), and many actin filaments turn over rapidly (Amato and Taylor, 1986; Theriot and Mitchison, 1991). Neither the rapid flux nor turnover appears to be compatible with the relatively slow dynamics of purified actin filaments i n vitro, so we do not yet understand how the polymer dynamics in vivo are related to nucleotide hydrolysis (for reviewsee Cooper, 1991). Excluded volume effects (Drenckhahn and Pollard, 1986) or accessory proteins could speed up subunit exchange in the cell. Recent observations of Janmey et al. (1990), showing a possible effect of nucleotide hydrolysis on actin filament structure, mechanical properties, and nucleotide exchange, suggested a particularlyexciting new hypothesis regarding the function of the nucleotide. Janmey et al. presented evidence that filaments assembled from ADP-actin differed from filaments assembled from ATP-actin in several ways eventhough both consist of ADP-actin subunits. Their ADP-actin filaments were irregular in contourand slightly larger in diameter thanATP-actin filaments. Nevertheless, solutions of the ADP-filaments had a much higher dynamic elastic modulus than ATP-actin filaments, a difference that the authors at-
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Actin monomers with bound ATP, ADP, or fluorescent analogues of these nucleotides exchange the nucleotide on a second time scale, whereas filaments assembled from each of these species exchange their nucleotide with the solution at least 1,000 times slower than monomers. Filaments assembled from either ATP-actin or ADP-actin are indistinguishable by electron microscopy after negative staining. The dynamic elasticity and viscosity of filaments assembled from ATP-actin or ADP-actin or mixtures of these two species are the same over a wide range of frequencies. These observations do not support a recent suggestion (Janmey, P. A., Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., and Hartwig, J. H. (1990) Nature 347, 9599) that ATP hydrolysis within actinfilaments stiffens the polymer and alters both their structure and affinity for nucleotides. The difference in observations between these two studies may be related to time-dependent changes in ADP-actin prepared in slightlydifferent ways.
20340
Nucleotides
Actin
1983) and a two-step mechanism EXPERIMENTALPROCEDURES
Protein Preparation-Actin was purified from rabbit skeletal muscle and gel filtered in Buffer G on Sephadex G-150 (MacLean-Fletcher and Pollard, 1980). Buffer G consists of 2 mM Tris-C1, pH 8.0, at 25 "c,0.2 mM ATP, 0.5 mM dithiothreitol, 0.1 mM CaC12, 1 mM sodium azide. Mg-ADP-actin was made by the method of Pollard (1986) using soluble hexokinase or, for the nucleotide exchange experiments, by a modification using hexokinase-agarose beads as follows. One milliliter of actin at a concentration of 30-50 yM was mixed gently with 50 pl of Dowex 1 (Bio-Rad AG-X2 washed in 2 mM Tris, p H 8.0) for 5 min at 4 "C. The Dowex binds free nucleotides. The Dowex was removed by centrifugation at 14,000 X g for 30 s, and 852 p1 of nucleotide-free actin monomers from the supernatant was added t o a microcentrifuge containing 100 pl of packed hexokinase-agarose beads (Sigma H-2005; washed with 2 mM Tris-C1, pH 8.0, 0.5 mM dithiothreitol; final concentration 2 units/ml), 5 or 10 pl of 100 mM ADP (final concentration 0.5 or 1 mM), 8 p1 of 10 mM MgCl, (final concentration 80 PM), 20 p1 of 10 mM EGTA' (final concentration 200 yM), and 10 pl of 100 mM glucose (final concentration 1 mM). The sample was mixed gently for 4 h at 0-4 "C. After pelleting the hexokinase-agarose beads, the Mg-ADP-actin in the supernatantwas stored on ice and used the same day. Etheno-ADP (Molecular Probes, Inc.) was exchanged onto the actin in the same way using a final concentration of etheno-ADP of 0.5 mM. Etheno-ADP-actin is not stable for more than a few hours at 0 "C based on a progressive loss in theamplitude of the signal when ATP is exchanged for the ethenoADP. Etheno-ATP was exchanged onto the actin in the same way with the omission of the hexokinase beads using a finalconcentration of 0.5 mM etheno-ATP. Actin-Nucleotide Exchange Experiments-The binding or dissociation of etheno-ATP or etheno-ADP was measured at 25 "C by fluorescence (Waechterand Engel, 1975) with a Perkin-Elmer-Cetus Instruments 640-A spectrofluorometerusing an excitation wavelength of 365 nm and emission wavelength of 410 nm. The fluorescence of the etheno-nucleotides is higher when bound to actin than when free. The relationship of the fluorescence intensitytothe absolute extent of a reaction was established as follows. First, we measured the fluorescence change at the end of each reaction as a function of the actin monomer concentration (as in Fig. 1). The fluorescence intensity was always directly proportional to the actin monomerconcentration, Second, from the initial fluorescence in experiments such as Fig. 2 0 with mixtures of filaments and monomers, we established that thefluorescence of etheno-ADP is the same bound to monomeric and polymeric actin. (A similar comparison is not possible for etheno-ATP, since it is hydrolyzed during polymeri-
eN
+ A + eN*A
N+AeNA
where e N is etheno-ATP or etheno-ADP, A is actin, eN* is the high fluorescence form of the nucleotide analog bound to actin, and N is ATP or ADP. The simulations were initiated by adding the second nucleotide to anequilibrium mixture of actin and thefirst nucleotide. The association rate constantswere assumed to be 10 PM-' s" (Nowak et al., 1988). We searched by trial and error for the single set of dissociation rate constants, one for each of the four nucleotides, that allowed the kinetic simulations to fit simultaneously the full-time courses of the fluorescence changes in all four types experiments and all actin concentrations in Fig. 1. Initially the ratios of these dissociation rate constants were constrained by the ratios of the corresponding Kd values as determined above but had to be adjusted slightly during the simulations to achieve a good fit to all of the data. The optimal rate constants are given in the legend of Fig. 1, and simulations are illustrated in Fig. 1 as continuous curves. Actin Polymerization-Some of the actin was modified with pyrenyl-iodoacetamide(MolecularProbes, Inc.) and used to measure actin polymerization (Pollard, 1984). The critical concentration for polymerization was determined from the dependence of the steadystate fluorescence or the initial rate of nucleated actin polymerization on the concentration of actin (Pollard, 1984). Having establishedthat actin filaments do not exchange bound nucleotide at an appreciable rate, we also used the extent of fluorescent nucleotide exchange as a function of the concentration of actin as a new method to measure the critical concentration of monomers in the presence of filaments. Electron Microscopy-Actin filaments on glow-discharged carbon films were negatively stained with 1% uranyl acetate (Cooper and Pollard, 1982). Four grids each of ATP-actin andADP-actin filaments were coded and randomized before one author took micrographs at 46,000 X of one area of eight grid squares with well spread and evenly stained filaments. Coded prints were scored for straight filaments by two blinded observers. Rheological Measurements-The dynamic viscosity and elasticity were measured in a cone and plate rheometer with small amplitude oscillations as described by Sato et al. (1985). ADP-actin was made by the method of Pollard (1986). Samples were degassed to avoid bubbles during the long incubations. Actin stocks were warmed to 25 "C and diluted with ADP or ATP buffer and concentrated salts to give final concentrations of 34 p M actin, 50 mM KCl, 1 mMMgC12, 1 mM EGTA, 0.2 mM nucleotide, 10 mM imidazole, pH 7.0. Samples were immediately transferred to the rheometer and incubated at 25 "C The abbreviation used is: EGTA, [ethylenebis(oxyethyleneni- for 8-12 h to attain an equilibrium configuration before measurements were made of the dynamic viscosity and elasticity. tri1o)jtetraacetic acid.
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tributed to the greaterflexibility of the ADP-actin filaments, zation.) Third, we titrated 4 PM etheno-ATP-actin or etheno-ADPwhich caused them to be more entangled than the stiff rod- actin with ATP. From the titration data we calculated the fluoreslikeATP-actin filaments. TheADP-actinfilaments ex- cence of the etheno-nucleotides when free and when bound to actin. changed their bound ADP faster than the ADPof filaments For etheno-ATP binding to actin increased the fluorescence 16 fold; for etheno-ADP binding increased the fluorescence 18-fold. From the assembled from ATP-actin. The addition of ATP stiffened dependence of the equilibrium fluorescence on the concentration of the ADP-actin filaments andgave them properties more like ATP at each point in the titration, we calculated the relative values ATP-actinfilaments. It was postulated that hydrolysis of of the dissociation constants for the etheno-nucleotides and ATP ATP within an actin filament "traps . . . the monomers . . . (similar to themethod of Waechter and Engel, 1975). The ratios were conformationally and stores elastic energy" that might be Kd ATP/Kd etheno-ATP = 0.3 and Kd ATP/Kd etheno-ADP = 0.015. "available for releaseby actin-binding proteins that transduceFrom the nucleotide concentrations and thesteady-state fluorescence at the end of the experiments in (Fig. 1C) we calculated the ratio of force or sever actin filaments." Kd ADP/Kd etheno-ATP to be 1.5. These relative equilibrium conSince this is the most interesting suggestion in years restants and thenucleotide concentrations were used to scale all of the garding the function of the bound nucleotide of actin, we fluorescence data. thought that it is important to confirm the key observations For exchange experiments with polymerized actin, we first deterof Janmey et al. (1990). In this paper we report new data on mined the critical concentration for the actin species being tested nucleotideexchange, filamentstructure,andmechanical (see below) and then prepared the samples for exchange experiments properties of actin assembled from ADP-actin or ATP-actin. by mixing concentrated monomer and polymer stocks to give the We prepared the ADP-actinquickly in the cold by a modifi- criticalconcentration of monomer and variableconcentrations of cation of current methods. Using this ADP-actin and ATP- polymer. This avoided prolonged incubations that otherwise would been required to allow either monomers or polymers to attain actin, we found noevidence for rapidexchange of any nucleo- have steady state after dilution into the polymerizing buffer. tide between polymerized actin and the solution and were Kinetic Simulation Methods-Rate constants for the exchange of unable to detect significant differences in theoverall structure nucleotides on actin monomers were determined by kinetic simulation or mechanical properties of filaments assembled from ATP- using an Apple Macintosh version (HOPKINSIM, Daniel Wachsactin or ADP-actin. stock, Johns Hopkins Medical School) of KINSIM (Barshop et al.,
Nucleotides
Actin
20341
Although the exchange of actin-bound nucleotide is relatively slow in calcium-containing solutions (Gershman etal., Preparation of Actin-Nucleotide Complexes-Actin with 1989), ATP, ADP, etheno-ATP,and etheno-ADPall exbound Mg2+ and ADP or etheno-ADP prepared by exchange in a low ionic strength buffer containing M e , EGTA, glu- change on a second time scale in buffers with M e and EGTA cose, and hexokinase had a critical concentration10-20 times (Fig. 1).Under these conditions, M$+ is bound to the high et al., 1986) in the higher than the corresponding ATP-actins (Table I). The affinity divalent cation site (Gershman cleft of the actin molecule (Kabsch et al., 1990). The time hexokinase acts asa scavenger for any ATP dissociated from course is essentially the same when the Mg-EGTA buffer has the actin or synthesized from ADP by contaminating adenylate kinase (Pollard, 1986; Gershman et al., 1989). Soluble either a low ionic strength favoring depolymerization of the hexokinase was left in the ADP-actin preparations used for actin or high enough concentrations ofKC1 and M e to rheological studies to remove any ATP generated during the strongly favor polymerization of the actin molecules. Simulation of the time course of these exchange reactions equilibration and measurements. allowed us toselect dissociation rate constantsfor each of the To measure the exchange of ADP bound to the actin for ATP from the buffer, it was necessary to remove the hexoki- four nucleotides (Fig. 1 legend) that together account the nase from the actin. This was accomplished by a new method kinetic data from four different types of nucleotide exchange using hexokinase immobilized on agarose beads. The hexoki- experiments (Fig. 1).We used the two-step mechanism exnase activity that could be achieved in the exchange reaction plained under“Experimental Procedures.” The simulation us to model the reactions without simpliusing the commercial preparation of hexokinase-agarose was approach allowed fying assumptions about irreversibility used in earlier analytabout 10 times lower than theconcentration of soluble hexoical solutions (Waechter and Engel, 1975). Although the nukinase known to be effective in producing ATP-free actin in cleotide exchange rate constants are very sensitive to experiprevious studies (Pollard, 1986). To reduce the need for high mental conditions (Frieden and Patane, 1988), our complete hexokinase activity, free ATP was removed from the starting set of values for the rate constants is in general agreement sample of actin before adding ADP andthe immobilized with literature values obtained for subsets of these constants enzyme. In steady-state criticalconcentrationexperiments under somewhat differentconditions(Kuehl and Gergely, the resulting Mg-ADP-actin has the same critical concentra1969; Waechter and Engel, 1975; Nowak et al., 1988). tion that made with soluble hexokinase (Table I). Thus the Nucleotide Exchange by PolymerizedActin-Samples of two preparations of Mg-ADP-actin have the same ratio of polymerized actin assembled from monomers with bound dissociation and association rate constants for polymer elonATP, ADP, etheno-ATP, or etheno-ADP exchanged a small gation. It is formally possible that theabsolute values of these fraction of their nucleotide on a second time scale, but the rate constants may differ for the two preparations. Since the rate of exchange by most of the actin in these samples was critical concentration is very sensitive to low concentrations too slow to detect in 20 min (Fig. 2). This confirms for all of ATP-actin in ADP-actin (Ohm and Wegner, 1991), these nucleotides previous observations onfilaments assembled results are strong evidence that the hexokinase-agarose effi- from ATP-actin (Martonosi et al., 1960). ciently removes all of the ATP from the ADP-actin preparaThe time course for the exchangeable fraction of nucleotide tion like soluble hexokinase (Pollard, 1984). in each of these polymerized actin samples was similar to Nucleotide Exchange on Actin Monomers-We first carried samples of monomer without filaments, and theamplitude of out a comprehensive set of nucleotide exchange experiments the signal was equal to or slightly less than that predicted by with actin monomers (Fig. 1) to establisha quantitative the critical concentration of monomers measured independmechanism that would allowus to interpretexperiments with ently by steady-state or kinetic methods. Beyond this signal more complex mixtures containing both monomers and fila- arising from exchange of nucleotide by the monomers, there ments. Others have investigated nucleotide exchange by actin was no additional reproducible signal attributable to nucleomonomers (see for example Kuehl and Gergely, 1969; tide exchange by the polymeric actin over the time course of Waechter and Engel, 1975; Frieden and Patane, 1988; Nowak our experiments. In the experiments with ATP-actin plus et al., 1988), but not for all of the relevant nucleotides and etheno-ATP, we detected no exchange of nucleotide by polynot in a buffer than approximates physiological ionic condi- mer concentrations up to 50 times the critical concentration tions. of monomers, where exchange, even at a low rate, would have been obvious (Fig. 2 A ) . The signal to noise ratio is even better TABLE I with etheno-ATP-actin reacted with ATP. In these experiCritical concentrationsfor actin polymerization ments, polymer concentrations 60 times higher than the critConditions: 50 mM KCI, 1 mM MgCl,, 1 mM EGTA, 0.2 mM ical concentration did not exchange over a period of 20 min nucleotide, 0.5 mM dithiothreitol, 10 mM imidazole (pH 7.0). The underlined values were obtained with samples of actin prepared with (Fig. 2B). In the experiments with ADP-actin filaments the hexokinase-agarose beads. The other ADP-actin samples were pre- critical concentration is much higher, so the highest polymer pared with soluble hexokinase. The samples used for elongation rate concentration tested was six times the monomer concentraand steady-state polymer concentration measurements contained 5% tion. Nevertheless, no nucleotide exchange of the polymerized pyrene-labeled actin. actin was detected over times greater than 10 half-times for exchange of nucleotide on the monomers in the same sample Method ATP-actin ADP-actin (Fig. 2C). Similarresults were obtained with filaments of PM PM PM etheno-ADP-actin where the signal to noise ratio was even Elongation rate 0.04 1.35 better (Fig. 2 0 ) . 0.90 0.05 Given the sensitivity of these assays, the half-time for the 0.05 exchange of nucleotide on the subunits of actin filaments is Steady-state polymer 0.10 1.20 concentration 0.05 more than 1,000 times greater than the half-times for the 1.15 1.35 exchange of the same nucleotide bound to actin monomers. Nucleotide exchange 1.0 This conclusion holds true for filaments assembled from ATP0.9 actin, ADP-actin, etheno-ATP-actin, or etheno-ADP actin. RESULTS
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Ethe::iy-
20342
Actin Nucleotides 1 .o
4
TA+eT==eTA+T
A o'8
0.6
t1
J
1000
500
0
1500
2000
3
a 4 I-
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DA + eT==eTA + D ,
a
200 D
400
600
800
eDA + T == TA +eD
2
1
1
~
~
100
"
"
200
"
"
300
"
'
400
0
500
600
0
50
100
150
200
Seconds FIG. 1. Time course of nucleotide exchange by actin monomers. Experimental data are given as open symbols and dashed line. Theoretical kinetic curves shown as continuous lines were calculated using KINSIM and the following rate constants: k , = 10 pL"' s-' for all nucleotide binding reactions; k- = 0.0045 s" for ATP, 0.015 s-' for etheno-ATP, 0.023 s-l for ADP, and 0.09 s" for etheno-ADP. Conditions: 2 mM Tris-C1, pH 7.5; 500 pM dithiothreitol; 200 pM EGTA, 80 p~ MgCI,. All samples contained nucleotides and etheno-adeninenucleotides as indicated and low concentrations of CaCl, that varied with the actin concentrations in the range of 3-10 p ~ Panel . A , exchange of ATP bound to actin for etheno-ATP. ATP-actin concentrations were 0.2 p M monomers plus 1 F M filaments (0) and 0.5 p~ monomers (0).The final ATP concentrations were 0.8 and 2 p ~ The . etheno-ATP concentration was 200 p ~ Panel . B , exchange of etheno-ATP bound to actin for ATP. The etheno-ATP-actinmonomer concentrations were 1 p~ (0), 2 /IM( O ) ,and 3 p~ (A). The final etheno-ATP concentrations were 17, 34, and 51 p ~ The . ATP concentration was 1,000 PM. Panel C , exchange of ADP hound to actin for etheno-ATP. ADP-actin monomer concentrations were 1 F M (O),2 pM (El),and 3 FM (A). The final ADP concentrations were 31,62, and 93 p ~ The . etheno-ATPconcentration was 200 p ~ Under . these conditions there was a significant concentration of ADP-actin in equilibrium with etheno-ATP-actin and the free nucleotides at the end of the reaction. Punel D, exchange of etheno-ADP bound to actin for ATP. The etheno-ADP-actin monomer concentrations were 1 p~ (O),2 p~ (El), and 3 p~ (A). The final etheno-ADP concentrations were 17,34, and51 p M . The ATP concentration was 1,000 pM.
Electron Microscopy of Actin Filaments Assembled from DISCUSSION ATP- or ADP-Monomers-Although there is alwayssome In a physiological buffer containing M e , KCl, and EGTA, heterogeneity in the appearance of actin filaments prepared a single set of rate constants can account for the timecourses for electron microscopy by negative staining, we could not detect any systematic differences in the gross appearance of of a wide range of nucleotide exchange experiments (Fig. I), values are reliable. All are actin filaments assembled from ATP-actin (Fig. 3, A-C) or so we are confident that these ADP-actin (Fig. 3, D-F). In particular, all of the grids con- based on a reasonable assumption that the association rate s-', a value citedby Nowak et al. (1988) tained filaments with smooth, gently curving contours. Most constants are 10 p"' from unpublishedwork. ofthe grids alsocontained someirregular filaments,but Four adeninenucleotides exchanged on a second time scale blinded observers could not distinguish two populations of of the from actin monomers, but we detected very little exchange on grids made with ATP-actin or ADP-actin. The quality stain varied from one grid square to the next but was not actin filaments over much longerperiods of time even in related to the nucleotide. One of four grids with ADP-actin experiments with high concentrations of filaments (Fig. 2). had considerable lateral aggregation of the filaments, but this Conservatively, we estimate that the nucleotide bound to actin was not characteristic of the other ADP grids. These obser- filaments polymerized from either ATP- or ADP-actin exvations rule out gross differences in filaments of ATP-actin changes 1,000 times slower than ATP or ADP on actin monand ADP-actin but do not preclude subtle differences in the omers. This exchange rate for subunits within filamentsis an packing or shape of the subunits that might be detected by upper limit, since some nucleotide exchanges into polymers high resolution reconstructions. Mechanical Properties of Actin Filaments Assembled from indirectly because of nucleotide exchange on monomers that off the endsof filaments. Theslow rate ATP- or ADP-Monomers-We detected no reproducible dif- then exchange on and of nucleotide exchange by polymerized actinisconsistent ferences in the viscous or elastic modulii of actin filaments assembled from ATP-actin or ADP-actin over a wide range with the atomic model of the actin filament (Holmes et al., long pitch helix appear of frequencies (Fig. 4). Filaments assembled from mixturesof 1990) wheresubunit contacts along the well ATP- and ADP-actin (made by converting most but not all to block the exitof the nucleotide from its binding site as as constraining the type of interdomain motions that open of the buffer ATP to ADP) had similar mechanical properties.
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2
0
0
3
3 t
0
eTA+T==TA+eT
B
20343
Actin Nucleotides TA+eT==eTA+T
0.6
"*-
-
0.2 uM monomer +1 uM filaments
0.1 uM monomer +1 uM filaments
"0"+2 uM filaments +3 uM filaments
-
+3 uM filaments
0.4
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E
o
loo0
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1500
2000
0.0 0
200
. ,
1.6
D
DA+eT==eTA+D
400
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600 ,
,
1000
800
,
.
.
eDA+T==TA+eD
0.8
0.4
1 uM monomer
o . o ~ " ' " ' ~ " r
0.0
0
100
200
300
400
0
500
100
200
300
400
500
. these conditions there was a significant concentration of ADP-actin in equilibrium with etheno-ATPwere 31, 62, 93, and 124 p ~ Under actin and thefree nucleotides at the endof the reaction, accounting for the incomplete exchange of ADP. Panel D,exchange of etheno-ADP bound to actin for ATP. All samples contained 1.0 p~ etheno-ADP-actin monomers and 1,000 p~ ATP. The etheno-ADP-actin filament 2 p M (O), and 3 pM (A). The final etheno-ADP concentrationswere 17, 34, 51, and 68 p ~ . concentrations were 0 (bold line), 1 p~ (0),
the nucleotide binding site in hexokinase. Our conclusions are infull agreement with the long accepted belief that ADP is tightly bound to the subunits in actin filaments (Martonosiet al., 1960) but differ from two previous studies of nucleotide exchange by actin filaments using etheno-ATP fluorescence. Wang and Taylor (1981) followed the exchange of etheno-ATP for ATP and the reverse reaction with 5 p~ actin in a polymerization buffer with 2 mM MgC12 & 100 mMKC1. They observed two phases of fluorescence change. Initially, fluorescence changed rapidly with an amplitude of about 20% (-1 p M actin) and half-time of 5-10 min ( k 0.001 s-l). They interpreted thisfirst phase as therapid exchange of nucleotide on monomers and addition of some of these monomers to theends of filaments. They did not measure the critical concentration, but the amplitude of their fast phase was about 10 times larger than expected from later determinations under similar conditions (Table I).We cannot explain this difference from our experiments. The rate of the second phase of the fluorescence change was variable. In 100 mMKC1 with 50 pM MgCI2 the rate was near zero. In 2 mM MgC12 the maximum rate was about 0.1 nM s" and was attributed to the flux of subunits throughfilaments. Our calculation of their subunit exchange rate (0.1 nM subunits/ s/2 nM filamentends = 0.05 subunits/filament end/s) is consistent with the rate of subunit flux (about 0.1 s-l) calculated from the elongation rate constants (Pollard, 1986), so it is not necessary to postulate rapid exchange of nucleotide by subunits within the polymers. These conclusions are consistent with ours. Janmey et al. (1990) reported that solutions of ADP-actin filaments exchange bound nucleotide more rapidly than ATPactin filaments. They capped the barbed end of the filaments
-
to eliminate the slow flux of subunits observed by Wang and Taylor (1981). From the the partial time courses of nucleotide exchange in polymeric actin reported by Janmey et al. we estimate first-order rate constantsof 0.005 s-l for ADP-actin and 0.001 s" for ATP-actin.Their signal came from the binding of etheno-ATP, butthese values should approximate the dissociation rate constantsfor ADP and ATP. Both values are about &fold lower than our dissociation rate constants for ADP and ATP from monomeric actin, a difference that is expected because of the Ca2+in their buffer which slows the rate of nucleotide dissociation (Frieden and Patane, 1988). We suggest that their signal arose in large part from the critical concentration of monomers in their filament samples, as in our experiments in Fig. 2. The reason is that Janmeyet al. calibrated their fluorescence signal by assuming that 100% of the nucleotide in samples of filaments exchanged in 20 h. They calculated the fluorescence increment per molecule of actin-bound nucleotide from the totalactin concentrationand the fluorescence change at that time. Twenty hours is probably inadequate for complete equilibration of nucleotide with filaments capped on their rapidly exchanging ends by gelsolin. Consequently, they may have underestimated the magnitude of the fluorescence change. This explains why their experiments appear to show extensive exchange of nucleotide on polymerized actin on a minute time scale. The presence of gelsolin may also account for the relatively small difference in the amplitudes of exchange that they observed for ATPactin andADP-actin. Without gelsolin the fluorescence signal arising from the monomers present ina filament sample is 10 times higher for ADP-actin than ATP-actin (Fig. 2) because of the large difference in their critical concentrations at the barbed end. However, with the barbed ends capped by gelsolin,
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Seconds
FIG. 2. Time course of nucleotide exchange by mixtures containing a critical concentration of actin monomers and a range of concentrations of actin filaments. Note that therewas no exchange by the filaments above that expected from the critical concentrations of actin monomers. Panel A , exchange of ATP bound to actin for etheno-ATP. All samples contained 0.2 p~ ATP-actin monomers and 200 f i etheno-ATP. ~ ATP-actin filament concentrations were 1p M (0) and 3 pM (A).The final ATP concentrations were 7.2 and 20.4 p ~ Panel . B , exchange of etheno-ATP bound to actin for ATP. All samples contained 0.1 @M etheno-ATP-actin monomers and 1,000 PM ATP. The etheno-ATP-actin filament concentrations were 0 (bold line), 1 pM (O),2 pM (U), and 3 pM (A). The final etheno-ATP concentrations were 17, 19, 36, and 53 p M . Panel C , exchange of ADP bound to actin for etheno-ATP. All samples contained 1.0 p~ ADP-actin monomers and 200 p~ etheno-ATP. ADP-actin filament concentrations were 0 (bold line), 1 p~ (0), 2 p M (O), and 3 p M (A). The final ADP concentrations
20344
Nucleotides
Actin
0
ATP. q' ATP. G
0
ADP-q' ADP.G
8
E
100
0 1 FIG.4. Comparison of the rheological properties of ATPand ADP-actin filaments. Actin filaments at were analyzedby oscillation in a cone-plate rheometer at 25 "C over a wide range of frequencies. Conditions:34 PM ATP-actin (circles);34 p~ ADP-actin (squares). The filled symbols represent the elastic modulus ( G ' ) is dynes/cm2, and the open symbob represent the dynamic viscosity in poise.
the critical concentrationsare determined by the pointed ends where the ATP-actin and ADP-actin differ only by a factor of 2-3 (Coue and Korn, 1985; Pollard, 1986). We used nucleotide exchange by monomers and competitive nucleotide bindingexperiments (see "Experimental Procedures") to calibrate the absolute fluorescence increment for each nucleotide exchange pair. In our experiments with mixtures of filaments and thecritical concentrationof monomer, only the critical concentration of actin monomers exchanged rapidly. Even without capping the barbed ends, no nucleotide exchange pair gave a substantial fluorescence signal from filaments during the 20-min duration of our experiments. Because the nucleotide exchange rate by polymerized actin is so slow, a new experimental design will be required to detect any subtle differences in nucleotide exchange rates of filaments assembled from ADP- and ATP-actin. We found no difference in the electron microscopic appearance (Fig. 3) or mechanical properties (Fig. 4) of actin filaments assembled from ADP- or ATP-actin. Our observations do not exclude small differences in the structureof ADP- and ATP-actin filaments that may exist at the atomic level, but both the microscopy and rheology differ from the report of Janmey et al. (1990). First, our ADP-actin filaments have smooth, not irregular, profiles just like ATP-actin filaments.
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FIG. 3. Electron micrographs of negatively stained actin filaments. A-C, filaments assembled from ATP-actin. D-F, filaments assembled from ADP-actin. Magnifications:A, R, D,and E = 113,000 X; C and F = 173,000 X. Bars = 0.1 um.
20345
Actin Nucleotides
through the filaments and to preparefilaments for rapid disassembly at a time subsequentto theirassembly. Acknowledgments-WethankDr. Pascal Goldschmidt-Clermont for suggesting the use of the hexokinase-agarose beads for making ADP-actin andDr.PaulJanmeyfor exchanging ideas, data, and proteins in our ongoing effort to reconcile our experimental results. REFERENCES Amato, P. A., and Taylor, D. L. (1986) J. Cell Bid. 102,1074-1084 Barshop, B. A,, Wrenn, R. F., and Frieden,C. (1983) Anal. Biochem. 1 3 0 , 134145 Carlier, M.-F. (1991) J. Bid. Chem. 2 6 6 , 1-4 Carlier, M.-F., and Pantaloni, D. (1986) Biochemistry 26,7789-7792 Carlier, M.-F., and Pantaloni, D.(1988) J. Biol. Chem. 263,817-825 Carlier, M.-F., Pantaloni, D., and Korn, E.D. (1987) J. Bid. Chem. 262,30523059 Carlier, M.-F., Pantaloni, D., Evans, J. A., Lambooy, P. K., Korn, E. D., and Webb, M. R. (1988) FEBS Lett. 236,211-214 Cooper, J. A. (1991) Annu. Reu. Physiol. 5 3 , 585-605 Cooper, J. A., and Pollard, T. D. (1982) Methods Enzymol. 85,182-210 Coue, M., and Korn, E. D. (1985) J. Biol. Chem. 260,15033-15041 Drenckhahn, D., and Pollard, T. D. (1986) J. Bid. Chem. 261,12754-12758 Drewes, G., and Faulstich, H. (1991) J. Bid. Chem. 2 6 6 , 5508-5513 Forscher, P., and Smith,S. J. (1988) J. Cell Biol. 107,1505-1516 Frieden, C., and Patane, K. (1988) Biochemistry 2 7 , 3812-3820 Gershman, L., Selden, L., and Estes,J. (1986) Biochem. Biophys. Res. Commun. 135,607-614 Gershman. L.. Selden., L... Kinosian. H. J.. and Estes., J . (1989) Biochim. Bioohvs. . . ” Acta 996,109-115 Holmes, K., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 3 4 7 , 4449 Janmey, P.A., Hvidt, S., Peetermans, J., Lamb, J., Ferry, J. D., and Stossel, T. P. (1988) Biochemistry 27,8218-8227 Janmey, P. A,, Hvidt, S., Oster, G. F., Lamb, J., Stossel, T. P., andHartwig, J. H. (1990) Nature 347,95-99 Kabsch, W., Mannherz, H. G., Suck, D., Pai, E. F., and Holmes, K. C. (1990) Nature 347,37-44 Korn, E. D., Carlier, M.-F., and Pantaloni, J. (1987) Science 238,638-644 Kuehl, W. M., and Gergely, J. (1969) J. Biol. Chem. 244,4720-4729 MacLean-Fletcher, S., and Pollard, T. D. (1980) Biochem. Biophys. Res. Commun. 9 6 , 18-27 Martonosi, A,, Gouvea, M. A,, and Gergely, J. (1960)J. Bid. Chem. 236,17001711 Nowak, E., Strezelecka-Golaszwska,H., and Goody, R. S. (1988) Biochemistry 2 7 , 1785-1792 O’Donoghue, S. I., Miki, M., and dosRemedios, C.G. (1992) Arch. Biochem. Biophys. 2 9 3 , 110-116 Ohm, T., and Wegner, A. (1991) Biochemistry 3 0 , 1193-1197 Pollard, T. D. (1984) J. Cell Biol. 9 9 , 769-777 Pollard, T. D. (1986) J. Cell Biol. 103,2747-2754 Pollard, T. D. (1990) Curr. Opin. Cell Biol. 2 , 33-40 Pollard, T.D., and Weeds, A. G. (1984) FEBS Lett. 170,94-98 Rickard, J. E., and Sheterline, P. (1988) J. Mol. Bid. 201,675-681 Sato. M..Leimbach. G.. Schwarz. W. H.. and Pollard. T. D. (1985) J. Bid. , , Chem. 260,8585-8592 Straub, F. B., and Feuer, G. (1950) Biochim. Biophys. Acta 4,455-470 Theriot, J. A., and Mitchison, T. J. (1991) Nature 3 5 2 , 126-131 Waechter, F., and Engel, J. (1975) Eur. J. Biochem. 57,453-459 Wang, Y:L. (1985) J. CellBiol. 1 0 1 , 597-602 Wang, Y.-L., and Taylor, D. L. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,55035507 Wanger, M., and Wegner, A. (1987) Biochim. Biophys. Acta 9 1 4 , 105-113 ,
I
,
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Second, our ADP-actin filaments and ATP-actin filaments had similar mechanical properties, whereas the ADP-actin filaments of Janmey et al. were about 10 times more rigid than their ATP-actin filaments. Furthermore, the absolute values of the elastic modulii of filaments prepared from fresh actin are much lower in our laboratory than Janmey’s, an unresolved, long-standing difference (see Sato et al., 1985; Janmey et al., 1988). The experimental design differed in two ways: ( a ) methods to prepare the actin, and ( b ) the inclusion of gelsolin in the rheological experiments of Janmey et al. to control the length of the filaments. Differences in the actin rather than the gelsolin seem more likely to account for the difference in rigidity. The differences in the observations between the two laboratories have not yet been resolved but may lie in the preparation of the ADP-actin. Our current methods take advantage of rapid exchange of nucleotide by Mg-actin (Gershman et al., 1989) to make ADP-actin quickly in the cold in an effort to avoid an inevitable, time-dependent (k = 2 X loT5s-l at 0 “C) conformational change that can be detected in ADP-actinby sulfhydryl titration of Cys-10 (Drewes and Faulstich, 1991). We never freeze purified actin preparations. Janmey et al. used a slower, early method from our laboratory (Pollard, 1984) to prepareADP-actin from frozen stocks. Ca-ATPactin is incubated with hexokinase and glucose at room temperature for several hours. These ADP-actin preparationsare not completely stable (Pollard, 1984), perhaps because of the conformational change. It was necessary to incubate their ADP-actin for 2 h at room temperature before the differences with ATP-actinbecame apparent. TheCys-10 conformational change should have occurred on part of the ADP-actin prepared by this method and may account for the substantial differences in the properties of the ADP-actin in the two laboratories. It is particularly interesting that the structural differences in ADP-actin filamentsobserved by Janmey et al. (1990) were reversed rapidly by ATP just like the Cys-10 conformational change (Drewes and Faulstich, 1991). O’Donoghue et al. (1992) also observed that ADP-actin prepared by our 1984 method has a higher viscosity than ATP-actin. Given that ADP- and ATP-actinfilaments are the same by our criteria, new evidence will be necessary to confirm the hypothesis that nucleotide hydrolysis within actin filaments is used to modulate their mechanical properties. Rather, we expect that nucleotide hydrolysis is used to drive subunit flux
~
