©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding of Divalent Cation and Nucleotide to G-actin in the Presence of Profilin (*)

(Received for publication, July 8, 1994; and in revised form, October 10, 1994)

Irina Perelroizen Marie-France Carlier Dominique Pantaloni

From the Laboratoire d'Enzymologie du C.N.R.S., 91198 Gif-sur-Yvette, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, has been investigated. 1) In low ionic strength buffer, profilin increases the rates of Ca and Mg dissociation from G-actin 250- and 50-fold, respectively. On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divalent metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion. 2) Under physiological ionic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is increased by profilin from 0.05 s to 2 s, the rate of MgADP dissociation is increased from 0.2 s to 24 s. The dependences of the exchange rates on profilin concentration are consistent with a high affinity (5 times 10^6 to 10^7M) of profilin for ATP-G-actin, and a 20-fold lower affinity for ADP-Gactin. Profilin binding to actin lowers the affinity of metal-nucleotide by about 1 order of magnitude. These results restrain the possible roles of profilin in actin assembly in vivo.


INTRODUCTION

Monomeric actin (G-actin) tightly binds a nucleotide (ATP or ADP) in complex with a divalent metal ion (Ca or Mg). The metal-ATP is bound as a beta,-bidentate chelate in the -configuration(1, 2, 3, 4) ; conversely, the metal-ADP is bound as a betamonodentate. The interaction between bound nucleotide and metal ion is responsible for the long-recognized regulation of the binding of nucleotide to G-actin by divalent metal ions (see Refs. 5 and 6, for review). Kinetic data of metal ion and nucleotide exchange on G-actin are consistent with Fig. SI, according to which dissociation of the tightly bound divalent cation (M) is kinetically limiting for nucleotide(N) dissociation and binding of the divalent metal ion to actin (A) increases the affinity of nucleotide by 4-6 orders of magnitude.


Scheme I: Scheme I



Recent extensive kinetic analysis of Ca or Mg dependences of the rates of nucleotide exchange in low ionic strength buffer (6) have brought full confirmation for the validity of this scheme with either ATP or ADP as bound nucleotide. The ratio of the dissociation rate constants for M (k) and MN (k) is 30 for CaATP, but only about 2 for MgATP, which indicates that the kinetics of nucleotide exchange are much less affected by Mg ion than by Ca ion. Under all conditions, ADP dissociates from G-actin about 10-fold faster than ATP (6) . It should be noted that all nucleotide exchange measurements have been thoroughly performed only at low ionic strength; available scattered evidence indicates that rates of exchange are about 1 order of magnitude higher under physiological ionic conditions(7) .

Whether nucleotide exchange on G-actin, which partly regulates the ATP-G-actin:ADP-G-actin ratio, is important in the regulation of actin assembly in living cells, is not known.

Many G-actin binding proteins are known to influence the rate of nucleotide exchange on G-actin. Cofilin(8) , thymosin beta(4)(9) , ADF(10) , and actophorin (11) reduce it, while in contrast, profilin accelerates it(8, 9, 12) . This property of profilin is corroborated by the observed 5 ° rotation of the two domains of the G-actin molecule crystallized in complex with profilin(4) .

A model was proposed (9, 13) according to which profilin would catalyze the formation of polymerizable ATP-G-actin off a pool of ADP-G-actin maintained unpolymerizable by interaction with thymosin beta(4). However, experimental evidence thus far does not support this model, since thymosin beta(4), a major G-actin sequestering agent in vertebrates(14, 15) , has been shown to bind ATP-G-actin with high selectivity over ADP-G-actin(7) . Hence in vivo Tbeta(4) essentially sequesters ATP-G-actin, which inevitably increases the overall ratio of ATP/ADP bound to G-actin, and prevents the formation of ADP-G-actin under conditions leading to ATP depletion in cells. On the other hand, it has been shown that the ability of profilin to promote actin assembly in the presence of a pool of sequestered actin (16) was a consequence of the reported(17, 18) participation of the profilin-actin complex to filament elongation at the barbed ends. In this process, the free energy of ATP hydrolysis associated to actin assembly is used by profilin to facilitate its dissociation from the barbed end following incorporation of an actin subunit(16) . Hence profilin might bind less well to an F-ADP than to an F-ADP-P(i) end, or profilin could accelerate P(i) release from an F-ADP-P(i) end.

To definitely assess whether the property of profilin to accelerate nucleotide exchange on G-actin is relevant in profilin function in living cells, it is necessary to provide a quantitative description of the mechanism of metal-nucleotide exchange on profilin-actin under physiological conditions. We have measured the change in affinity and in the kinetics of metal ion and nucleotide exchange on G-actin as a function of profilin concentration, at low ionic strength and in physiological ionic conditions. The results show that the effect of profilin on nucleotide binding to actin cannot account for the function of profilin in actin assembly.


MATERIALS AND METHODS

Chemicals

BAPTA, (^1)EGTA, ATP, 8-hydroxyquinoline sulfonate (8OH-Q), Quin2 were from Sigma. Dithiothreitol (DTT), ATP, and ADP were from Boehringer Mannheim. 8-[^3H]ATP was from Amersham.

Proteins

Actin was purified from rabbit muscle (19) and isolated as CaATP-G-actin by Sephadex G-200 chromatography (20) in standard G buffer (5 mM Tris-Cl, pH 7.5, 0.1 mM CaCl(2), 0.2 mM ATP, 0.2 mM DTT, 0.01% NaN(3)). Profilin was purified from bovine spleen as described previously (21) and stored on ice at 200-300 µM in 5 mM Tris-Cl, pH 7.5, 1 mM DTT.

The concentrations of G-actin and profilin were determined spectrophotometrically using extinction coefficients of 0.617 mg cm^2 at 290 nm for G-actin (22) and 15,000 M cm at 277 nm for profilin(23) .

CaATP-G-actin was prepared as follows. The CaATP-G-actin complex (30 µM) freed of unbound ATP by Dowex 1 treatment was incubated overnight on ice in the presence of 0.25 mM ATP. About 80-90% of free nucleotide was removed by one rapid Dowex 1 treatment (0.2 ml of a 50% Dowex 1 suspension/ml of actin solution). The slurry (1-1.5 ml) was immediately filtered onto a Millipore (0.22 µm) filter and loaded on a Sephadex G-25 column (PD-10, Pharmacia Biotech Inc.) equilibrated in G buffer containing the desired amounts of CaCl(2) and ATP (5-100 µM, as indicated in text).

MgATP-G-actin was prepared as described (24) by simultaneous addition of 0.2 mM EGTA and 50 µM MgCl(2) to a solution of G-actin (leq20 µM) in standard G buffer. After 3 min incubation, the MgATP-actin solution was equilibrated by gel filtration (PD-10) at 0 °C in G buffer containing no CaCl(2), 25 µM EGTA, and the desired concentrations of MgCl(2) and ATP. The concentration of MgCl(2) in buffer was at most 10 µM in excess of ATP, to avoid formation of actin oligomers(25) . MgADP-G-actin and MgADP-G-actin were prepared as described previously(26) .

Fluorescence Measurements

Static fluorescence measurements or slow kinetics were carried out at 20 °C using a Spex Fluorolog 2 spectrofluorimeter. Excitation and emission wavelengths were 350 and 410 nm, respectively, for ATP, 340 and 500 nm for Quin2, 295 and 330 nm for tryptophan. Small square quartz cells (3-mm light path) were used to minimize the inner filter effect, which nevertheless was taken into account and corrected at high concentration (geq100 µM) of Quin2, by performing titration curves of fluorescence change versus [Ca] at different concentrations of Quin2.

Rapid Kinetics

The kinetics of metal ion or nucleotide exchange on G-actin were carried out using a stopped-flow apparatus (DX.17 MV, Applied Photophysics) operated either in absorbance or fluorescence mode. The instrument was thermostatted at 20 °C. The excitation slit was 1 mm in absorbance mode, and 0.5 mm in fluorescence mode. The dead time of the instrument was 0.8 ms. The rate of Ca dissociation from CaATP-G-actin in the presence of different amounts of profilin was monitored using the change in absorbance at 254 nm due to the formation of the BAPTA-Ca complex(27) . It was checked that the reaction of 50 µM BAPTA with Ca, at concentrations as low as 0.25 µM, was complete within the stopped-flow dead time. The specific absorbance change of BAPTA was 0.010 cm µM. The experiment was conducted as follows. The two solutions containing the reagents to be mixed were placed in drive syringes A and B. Syringe A contained CaATP-G-actin (5-12 µM, equilibrated in G buffer containing 20 µM ATP and 5 µM CaCl(2)) and variable amounts of profilin in the range 0 to 30 µM. Syringe B contained 120 µM BAPTA and 20 µM MgCl(2) in 5 mM Tris-Cl, pH 7.6, buffer. Identical volumes of A and B were mixed.

The rate of Mg dissociation from MgATP-G-actin in the presence of profilin was monitored spectrophotometrically using the change in fluorescence of 8OH-Q upon reaction with Mg as described(28) . The excitation wavelength was 327 nm, and a 430-nm cut off filter was placed on the emission beam.

The rate of ATP dissociation from G-actin in the presence of profilin was monitored by fluorescence. A KV 380 Schott cut-off filter was placed on the emission beam. Syringe A contained CaATP-G-actin (2-5 µM) in G buffer containing 10 µM ATP and CaCl(2) as indicated. Syringe B contained 100-400 µM ATP in 5 mM Tris-Cl, pH 8.0. The final free Ca ion concentration was calculated taking into account the formation of the CaATP complex with an equilibrium dissociation constant of 5 µM(29) . The rate of ATP dissociation from MgATP-G-actin was studied in a similar fashion as follows. A stock solution of CaATP-G-actin was prepared at 20-30 µM in 5 mM Tris, 0.2 mM DTT, 5 µM CaCl(2), 5 µM ATP, pH 8.0. At time 0, actin was diluted to 1.5 µM (1 ml) in a buffer containing 5 mM Tris, 0.2 mM DTT, 100 µM EGTA, 20 µM MgCl(2), 10 µM ATP, pH 8.0, and a given amount of profilin. After 2 min incubation for calcium-magnesium exchange, this solution was placed in syringe A of the stopped-flow. Syringe B contained 5 mM Tris, 0.2 mM DTT, 100 µM EGTA, 20 µM MgCl(2) (or as indicated), and 200-2000 µM ATP, pH 8.0. When physiological ionic conditions were investigated, syringe B (ATP chase) also contained 1 mM MgCl(2) and 0.1 M KCl, and the same amounts of MgCl(2) and KCl were added, from a [4 M KCl, 40 mM MgCl(2)] stock solution, to the MgATP-G-actin-profilin sample immediately prior to filling the drive syringe A. For each (profilin + MgATP-G-actin) sample, 4 to 6 shots were performed, the traces were checked to be superimposable, averaged, and analyzed within monoexponentials using the software attached to the DX.17 MV instrument.

The rate of MgADP dissociation from G-actin in the presence of profilin was investigated in a similar fashion. MgADP-G-actin was in the presence of 20 µM ADP in syringe A, and ADP (200 µM) was present in syringe B.

The analysis of the kinetics and amplitude of either metal ion or nucleotide exchange routinely revealed the existence of a small proportion (10-25%) of the G-actin molecules which did not bind profilin, presumably due to oxidation of Cys-374(31) . This part of the population exchanged metal ion and nucleotide at the slow rate specific of G-actin alone, at all concentrations of profilin. All data shown in this article concern the 75-90% proportion of actin which truly binds profilin.

It should be noted that since most kinetic experiments of metal-nucleotide exchange on G-actin in the presence of profilin were carried out in the stopped-flow, the pH was chosen to be 7.5-8.0 in most cases, because the rates of divalent metal ion and nucleotide are known to be about 1 order of magnitude faster at pH 8.0 than at pH 7.0 (30) , which made experiments technically more convenient at 0 or low amounts of profilin. Additional controls showed that the main conclusions concerning the effect of profilin on the rate constants apply at pH 7.0 too, with lower rate constants.


RESULTS

Analysis of the Profilin Concentration Dependence of the Kinetics of Ca Dissociation from G-actin

The time course of Ca dissociation from G-actin was monitored by the change in BAPTA absorbance using the stopped-flow. In the absence of profilin, the release of Ca upon simultaneous addition of Mg and BAPTA was a simple exponential process, with rate constant k = 0.087 ± 0.005 s at pH 8.0 and 20 °C, in satisfactory agreement with published values (6, 28, 32, 33, 34) . When profilin was present with G-actin, the release of Ca was faster, as previously reported(9, 34) . At saturation by profilin, a higher limit rate constant of 19 ± 2 s was obtained (Fig. 1), which corresponded to the rate constant k` for dissociation of Ca from the profilin-actin complex. The rate of Ca dissociation from G-actin is known to depend on pH(28, 29) . This dependence was also observed on the profilin-actin complex. Typically the rate constants k and k` were 0.05 s and 12 s at pH 7.5, and 0.04 s and 10 s at pH 7.0. Hence at all pH values profilin accelerated 250-fold the release of Ca from G-actin. However, the process was truly first order only in the absence and presence of saturating amounts of profilin. In the concentration range where profilin partially saturated G-actin, the dissociation process of Ca was not very well fitted by a single exponential and appeared biphasic, as can be seen in Fig. 1; in addition, the profilin concentration dependence of the reciprocal of t was not a simple Michaelian titration curve, rather it exhibited a strongly sigmoidal behavior, displayed in Fig. 2. These results indicate that one cannot consider that profilin is in rapid equilibrium with G-actin, in which case the release of Ca would be a monoexponential process of rate constant.


Figure 1: Time course of Ca dissociation from G-actin in the presence of profilin. The change in BAPTA absorbance upon reaction of 60 µM BAPTA and 10 µM MgCl(2) with 3 µM G-actin in the presence of 1.5 µM profilin was recorded. The noisy curve is the experimental time course; the thin curve is the calculated monoexponential best fit to the data (k = 1.763 s); the thick curve is the simulated time course, within Fig. SIII, using the following (best fit) values of the rate constants: k = 0.08 s; k` = 15 s; k = 45 µM s; k = 10 s; k = 2.5 s. Inset, expanded view of the early stages of the Ca dissociation process, emphasizing the biphasic nature of the reaction.




Figure 2: Profilin concentration dependence of the rate of Ca and ATP dissociation from G-actin. The kinetics of dissociation of Ca from G-actin was monitored by the change in BAPTA absorbance using the stopped-flow as described under ``Materials and Methods.'' Dissociation of ATP was monitored by fluorescence. Syringe A contained CaATP G-actin (6 µM) in 5 mM Tris-Cl, 0.2 mM DTT, 20 µM CaCl(2), 10 µM ATP, and profilin at different concentrations. Syringe B contained 120 µM BAPTA, 20 µM MgCl(2), and 50 µM ATP in 5 mM Tris-Cl, 0.2 mM DTT, pH 8.0, buffer. The reciprocal of the dissociation half-time is plotted versus final total profilin concentration. Crosses and circles correspond to measurements of Ca and ATP dissociation, respectively, carried out in parallel experiments. The three curves are theoretical and have been calculated as follows. The left curve (thin line) corresponds to the case where profilin is in rapid equilibrium with G-actin and is calculated according to in which k = 0.08 s, k` = 19 s, K = 0.33 µM, and

The middle curve is calculated within Fig. SIIwith k = 0.08 s, k` = 19 s, k = 45 µM s, k = 15 s. The rightmost curve (thick line) is calculated within Fig. SIII using the same values as above of all rate constants and k = 0.5 s. Different values of k could be found that would better fit each time course separately, however, the value of 0.5 s provides the best global fit to all time courses.




Scheme II: Scheme II



In , K(P) represents the equilibrium dissociation constant for binding profilin to G-actin, [P] the free profilin concentration, and k and k` the rate constants for Ca dissociation from actin and profilin-actin, respectively. In the present case, the rate constants for profilin association to and dissociation from G-actin are known to be 45 µM s and 10 ± 3 s, respectively(21) , so that the overall association-dissociation rates are not very fast as compared to the rates of Ca dissociation from profilin-actin; in particular at low saturation levels of G-actin by profilin, the shuttling process of profilin from one actin monomer to the other limits the rate of Ca release. The above kinetic Fig. SII(subscript P represents profilin) was suggested by the data.

The kinetics of Ca dissociation from actin in the presence of profilin, upon addition of BAPTA + Mg were simulated according to the above scheme using KINSIM (^2)with values of rate constants k(P) and k(P) experimentally determined in our previous work (21) and values of k and k` measured in the present work. The simulated exchange kinetics did show a biphasic appearance in the presence of profilin, and the reciprocal of t of simulated time courses varied in a sigmoidal fashion with profilin concentration, however, the resulting curve did not fit experimental data satisfactorily (Fig. 1, thin curve). Comparison with the data indicates that at substoichiometric amounts of profilin, the shuttling of profilin from one actin molecule to the other is slower than expected within Fig. SII. Using lower values of k(P) yielded a plot of simulated values of 1/t which was more steeply cooperative than the latter (with k(P) = 10 s) but did not fit the data either. A better fit to the data was obtained by introducing a supplementary slow step (k(i)) in the recycling of profilin, as described in Fig. SIII (subscript P represents profilin).

Fig. 2shows that the fit was improved by setting k(i) < 2.5 s. The slow step (k(i)) may be either the slow binding of Mg ions to the profilin-actin complex following dissociation of Ca, or the isomerization of profilin-actin before or after Mg binding.

It is well established that at low Ca concentration the dissociation of Ca is rate-limiting in nucleotide dissociation from G-actin(32) . In order to determine whether this compulsory order dissociation of Ca followed by ATP also takes place on the profilin-actin complex, an experiment was designed in which the kinetics of either ATP or Ca dissociation from CaATP-G-actin (6 µM) were measured at different profilin concentrations. The experiment was carried out with CaATP-G-actin and profilin present in syringe A in G buffer containing 10 µM ATP and 20 µM CaCl(2). Syringe B contained 120 µM BAPTA, 20 µM MgCl(2), and 50 µM ATP. Either the changes in BAPTA absorbance or ATP fluorescence were recorded, in a parallel series of experiments in which profilin concentration was varied. The time courses of ATP or Ca dissociation, once expressed in concentration units, were strictly superimposable. Fig. 2shows that the values of 1/t were the same for ATP or Ca dissociation, at all concentrations of profilin. Therefore, both in the presence and absence of profilin, the dissociation of nucleotide from calcium-G-actin is kinetically limited by the dissociation of Ca. This conclusion was further documented by examining the profilin concentration dependence of the rate of ATP dissociation from G-actin, at different free Ca concentrations shown in Fig. 3. At all Ca concentrations, the rate constant for ATP dissociation increased with profilin and reached a higher limit at saturation by profilin, which represented the apparent rate constant for ATP dissociation from the profilin-actin complex. The maximum increase in ATP dissociation rate was 150-200-fold, at all Ca concentrations. The exchange rate constant k was a hyperbolic function of profilin concentration leading to a value of 0.13-0.15 µM for K(P), the equilibrium profilin binding constant, in good agreement with the value derived from fluorescence measurements(21) . The value of the rate constant for dissociation of ATP from profilin-actin varied with free Ca concentration, as has long been observed in the absence of profilin (28, 32, 36, 37) . Fig. 3b shows that the reciprocal of k at saturation by profilin varied linearly with free Ca concentration in the range 0-1 µM.


Figure 3: Change in the apparent rate constant for ATP dissociation from G-actin versus profilin concentration, at different concentrations of Ca. The kinetics of exchange of ATP for bound ATP on calcium-G-actin was monitored using the stopped-flow as described under ``Materials and Methods.'' Panel A, syringe A contained 1.6 µM CaATP-G-actin in 5 mM Tris, 0.2 mM DTT, 10 µM ATP, 5-150 µM CaCl(2), and different amounts of profilin. Syringe B contained 200 µM ATP in the same buffer. The concentration of free Ca was calculated in each series of experiments, as follows: a, 5 µM CaCl(2), [Ca] = 0.2 µM; b, 10 µM CaCl(2), [Ca] = 0.5 µM; c, 20 µM CaCl(2), [Ca] = 1 µM; d, 75 µM CaCl(2), [Ca] = 10 µM. The first-order rate constant for ATP dissociation is plotted versus final profilin concentration. Panel B, Ca concentration dependence of the rate constant for ATP dissociation from the profilin-CaATP-G-actin complex. Syringe A contained 1.6 µM CaATP-G-actin (same buffer conditions as in Panel A) and a saturating amount (5 µM) of profilin. Syringe B contained different concentrations of ATP in 5 mM Tris-Cl. The reciprocal of the observed rate constant is plotted versus the concentration of free Ca calculated at each point. The square symbol is the reciprocal of k` coming from an independent experiment using BAPTA. Inset, expanded view of the data at low [Ca].



In conclusion, the general scheme for Ca and nucleotide dissociation from G-actin is not qualitatively different, on the profilin-actin complex, from Fig. SI, which has been established for G-actin alone(5, 6) . The following equation, initially proposed (31) to describe the apparent rate constant for nucleotide exchange on G-actin as a function of free Ca in a range of low Ca concentrations, also applies to the profilin-actin complex.

where k` represents the rate constant for calcium dissociation from the CaATP-G-actin-profilin complex, K` the equilibrium dissociation constant for Ca binding to ATP-G-actin-profilin and k` the rate constant for dissociation of ATP from ATP-G-actin-profilin following Ca release. The value of k` derived from the plot of 1/kversus [Ca] in Fig. 3b, was 12 s at pH 7.5 in agreement with the value derived from direct measurement of Ca dissociation from the profilin-actin complex using BAPTA. Measurement of the slope of the plot allows the determination of k`, providing that the value of K` is known.

In the presence of saturating amounts of profilin, as in the absence of profilin(6) , the dissociation rate constant of ATP decreased upon increasing the concentration of free Ca ions. A lower value of 0.17 s corresponding to the dissociation of CaATP from the profilin-actin-CaATP complex, was obtained at saturation by Ca ions. In the absence of profilin, a value of 0.002 s was obtained (data not shown). Hence the rate of dissociation of CaATP is also increased 100-fold by profilin.

Change in Affinity of Ca for ATP-G-actin Upon Binding Profilin

The affinity of Ca for ATP-G-actin in the absence and presence of saturating amounts of profilin was measured at pH 7.0 using Quin2, essentially as described by Gershman et al.(38) . In full agreement with these authors, the partial dissociation of Ca from G-actin observed in the presence of a large excess of Quin2 (500 µM) over G-actin (Fig. 4) was consistent with G-actin having a 50-fold higher affinity for Ca than Quin2. The equilibrium dissociation constant of the Quin2-calcium complex, K was measured at low ionic strength by fluorescence titration of 10 µM calcium by Quin2 in the presence of a series of concentrations of EGTA as a competitive inhibitor. The data indicated that K = 0.5-0.6 times K. Using K = 140 nM at pH 7.0 and low ionic strength(39) , a value of 70-80 nM was derived for K, in reasonable agreement with the 60 nM used by Gershman et al.(38, 40) . Hence the equilibrium dissociation constant for binding Ca to ATP-G-actin is 1.4 nM.


Figure 4: Profilin binding to G-actin is accompanied by a decrease in the affinity of tightly bound Ca. The change in Quin2 fluorescence was used to monitor the release of Ca from G-actin (20 µM) in the absence (thin line) and presence (thick line) of a saturating amount (30 µM) of profilin in a buffer containing 5 mM MOPS, 0.2 mM ATP, and 10 µM CaCl(2) at pH 7.0. The concentration of Quin2 was 500 and 50 µM in the absence and presence of profilin, respectively. The fluorescence change was converted in concentration of Ca released taking into account the inner filter effect of Quin2 which was different in both curves. The first arrow indicates the addition of Quin2 to the G-actin ± profilin solution leading to partial dissociation of bound Ca. The second arrow indicates the subsequent addition of 50 µM MgCl(2) leading to total exchange of Mg for bound Ca.



In the presence of saturating amounts of profilin, a 10-fold lower amount of Quin2 (50 µM) caused a larger dissociation of Ca from profilin-actin than the one elicited by 500 µM Quin2 in the absence of profilin. Although Ca dissociation was complete within the mixing time (5 s), the measurements of fluorescence intensities obtained upon addition of Quin2, then of Mg, were accurate enough to derive the relative amounts of profilin-ATP-actin and profilin-CaATP-actin after addition of Quin2. A quantitative analysis of the data shown in Fig. 4, and of similar data obtained at different concentrations of Quin2, led to the conclusion that the affinity of Ca for ATP-G-actin decreased 35-fold upon binding profilin, leading to K` = 49 nM at low ionic strength and pH 7.0. Since the rate constant for Ca dissociation increased 250-fold at the same pH, we understand that the rate constant for association of Ca to ATP-G-actin increases 250/35 = 7.5-fold upon binding profilin. Using a value of 49 nM for K`, the value of the rate constant for ATP dissociation from the profilin-actin complex (following Ca release) could be derived from the data shown in Fig. 3. A value of 30 s was obtained. A corresponding value of 4.2 s was derived from data (not shown) obtained in the absence of profilin. In conclusion, binding of profilin to G-actin causes a 2 order of magnitude increase in the rate constants for dissociation of Ca ion from G-actin and a 1 order of magnitude increase in the rate of association of Ca, which ends up with an overall decrease in the affinity of Ca for ATP-G-actin.

Change in the Rate of Mg Dissociation from G-actin upon Binding Profilin

The rate of Mg dissociation from G-actin was monitored using 8-hydroxyquinoline (sulfonate) as a magnesium-indicator as described under ``Materials and Methods.'' Data displayed in Fig. 5show that, in the absence of profilin, Mg dissociation from MgATP-G-actin, elicited by the simultaneous addition of Ca ions and 8-hydroxyquinoline, occurred as a slow monoexponential process of rate constant 0.025 s at pH 8.0, in good agreement with previous reports(28) . In the presence of profilin, the release of Mg from G-actin was accelerated. A higher limit of 1.25 ± 0.1 s was reached at saturation by profilin. Hence the dissociation of Mg is also accelerated about 50-fold by profilin.


Figure 5: Profilin binding to MgATP-G-actin increases the rate of Mg dissociation from G-actin. The release of Mg from MgATP-G-actin was elicited by the rapid mixing 8-hydroxyquinoline and (200 µM final) CaCl(2) (25 µM) to a solution of MgATP-G-actin (8 µM) in 5 mM Tris-Cl, 0.2 mM DTT, 12.5 µM EGTA, 5 µM MgCl(2), 5 µM ATP, and profilin as indicated. The change in fluorescence was monitored in the stopped-flow. The solid line is calculated using with K = 0.15 µM, k = 0.025 s, k` = 1.25 s; and total actin, 5.5 µM (i.e. 70% of actin bound profilin).



Change in the Rate of Nucleotide Exchange on MgG-actin in the Presence of Profilin at Low Ionic Strength and Under Physiological Ionic Conditions

The increase in the rate of ATP dissociation from MgATP-G-actin in the presence of profilin was examined using the stopped-flow as described under ``Materials and Methods.'' In low ionic strength buffer, and at very low Mg concentration (in the presence of 20 µM MgCl(2) and 0.6 mM ATP, free Mg concentration was 0.02 µM), the apparent rate constant for dissociation of ATP was 0.019 s, which represented the Mg dissociation rate constant from G-actin. At saturation by profilin this rate constant reached an upper limit of 0.92 ± 0.1 s corresponding to a 45-fold increase, in reasonable agreement with the value obtained from direct measurements of Mg dissociation using 8OH-Q. In the presence of saturating amounts (100 µM) of MgCl(2), in low ionic strength buffer, the rate constant for dissociation of ATP (i.e. the dissociation rate constant of MgATP) from the profilin-actin complex was 0.49 s, about only twice lower than the rate constant for Mg dissociation, similar to the result obtained for G-actin alone by Kinosian et al.(6) .

Under physiological ionic conditions (Mg-actin, 0.1 mM EGTA, 1 mM MgCl(2), 0.1 M KCl), the rate constant for dissociation of ATP was 0.054 s in the absence of profilin. The rate of ATP dissociation increased with profilin to an upper limit of 2.1 ± 0.2 s at saturation by profilin. The data shown in Fig. 6were consistent with the rapid association-dissociation equilibrium of profilin with Mg-actin as compared to the rates of nucleotide dissociation, leading to a hyperbolic profilin concentration dependence of the apparent rate constant for ATP dissociation, as described by . Within this analysis, the equilibrium dissociation constant for profilin binding to G-actin under physiological conditions was K = 0.16 µM ± 0.02 µM, in reasonable agreement with the value of 0.3-0.5 µM derived from the shift in critical concentration plots when barbed ends are capped(16) . Hence the present experiments demonstrate that the affinity of profilin for G-actin is in the 10^7M range in physiological as well as in low ionic strength buffer.


Figure 6: Change in the rate of nucleotide dissociation from G-actin upon binding of profilin under physiological ionic conditions. The kinetics of ATP (respectively, ADP) exchange for bound ATP (respectively, ADP) in the presence of different concentrations of profilin was monitored using the stopped-flow as described under ``Materials and Methods.'' Squares, exchange of ATP for bound ATP. The final G-actin concentration was 0.7 µM, and profilin varied as indicated. Solid line is the theoretical curve calculated using and the following values of the parameters: k = 0.05 s; k` = 2 s; K = 0.16 µM; total actin = 0.6 µM (i.e. 86% of the actin bound profilin). Circles, exchange of ADP for bound ADP. Final MgADP-G-actin was 1.25 µM. The curve is calculated using and k = 0.2 s, k` = 24 s, K = 3.3 µM; total actin, 0.9 µM (i.e. 72% of the actin effectively bound profilin).



Under physiological conditions, the rate of MgADP dissociation from G-actin was also accelerated by profilin from 0.2 s to 24 s (Fig. 6). The profilin concentration dependence of the dissociation rate () was consistent with an equilibrium dissociation constant of 3.3 µM for profilin binding to ADP-G-actin. Hence, in full agreement with previous independent measurements(16) , profilin displays a 1 order of magnitude higher affinity for ATP-G-actin than for ADP-G-actin under physiological ionic conditions.

This conclusion was comforted by the results of the following experiment. MgATP-G-actin was diluted to 0.5 µM in physiological polymerization buffer (0.1 M KCl, 1 mM MgCl(2)) containing 5 µM ATP and variable amounts of ADP in the concentration range 0-250 µM, either in the absence or presence of 5 µM profilin. The relative amounts of ATP-G-actin and ADP-G-actin were derived from the measurement of ATP fluorescence as a function of ADP concentration. The ratio [G-ATP]:[G-ADP] was found equal to 1 at 12 µM ADP in the absence of profilin and 45 µM ADP in the presence of profilin. Hence the ratio K/K was 12:5 = 2.4 in the absence of profilin and 45:5 = 9 in the presence of profilin. In other words profilin increased the preference of G-actin for ATP over ADP, under physiological conditions, in agreement with the conclusions from kinetic data.

Decrease in ATP and ADP Affinity for G-actin in the Presence of Profilin

The three-dimensional structure of G-actin at atomic resolution(2, 3, 4) , as well as biochemical evidence (1, 32) clearly indicate that the tight binding of the metal-triphosphate moiety of the metal-nucleotide complex to amino acid residues of the two domains of actin is responsible for the very high affinity of ATP. The results described above, showing that profilin binding weakens the binding of the metal ion, therefore let us anticipate that profilin causes a decrease in the overall affinity of ATP or ADP for G-actin, especially at low concentrations of divalent metal ion. The following experiments were carried out to address this issue.

[^3H]ADP-G-actin was prepared by polymerization of [^3H]ATP-G-actin 1:1 complex with 1 mM MgCl(2) and resuspension in G buffer containing 2.5 µM ADP, no CaCl(2), 100 µM MgCl(2), 25 µM EGTA, 1 µM Ap(5)A. Following equilibration between labeled and unlabeled ADP on G-actin (9 µM), the solution was split into two samples, one of which was supplemented with 15 µM profilin. Each sample (1 ml) was chromatographed on Sephadex G-25 (PD-10 Pharmacia) pre-equilibrated in the actin buffer without ADP. Fig. 7shows that [^3H]ADP remained 85% bound to G-actin eluted from the column in the control sample, while it was completely dissociated from G-actin in the presence of profilin. On the other hand, when the same experiment was done with [^3H]ATP-G-actin in the presence of as low as 5 µM CaCl(2) or MgCl(2) in G buffer, no appreciable dissociation of [^3H]ATP from G-actin was observed upon addition of profilin(21) . These results indicate that the affinity of ADP for G-actin is decreased upon binding profilin to a value allowing complete dissociation of the nucleotide in the micromolar range, while in the same range of concentration ATP does not dissociate appreciably from the profilin-actin complex.


Figure 7: Binding of profilin to MgADP-G-actin is accompanied by a decrease in the affinity of ADP. Mg-[^3H]ADP-G-actin was prepared as described under ``Materials and Methods'' and chromatographed on Sephadex G-25 (PD-10) in 5 mM Tris-Cl, 0.2 mM DTT, 100 µM MgCl(2), 25 µM EGTA buffer without nucleotide. One milliliter of the ADP-G-actin (9 µM) in the same buffer supplemented with 2.5 µM ADP without (Panel a) or with (Panel b) 15 µM profilin was loaded on the column. Elution was monitored by protein measurement at 280 nm (circle) and [^3H]ADP radioactivity measurement (bullet).



The extent to which the affinity of ATP for G-actin decreased upon binding profilin was therefore examined at a lower actin concentration using ATP fluorescence as follows. CaATP-G-actin was prepared as described under ``Materials and Methods'' and isolated by Sephadex G-25 chromatography at 23.8 µM in 5 mM Tris, 0.2 mM DTT, 5 µM ATP, 5 µM CaCl(2) buffer. This actin stock solution was diluted to different final concentrations (0.3-1.6 µM) in 5 mM Tris buffer containing different concentrations of CaCl(2) (0-5 µM) and no nucleotide. The ATP fluorescence of each sample was measured before and immediately (5 s) after addition of 4 µM profilin. The addition of profilin caused the rapid partial (17-47%) dissociation of bound ATP, as shown by an instantaneous drop in fluorescence. When 2 µM ATP was present in the buffer, no change in ATP fluorescence was observed upon addition of profilin, which confirmed that the decrease observed above was due to the actual dissociation of ATP from profilin-actin and not a simple quenching of its fluorescence linked to profilin binding to G-actin. The concentrations of PA-CaATP and PA complexes, free ATP, and Ca at equilibrium could be derived from the measured fluorescence decrease in each sample. The concentration of free CaATP was then calculated using a value of 5 µM for the equilibrium dissociation constant of CaATP(29) . The binding constant of CaATP to the profilin-actin complex was K = ([PA]bullet[CaATP])/([PA-CaATP]). Data coming from a series of measurements are shown in Table 1. A consistent value of 0.055 µM ± 0.020 µM was obtained for K. These results therefore demonstrate that the affinity of CaATP for G-actin is lowered to 2.10^7M, i.e. by about 2 orders of magnitude, upon binding profilin. All equilibrium and rate parameters for metal ion and nucleotide interaction with profilin-actin are summarized in Table 2.






DISCUSSION

We have examined how the thermodynamics and kinetics of metal ion and nucleotide binding to G-actin were affected by profilin, in order to quantitatively assess whether the changes were relevant in profilin function in vivo.

Profilin increases the rates of dissociation of both metal ion and nucleotide from G-actin as previously reported(8, 9, 12) . However, the basic mechanism (Fig. SI) for metal ion-nucleotide exchange is not changed by profilin, i.e. at low concentrations of divalent metal ion, dissociation of the metal ion is rate-limiting in nucleotide exchange on profilin-actin. At low ionic strength, the rates of Ca and Mg dissociation are increased 250- and 50-fold, respectively, by profilin. Our results differ from a previous report (35) stating that the rate of Ca release was increased 10-fold by profilin, while the rate of nucleotide exchange was increased 1000-fold, which implicity means that the mechanism of metal-nucleotide exchange would be modified by profilin. However, in the absence of rapid kinetics apparatus, the authors could not have access to the actual rate constants at saturation by profilin, and the values that they gave were derived from adjustment of kinetic curves to a model for binding of ATP and profilin to actin in which detailed balance was not respected ((35) , Table III). Hence we hope that the present experimental determinations are useful.

At very low Ca concentration (<0.1 µM), the rate-limiting process in metal dissociation varies with the saturation by profilin. At subsaturating amounts of profilin, Ca dissociation is rate-limited by the shuttling of profilin from one actin molecule to the other; the nature of the kinetically limiting reaction (0.5 s), either binding of Mg to profilin-actin following Ca release, or isomerization of profilin-actin prior to or after Mg binding, is not known. On the other hand, at saturation by profilin, the observed metal-nucleotide exchange reaction is the dissociation of Ca from the profilin-actin complex.

For the first time, the exchange of ATP and ADP has been studied under physiological ionic conditions. In support to a previous report showing that binding of cations to low affinity sites increased the rate of dissociation of the tightly bound metal ion (41) we find that MgATP, the physiological ligand of G-actin, dissociates 1 order of magnitude faster (0.054 s) than at low ionic strength. Profilin accelerates dissociation of MgATP up to 50-fold (2.1 ± 0.2 s). The rate of MgADP is also increased by profilin from 0.2 to 24 s under physiological ionic conditions. The rate constant of MgATP or MgADP dissociation from actin varies as a hyperbolic saturation function with profilin concentration, from which the affinity of profilin appeared to be 5.10^6-10^7M for MgATP G-actin, and 2-fold lower for MgADP G-actin, consistent with recent independent measurements of the sequestering activity of profilin(16) . The spleen profilin species used here and in our previous work (16, 21) is profilin I, also widely found in platelets, neutrophils, thymus, and lung. The affinity of profilin I for ATP-actin was previously thought to be 10-100-fold lower (10^5-10^6M, see (42) for a review of previous works), based on measurements of the shift in critical concentration or the inhibition of filament growth when barbed ends are free. These methods have been demonstrated to be inadequate (16, 44) because profilin effects the thermodynamic of assembly at the barbed ends in the presence of ATP. All direct measurements of profilin I binding to ATP-actin confirm the very high affinity (10^7M) that can be derived from the shift in critical concentration or from inhibition of growth at the pointed ends ( (16) and (21) , and the present work). The very high affinity of mammalian profilin I for ATP-actin has profound consequences regarding its function in vivo. The following conclusions can be drawn from our data.

Profilin is a potent ATP-G-actin sequestering agent when the profilin-actin complex does not participate in filament assembly, i.e. when barbed ends are capped(16) , a situation thought to take place when cells are not stimulated, e.g. in resting platelets(43) . Profilin appears to bind G-actin 10-fold more tightly than thymosin beta(4). The relative amounts of actin sequestered as profilin-actin (PA) and thymosin beta(4)-actin (TA) can be calculated as follows. Typically in a cell containing 100 µM Tbeta(4) and 50 µM profilin, assuming these two proteins to have free access to actin, and the free G-actin concentration to be buffered, by the filament pool, to the critical concentration at the pointed ends (C

Hence the amount of G-actin sequestered by profilin is greater than previously estimated. Because profilin has a strong preference for ATP-G-actin versus ADP-G-actin, like other G-actin binding proteins, e.g. ADF (10) and beta thymosins(7) , it contributes to increase the pool of G-actin sequestered in the ATP form in the cytoplasm.

It has been demonstrated (45) that because exchange of ATP for bound ADP on G-actin occurs at a finite rate (k), ADP-G-actin accumulates to a steady state level in F-actin solutions in the presence of ATP, as a consequence of the combined production of ADP-G-actin by dissociation of ADP subunits from filaments (k), disappearance of ADP-G-actin by medium ATP exchange for bound ADP (k), and by association to filament ends (k). The following equation (45) describes the resulting amount of ADP-G-actin at steady state.

Assuming that a living cell typically contains 20 nM filaments, and using values of 5 µM s for k and 10 s for k, and the values of k determined here under physiological conditions (k = 0.2 s in the absence of profilin and k = 24 s at saturation by profilin), we come to the conclusion that the acceleration of ADP dissociation from G-actin by profilin causes a change in [G ADP] from 0.6 to 0.01 µM, a change much too small to have any physiological relevance as compared to the 5.10M change in the amount of sequestered actin actually occurring in cells upon stimulation. Hence our quantitative measurements do not support the model proposed (9, 12; recently reviewed in (46) and (47) ) according to which the acceleration of nucleotide exchange would be responsible for profilin function. In contrast, the control of free G-actin concentration at steady state by profilin, as a corollary of profilin-actin participation to assembly at the barbed ends, has been demonstrated to cause actin assembly off the pool of sequestered actin (16) .

The affinity of both metal ion and nucleotide for G-actin is reduced by about 30-fold upon binding profilin, which implies a greater increase in their dissociation than in their association rate constants. This decrease in affinity may be in relation with the observed 5 ° angle rotation of the two domains of the actin molecule in the profilin-actin crystalline complex(4) , as compared to the actin-DNase I or actin-gelsolin segment I complexes. It may also indicate that the environment of the bound metal ion in the actin cleft is changed upon binding profilin. In a cellular medium rich in ATP, this decrease in affinity of MgATP is not a priori likely to have any physiological significance. On the other hand, under conditions leading to ATP depletion in cells, ADP might dissociate from G-actin upon binding of profilin, leading to a profilin-actin (0) complex with no metal ion and nucleotide bound, in which the affinity of profilin would be increased in proportion with the decrease in nucleotide affinity. More detailed experiments and modelization studies would be necessary to understand the structure and function of this complex.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; 8OH-Q, 8-hydroxyquinoline (8-quinolinol); ATP, 1-N^6-etheno-ATP; ADF, actin depolymerizing factor; DTT, dithiothreitol; Ap(5)A, P^1,P^5-di(adenosine 5`)-pentaphosphate; PA, profilin-actin; MOPS, 4-morpholineethanesulfonic acid.

(^2)
KINSIM software versions were kindly supplied to us by C. Frieden and T. Pollard.


ACKNOWLEDGEMENTS

We are very grateful to Drs. Carl Frieden and Thomas Pollard for sending the two versions of the KINSIM software used in this work. We also thank Dominique Didry for help in the preparations of actin and profilin, and Laurent Blanchoin for his careful guidance in initial stopped-flow experiments.

Note Added in Proof-The kinetics of metal/nucleotide exchange on profilin-actin were not affected by poly-L-proline.


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