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Binding of phosphate ions to actin
Biochimica et Biophysica Acta 914 (1987) 105-113
105
Elsevier BBA 32887
Binding of phosphate ions to actin Michael Wanger and Albrecht Wegner Institute of Physiological Chemistry, Ruhr-University Bochum, Bochum (F.R.G.)
(Received18 February1987)
Key words: Actin; Gelsolin; Phosphate; Fluorescence;1,N6-Ethenoadenosine5'-triphosphate The decrease of the critical monomer concentration of ADP-actin by millimolar phosphate concentrations has been analysed in terms of equilibrium constants for binding of phosphate ions to ADP-actin. The decrease has been explained by a 10-fold greater affinity of phosphate ions to polymeric ADP-actin (binding constant I00 M - l ) than to monomeric ADP-actin (binding constant I0 M - t). Phosphate has an almost identical effect on the critical monomer concentration of the pointed ends of gelsolin-capped actin filaments in the presence of ATP. The phosphate concentration required for half-maximal decrease of the critical monomer concentration of the pointed ends has been determined to be about 15 mM. By using the fluorescent ATP-analogue, l,N6-ethenoadenosine 5'-triphosphate, phosphate ions have been found to bind also to monomeric ATP-actin, yet with a slightly higher affinity than to monomeric ADP-actin (binding constant 50 M - 1).
Introduction Since the discovery that actin hydrolyzes ATP during polymerization [1], many efforts have been made to better understand and quantitatively analyze this reaction. ATP hydrolysis has been found to cause actin filaments to treadmill due to the different critical monomer concentrations of the two ends [2-6]. ATP hydrolysis has been reported to lag behind assembly of monomers with filament ends [7-9]. ADP-actin wi'th tightly bound phosphate has been detected following ATP hydrolysis by chemically cross-linked actin dimers [10]. More recently, Rickard and Sheterline [11] reported that the critical monomer concentration of ADP-actin is decreased by millimolar phosphate concentrations. They proposed that ADPactin with a weakly bound phosphate ion may be Correspondence: A. Wegner, Institute of PhysiologicalChemistry, Ruhr-University Bochum, P.O. Box 102148, D-4630, Bochum, F.R.G.
an intermediate of the ATP hydrolysis reaction by actin, and that ATP-actin and ADP-actin with bound phosphate have a high affinity to filament ends whereas ADP-actin has a low binding affinity. In this paper we analyze quantitatively the equilibrium of binding of phosphate ions to ADP-actin and we investigate whether or not ATP and phosphate ions compete for binding to the surface of actin molecules. Materials and Methods Preparation of the proteins
Actin was prepared according to the method of Rees and Young [12]. The protein was applied to a Sephacryl S-200 column (2.5 × 90 cm). Part of the protein was modified with N-ethylmaleimide at cysteine-374 [13] and subsequently with 7-chloro4-nitro-2,1,3-benzoxadiazole at lysine-373 to produce a fluorescently labeled actir/ [14]. The concentration of actin was determined photometri-
0167-4838/87/$03.50 © 1987 ElsevierScience Publishers B.V. (BiomedicalDivision)
106
cally at 290 nm using an absorption coefficient of 24 900 M - 1. cm- 1 [2]. The gelsolin-actin complex was prepared from human platelet concentrate. Platelets were purified by differential centrifugation [15] with the modification that 20 nM prostaglandin E 1 was included in the suspension buffer to inhibit aggregation of the platelets [16]. Following the first resuspension, the purification was continued according to the method of Lind et al. [17]. The platelet extract was applied to an Agarose affinity column to which DNAase I (deoxyribonuclease I, EC 3.1.21.1) was covalently bound (1.5 x 12 cm, Affi-Gel 10, purchased from Bio-Rad). The column was equilibrated with monomeric actin to produce immobilized actin [18]. The gelsolin-actin complex was eluted from the affinity column by using 5 mM EGTA buffer [17]. The concentration of the gelsolin-actin complex was determined by the method of Lowry et al. [19] and Bradford [20]. For calculation of the molar concentration the molecular masses of gelsolin and actin were assumed to be 90 kDa and 42.3 kDa, respectively [21,22].
Preparation of the nucleotides 1,N6-Ethenoadenosine 5'-triphosphate (~ATP) was synthetized according to the method of Secrist et al. [23] with the modification that the crude product was apphed to a DEAE-Sephadex A-25 column (2.5 x 40 cm) and eluted with a linear NH4HCO 3 gradient (0.15-0.33 M) [24]. The cATP was analyzed as described previously [24]. ADP was separated from traces of ATP by chromatography on a DEAE-Sephadex A-25 column (2.5 x 40 cm). ADP was eluted with a linear NH4HCO 3 gradient (0.15-0.33 M). According to HPLC chromatography (RP 18 column, 1 mM tetrabutylammonium dihydrogenphosphate/3.3 % acetonitrile/65 mM KH2POa-H3PO4 (pH 2.2)), ADP contained less than 0.1% ATP. ADP-actin was prepared by exchange of ATP for ADP on a Sephadex G-25 Superfine column (2.5 x 50 cm) equilibrated with 500/aM A D P / 1 0 #M MgC12/5 mM triethanolamine-HC1 (pH 7.5)/200 mg/1 NaN 3.
Fluorescence Actin polymerization was followed by the
2.2-2.5-fold greater fluorescence intensity of polymeric actin compared to that of monomeric actin [14]. 5% of fluorescently labeled actin was copolymerized with unmodified actin. This low proportion of labeled actin does not significantly alter the polymerization rate or extent of assembly of unmodified actin [5]. The excitation wavelength was 436 nm, and the fluorescence intensity was measured at 530 nm. The changes of the fluorescence intensities were evaluated in terms of concentration of monomeric or polymeric actin. The fluorescence intensity of monomeric or polymeric actin was calibrated by measuring the fluorescence intensities of dilution series of monomeric or polymeric actin. The exchange of actin-bound nucleotides was measured by the increase of the fluorescence intensity on binding of cATP to monomeric actin [25,26]. The excitation wavelength was 360 nm and the emitted light was measured at 410 nm. The changes of the fluorescence intensities were evaluated in terms of free or actin-bound ~ATP. The fluorescence intensity of free or actin-bound ~ATP was calibrated by measuring the fluorescence intensities of dilution series of free and actin-bound ~ATP. Addition of phosphate (40 mM) did not significantly change the fluorescence intensity of the fluorescence label of actin or of cATP.
Experimental procedure The critical monomer concentration of ADPactin in the presence of various phosphate concentrations was measured both after depolymerization of polymeric ADP-actin (3 #M) and after polymerization of monomeric actin (2 #M) onto actin filaments (1 ~M). Samples were prepared by mixing a 20 mM MgC12 solution, a 1 M KCI solution, a 25 mM EGTA solution, a 100 mM KPOn solution, a 5 mM ADP solution, ADP-buffer (500 /~M ADP/10 ~M MgC12/5 mM triethanolamine-HCl (pH 7.5)/200 mg/1 NAN3), 25 /~M polymeric ADP-actin polymerized by the addition of 1 mM MgC12 and 100 mM KC1, and 25 /~M monomeric ADP-actin solution if necessary. The solutions were mixed in such a ratio that the final composition of the samples was 1 mM MgC12, 100 mM KC1, 0.5 mM EGTA. 500 /~M ADP, various concentrations of phosphate (0-45
107
mM), 5 mM triethanolamine-HC1 (pH 7.5), 200 mg/1 NAN3, polymeric ADP-actin and monomeric ADP-actin if necessary. Fluorescence intensities of the samples were measured at 10 min intervals until a constant final fluorescence intensity was reached (4 h). The critical monomer concentration of the pointed ends of actin filaments in the presence of ATP was determined similarly to the measurements on ADP-actin with the following alterations. ADP was replaced by ATP. Gelsolincapped actin filaments were used instead of polymeric ADP-actin. EGTA was replaced by 0.2 mM CaC12, which is necessary for tight binding of gelsolin to the barbed ends of actin filaments [27]. The exchange of actin-bound nucleotides was followed by titration of monomeric ADP- or ATP-actin (1 ~tM) with cATP. Samples were prepared by combining the solutions used for measurements of the critical monomer concentrations of ADP-actin with the following modifications. The concentration of ADP or ATP was in the range of 10-25/~M, and various CATP concentrations (0-250 /~M) were added. EATP was dissolved in 10/~M MgC12/5 mM triethanolamineHC1 (pH 7.5)/200 mg/1 NaN 3. First, the fluorescence intensity of (ATP was measured and then monomeric ADP- or ATP-actin was added. The exchange of ADP or ATP for cATP was followed by the increase in the fluorescence intensity. All experiments Were performed at 25 o C. Results
Binding of phosphate ions to monomeric and polymeric ADP-actin The effect of various concentrations of phosphate on the critical monomer concentration of ADP-actin is depicted in Fig. 1. Similar monomer concentrations were reached both after depolymerization of polymeric actin and after polymerization of monomeric actin onto actin filaments (Fig. 1). The monomer concentrations measured after depolymerization tended to be slightly lower than those measured after polymerization of monomers onto actin filaments. In agreement with Rickard and Sheterline [11], phosphate was found to decrease the critical monomer concentration. Half-maximal decrease occurred at about 13.5 mM
IK
~
2"
¢-I I
< + I
a_
1
0 .<
" ] ' ~ t ~ ~
l
0
i
i
I
........
i
|
5O
[P] /mM Fig. 1. Effect of various concentrations of phosphate on the critical monomer concentration of ADP-actin. [P], phosphate concentration; [ADP-G], concentration of monomeric ADPactin; [ADP-G-P], concentration of monomeric ADP-actin with bound phosphate. 1", monomer concentrations reached after polymerization of monomers onto filaments; Z, monomer concentration reached after depolymerization of actin filaments. - - , one-parameter fit of the equilibrium constant for binding of phosphate to monomeric ADP-actin K l ( K 1 =10 M - l ) . • . . . . . , two-parameter fit of K 1 ( K l = 8 M -1) and of the equilibrium constant for binding of ADP-actin with bound phosphate to a filament end K 4 ( K 4 = 5.5.10 6 M - ] ).
phosphate. The data summarized in Fig. 1 can be interpreted by the simple reaction scheme of binding of phosphate to ADP-actin depicted in Fig. 2. The four equilibrium constants defined in Fig. 2 are correlated with the concentrations of free phosphate [Pi], of monomeric ADP-actin [ADP-G], of monomeric ADP-actin with bound phosphate [ADP-G-P] and to the probability, ap, that the terminal filament subunit binds phosphate, in the following way: K1
[ADP-G-PI [ADP-G]- [Pi]
K1
(1 --
K3
[ADP-G]
K4
[ADP-G-P]
ap ap)"[Pi]
(1)
(2)
1-ap
(3)
ap
(4)
108
ADP~ +
p KI ~, ~ A O P ~ P P +
...."~'"..i
!...'"'..i p K2
ADP~
~"
~ ADP~P
Fig. 2. Reaction scheme of binding of phosphate to actin and of the assembly of monomeric actin with filament ends. P, phosphate.
The four equilibrium constants are not independent. K 2 c a n be expressed in terms of K~, K 3 and K4: K4
K2 = K33 "K1
(5)
The total monomer concentration, [ADP-G] + [ADP-G-P], can be calculated by using Eqns. 1-5: K1"[Pi]+ 1 [ADP-G] + [ADP-G-P] = K3 + K4"Ka"[Pi]
(6)
As the phosphate ions occur in great excess over actin monomers and filament subunits, the free phosphate concentration [Pi] is approximately equal to the total phosphate concentration. The equilibrium constant for binding of phosphate-free monomeric ADP-actin to actin filaments, K3, can be easily determined. In the absence of phosphate ( a p = 0 ) the monomer concentration ADP-G is equal to K31 (Eqn. 3). Since, according to Fig. 1 1.5 ~tM monomers coexist with polymeric actin in the absence of phosphate, K 3 turns out to be 6.7.105 M -1 (1/1.5 # M - l ) . It has been proposed [11] that ADP-actin with bound phosphate is similar to ATP-actin with respect to the polymerization reaction. In that case the critical monomer concentration of ADP-actin with bound phosphate would be expected to be similar to that of ATP-actin. The critical monomer concentration of ATP-
actin has been found to be 0.12 /~M under the same experimental conditions (Fig. 4). If this value was pertinent to ADP-actin with bound phosphate, K 4 would be 8 . 1 0 6 M -1 (1/0.12 /~M -1, a p = 1, Eqn. 4). The equilibrium constant for binding of phosphate to monomeric ADP-actin K 1 was fitted for K 3 = 6.7.105 M -1 and K 4 = 8 • 1 0 6 M -1. A curve calculated for K 1 = 10 M -1 is depicted in Fig. 1 as a continuous line. This calculated curve is in reasonable agreement with the experimental data. Generally, at great phosphate concentrations, the calculated monomer concentrations are lower than the measured monomer concentrations, suggesting that the critical monomer concentration of ADP-actin with bound phosphate is slightly higher than that of ATP-actin. We therefore determined K 1 and K 4 also by a twoparameter fit. The fitted line is depicted in Fig. 1 as a dotted line. According to this fit, the equilibrium constant for binding of phosphate to monomeric ADP-actin K 1 has been found to be 8 M-1 and the equilibrium constant for binding of monomeric ADP-actin bearing phosphate to a filament end K 4 has turned out to be 5.5.10 6 M -1 The critical monomer concentration of ADP-actin with bound phosphate is 0.18 /~M (1/K4). Both fits yield similar values of K 1 (10 and 8 M -1) and K l ( 8 . 1 0 6 and 5.5.106 M - l ) . The equilibrium constant of binding of phosphate ions to actin filaments K z can be calculated to be in the range of 100-a (Eqn. 5). Effect of phosphate on the critical monomer concentrations of the two ends of actin filaments in the presence of A TP The effect of various concentrations of phosphate on the critical monomer concentration of the pointed ends of actin filaments in the presence of ATP is depicted in Fig. 3. Actin filaments capped at the barbed ends were produced by polymerization of actin onto gelsolin-actin complex [16]. The monomer concentrations reached both after depolymerization of polymeric actin and after polymerization of monomeric ATP-actin onto the pointed ends of gelsolin-capped actin filaments are depicted in Fig. 3. A decrease in the critical monomer concentration of the pointed ends in the presence of phosphate has already been observed by Rickard and Sheterline [11]. In con-
109
::k
1.0
o.+ II+ I t~t x
+
t t:+I o i
so [P] !mM Fig. 3. Effect of various concentrations of phosphate on the critical monomer concentration of the pointed ends of gelsolin-capped actin filaments in the presence of ATP. [P], phosphate concentration; ~p, concentration of monomeric actin. T, monomer concentration reached after polymerization of monomers onto filaments; ±, monomer concentration reached after depolymerizationof actin filaments.
trast to the results of Rickard and Sheterline, who reported that under similar experimental conditions the critical monomer concentration of the pointed ends decreases half-maximally in the presence of 1 mM phosphate, we found that 15 mM phosphate is necessary for the same effect (Fig. 3). These results cannot be analyzed as an equilibrium because of the continuous ATP hydrolysis occurring during assembly and disassembly of the pointed ends [28,16]. In agreement with the results of Rickard and Sheterline [11] we found that phosphate has no significant effect on the critical monomer concentration of uncapped actin filaments in the presence of ATP (Fig. 4). As the association and dissociation reactions at the barbed ends are at least 10-fold faster than at the pointed ends [3,4,27], the critical monomer concentration is mainly determined by the barbed ends [3,5,29]. Thus, the determined critical monomer concentrations are approximately equal to the critical monomer concentration of the barbed end. One can conclude that in the presence of ATP, phosphate affects the pointed ends but not the barbed ends of actin filaments.
Effect of phosphate on the exchange of actin-bound nucleotide 3--
-0.1 -0.2
%0 ~~ w
0
w
.,~
gl
i
0.2
!
0.4
C/ilJM Fig. 4. Determination of the critical m o n o m e r concentration of actin in the presence of ATP. Various concentrations of monomeric actin (Cli) were added to 0.2 /~M polymeric actin. Subsequently the change in the monomer concentration ( A q ) was determined. Phosphate concentrations: e, zero; ©, 20 raM. 71, critical m o n o m e r concentration in the absence of phosphate.
As ATP-actin and ADP-actin with bound phosphate are similar with respect to the polymerization reaction, it is tempting to investigate whether ATP and phosphate ions compete for binding to actin. It is conceivable that phosphate binds near the binding site of the ~,-phosphate of the actinbound ATP. If ADP is bound to actin, the "~-phosphate-binding site of ATP would be free for binding of phosphate ions. On exchange of ADP for ATP, the phosphate ions would be displaced by the y-phosphate of ATP. We investigated this problem by analyzing the effect of phosphate on the exchange of actin-bound nucleotides. The exchange can not be measured directly, because no reliable method is available. This problem was circumvented by Waechter and Engel [26] who used cATP for observation of the exchange of actin-bound nucleotide. First the exchange of actin-bound ADP for ~ATP was determined in the absence of phosphate. A titration curve of monomeric ADP-actin with eATP is depicted in Fig. 5.
110
nucleotide concentrations: &
1.0
[ c ATP-G] [ ~ATP-G] + [ADP-G]
I--
[ c ATP-G] [G] [ ~ATP] [ADP] "Ks
0.5 ¸
(7)
[ c ATP] 1+ [ ~ - ' K s
/ go
/ s .o
0" ,o 0
2
l+
5
[~-ATP] / [ADP] Fig. 5. Titration of ADP-actin with cATP. [cATP], concentration of cATP; [ADP], concentration of ADP, which was kept constant at 20.1/~M. [~ATP-G], concentration of actin monomers with bound ~ATP; [G], total concentration of monomeric actin. - - , fit of the equilibrium constant of exchange of actin-bound ADP for cATP K s (K 5 = 1.4).
Actin-bound ADP (ADP-G) was displaced by EATP to yield actin-bound EATP (cATP-G):
[G] being the total concentration of actin monomers. The nucleotide concentrations were applied in great excess over bound nucleotides so that the free nucleotide concentrations were practically equal to the total nucleotide concentrations. Best agreement of titration curves calculated according to Eqn. 7 with the experimental data was obtained when K 5 was assumed to be 1.4 (Fig. 5). Titration of monomeric ATP-actin with c-ATP is depicted in Fig. 6. Actin-bound ATP (ATP-G) was displaced by ~ATP to yield actin-bound ~ATP (cATP-G): K6
ATP-G + cATP ~ cATP + ATP
g 5
ADP-G + c A T P ~ c A T P - G + A D P
The proportion of actin with bound c-ATP can be expressed in terms of K 5 and the ratio of the
1.0-
L~ I
12. I--
+
&
1.0
0.5
I
I---
jo
o_
f
/°
I
,O /o • ,o
0.5
/o
e
l
u
i
i
l
0
I
2
3
4
5
[(-ATP]I[ADP]
I
/
[~-ATP ] ! [ATP] Fig. 6. Titration of ATP-actin with cATP. [cATP], concentration of ~ATP; [ATP], concentration of ATP, which was kept constant at 12.8/~M. [cATP-G], concentration of actin monomers with bound cATP; [G], total concentration of monomeric actin. - - , fit of the equilibrium constant of exchange of actin-bound ATP for cATP K 6 ( K 6 = 0.6).
Fig. 7. Titration of ADP-actin with cATP in the presence of 40 mM phosphate. [cATP], concentration of ~ATP; [ADP], concentration of ADP which was kept constant at 20.1 ~tM. [EATP-G], concentration of monomeric cATP-actin; [cATPG-P], concentration of monomeric ~-ATP-actin with bound phosphate; [G], total concentration of monomeric actin. Middle continuous line, fit of the equilibrium constant for binding of phosphate to cATP-actin K 7 ( K 7 = 40 M - I ) . Upper and lower continuous line, test of the sensitivity of the fit to the choice of g 7 (upper line, K 7 = 1 0 0 M - I ; lower line, K 7 = 1 0 M-l).
111
~"
7 and 8 titration curves of ADP- or ATP-actin with cATP are depicted. The curves were quantitatively evaluated according to the reaction scheme displayed in Fig. 9. The proportion of actin with bound cATP can be expressed as a function of the ratio of the nucleotide concentrations, the phosphate concentration and the equilibrium constants K1, K 5 and K 7 to yield an equation for evaluation of titration of ADP-actin with eATP:
1.0-
& & L ÷
0.5-
A I O"
f l
0
!
w
|
!
10
20
[E-ATP] / [ATP] Fig. 8. Titration of ATP-actin with cATP in the presence of 40 mM phosphate. [cATP], concentration of cATP; [ATP], concentration of ATP which was kept constant at 12.8 #M. [~ATP-G], concentration of monomeric cATP-actin; [cATPG-P], concentration of monomeric cATP-actin with bound phosphate; [G], total concentration of monomeric actin. Middle continuous line, fit of the equilibrium constant for binding of phosphate to ATP-actin K 8 (K 8 = 50 M - l ) . Upper and lower continuous line, test of the sensitivity of the fit to the choice of K s (upper line, K s = 1 0 M - l ; lower line, Ks=100 M-l).
The equilibrium constant K 6 was fitted analogously to K 5. Best agreement of the calculated titration curves with the experimental data was obtained for K 6 = 016 (Fig. 6). The overall equilibrium constant for exchange of ATP for ADP can be calculated to be K s / K 6 = 2.3. The exchange of the nucleotide was followed also in the presence of 40 mM phosphate. In Figs.
[ ~ATP-G] + [ c ATP-G-P] [ADP-G] + [ADP-G-P] + [cATP-G] + [ ~ATP-G-P] [ c ATP-G] + [ c ATP-G-P]
[GI [ ~ATP] [ADP] 1 K l [ P i ] + l + [,ATP] K 5 Kv-[Pi] + 1 [ADP]
As the nucleotides and phosphate were present in excess over actin monomers, the free nucleotide and free phosphate concentrations were approximately equal to the total concentrations. The equilibrium constants K 1 and K 5 are known from determination of the critical monomer concentrations (Fig. 1) and from titration of ADP-actin with c-ATP (Fig. 5). The unknown equilibrium constant for binding of phosphate to ~ATP-actin K 7 was fitted to the experimental data. The best fit was achieved for K 7 = 40 M -1 (Fig. 7). The sensitivity of the fit to the choice of K v can be estimated from Fig. 7 where also titration curves
K5 £-ATP ADP
K6 ATP (-ATP
~-ATP
ATP c-ATP
ADP
~-ATP ADP A D P - ~ P ~' " c-ATP.~P ~-ATP
ADP
(8)
ATP ~-ATP ,' ~ " ATP.~P ATP ~-ATP
Fig. 9. Reaction scheme of binding of phosphate to actin and of exchange of actin-bound nucleotides. P, phosphate ions.
112
calculated for other values of K 7 are displayed. Phosphate ions tend to bind to eATP-actin more strongly (K 7 = 40 M -1) than to ADP-actin (K1 = 10 M-l). This result suggests that phosphate ions and y-phosphate of cATP do not compete for binding to actin. Titration of ATP-actin with cATP is depicted in Fig. 8. The equilibrium constant for binding of phosphate ions to ATP-actin K 8 was determined analogously with K 7. Best agreement of calculated with measured curves was obtained for K s = 50 M -t. The significance of the fit of K 8 can be judged from curves calculated for other values of K s (Fig. 8). It appears that ATP-actin binds phosphate slightly more strongly than ADP-actin. Competitive binding of phosphate ions and yphosphate of ATP can be excluded. Discussion
In this paper we determined the equilibrium constants for binding of phosphate ions to monomeric and polymeric ADP-actin. The decrease of the critical monomer concentration in the presence of phosphate was explained by the greater affinity of phosphate to polymeric ADP-actin than to monomeric ADP-actin (Eqn. 5). Our measurements on ADP-actin confirm the observations made by Rickard and Sheterline [11], who investigated the effect of phosphate on the critical monomer concentration of ADP-actin. However, our determinations of the critical monomer concentrations of the pointed ends in the presence of ATP disagree with the results of Rickard and Sheterline [11]. We found that 15 mM phosphate are necessary for a half-maximal decrease of the critical monomer concentration of the pointed end, whereas Rickard and Sheterline reported that 1 mM phosphate has the same effect. Both experiments were performed under almost identical conditions. We used gelsolin-actin complex for capATP
ATP
l-q
0
I
ATP ~
ATP
Oii;i!!: ii:!. i ~
2
ping of the barbed ends, whereas Rickard and Sheterline used cytochalasin. Cytochalasin may not only cap the barbed ends, but may also have other effects on actin [30]. According to most reliable nuclear magnetic resonance measurements, the physiologically occurring free phosphate concentrations are in the range of 5 mM [31]. If the in vitro experiments reported in this paper are pertinent to actin in a living cell, one can conclude that the physiological free phosphate concentration is not sufficient for abolition of the difference of the critical monomer concentrations of the two ends, or for inhibition of treadmilling of actin in vivo (see Fig. 3). Mockrin and Korn [10] have found that, following ATP hydrolysis by chemically cross-linked actin dimers, phosphate remains tightly bound to actin. They postulated that during actin polymerization an intermediate with tightly bound phosphate is formed. This rather stable intermediate is certainly not identical with the labile actin-phosphate complex investigated in this paper. Rickard and Sheterline [11] proposed that ADP-actin with weakly bound phosphate, is a later intermediate occurring after polymerization and ATP hydrolysis by actin. In that case, the release of the weakly bound phosphate would be the reaction by which the affinity for binding of actin to a filament end is affected. ATP-actin and ADP-actin with bound phosphate would have a high affinity and ADP-actin would have a low affinity of binding to filament ends. During the last years, the polymerization reaction of actin has been elucidated by the efforts of many laboratories. To date, six reaction steps have been characterized or postulated (Fig. 10): (1) activation of monomers by metal-ion binding [32-35]; (2) binding of activated ATP-actin to filament ends to form ATP caps [36,7-9]; (3) conformational change of bound actin molecules [37]; (4) hydrolysis of the actin-bound ATP to
3
ADP .~
/+
ADP
iiiii,i;: iiiii i: ~
P
5
.
P
p ADP ~
.
6
Fig. 10. Reaction steps occurring during actin polymerization: (1) monomer activation, (2) binding to a filament end, (3) conformational change, (4) hydrolysis of ATP and formation of tightly bound phosphate, (5) dissociation of the tight bond to yield weakly bound phosphate, (6) dissociation of the phosphate ion.
113
yield ADP-actin subunits with tightly bound phosphate [10]; (5) dissociation of the bond between actin and phosphate to yield weakly bound phosphate; (6) dissociation of the weakly bound phosphate from ADP-actin [11]. Acknowledgements We thank E. Werres for excellent technical assistance. We are indebted to Dr Wolter (Blutbank Hagen i. W.) for the generous gift of human platelet concentrate and to Dr Norma Selve for providing us with gelsolin-actin complex. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 168). References 1 Straub, F.B. and Feuer, G. (1950) Biochim. Biophys. Acta 4, 455-470 2 Wegner, A. (1976) J. Mol. Biol. 108, 139-150 3 Pollard, T.D. and Mooseker, M.S. (1981) J. Cell Biol. 88, 654-659 4 Bonder, E.M., Fishkind, D.J. and Mooseker, M.S. (1983) Cell 34, 491-501 5 Wegner, A. (1982) J. Mol. Biol. 161,607-615 6 Wegner, A. and Isenberg, G. (1983) Proc. Natl. Acad. Sci. USA 80, 4922-4925 7 Pardee, J.D. and Spudich, J.A. (1982) J. Cell Biol. 93, 648-654 8 Pollard, T.D. and Weeds, A.G. (1984) FEBS Lett. 170, 94-98 9 Carlier, M.-F., Pantaloni, D. and Korn, E.D. (1984) J. Biol. Chem. 259, 9983-9986 10 Mockrin, S.C. and Korn, E.D. (1981) J. Biol. Chem. 256, 9228-8233 11 Rickard, J.E. and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280 12 Rees, M.K. and Young, M. (1967) J. Biol. Chem. 242, 4449-4458
13 Faust, U., Fasold, H. and Ortanderl, F. (1974) Eur. J. Biochem. 43, 273-279 14 Detmers, P., Weber, A., Elzinga, M. and Stephens, R.E. (1981) J. Biol. Chem. 256, 99-105 15 Harris, G.L. and Crawford, N. (1973) Biochim. Biophys. Acta 291,701-719 16 Selve, N. and Wegner, A. (1986) J. Mol. Biol. 187, 627-631 17 Lind, S., Yin, H.L. and Stossel, T.P. (1982) J. Clin. Invest. 69, 1384-1387 18 Lazarides, E. and Lindberg, U. (1974) Proc. Natl. Acad. Sci. USA 71, 4742-4646 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. (1951) J. Biol. Chem. 193, 265-275 20 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 21 Yin, H.L. and Stossel, T.P. (1979) Nature (London) 281, 583-586 22 Elzinga, M., Collins, J.H., Kuehl, W.M. and Adelstein, R.S. (1973) Proc. Natl. Acad. Sci. USA 70, 2687-2691 23 Secrist, J.A., Barrio, J.R., Leonard, N.L. and Weber, G. (1972) Biochemistry 11, 3499-3506 24 Wanger, M. and Wegner, A. (1983) FEBS Lett. 162, 112-116 25 Miki, M., Ohnuma, H. and Mihashi, K. (1974) FEBS Lett. 46, 17-19 26 Waechter, F. and Engel, J. (1975) Eur. J. Biochem. 57, 453-459 27 Selve, N. and Wegner, A. (1986) Eur. J. Biochem. 160, 379-387 28 Cou6, M. and Korn, E.D. (1986) J. Biol. Chem. 260, 15033-15041 29 Wanger, M. and Wegner, A. (1985) Biochemistry 24, 1035-1040 30 Brenner, S.L. and Korn, E.D. (1981) J. Biol. Chem. 256, 8663-8670 31 Burt, C.T., Glonek, T. and Bhr~ny, M. (1977) Science 195, 145-149 32 Rich, S.A. and Estes, J.A. (1976) J. Mol. Biol. 104, 777-792 33 Rouayrenc, J.-F. and Travers, F. (1981) Eur. J. Biochem. 116, 73-77 34 Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886 35 Cooper, J.A., Buhle, E.L., Walker, S.B., Tsong, T.Y. and Pollard, T.D. (1983) Biochemistry 22, 2193-2202 36 Oosawa, F. and Kasai, M. (1962) J. Mol. Biol. 4, 10-21 37 Keiser, T., Schiller, A. and Wegner, A. (1986) Biochemistry 25, 4899-4906
105
Elsevier BBA 32887
Binding of phosphate ions to actin Michael Wanger and Albrecht Wegner Institute of Physiological Chemistry, Ruhr-University Bochum, Bochum (F.R.G.)
(Received18 February1987)
Key words: Actin; Gelsolin; Phosphate; Fluorescence;1,N6-Ethenoadenosine5'-triphosphate The decrease of the critical monomer concentration of ADP-actin by millimolar phosphate concentrations has been analysed in terms of equilibrium constants for binding of phosphate ions to ADP-actin. The decrease has been explained by a 10-fold greater affinity of phosphate ions to polymeric ADP-actin (binding constant I00 M - l ) than to monomeric ADP-actin (binding constant I0 M - t). Phosphate has an almost identical effect on the critical monomer concentration of the pointed ends of gelsolin-capped actin filaments in the presence of ATP. The phosphate concentration required for half-maximal decrease of the critical monomer concentration of the pointed ends has been determined to be about 15 mM. By using the fluorescent ATP-analogue, l,N6-ethenoadenosine 5'-triphosphate, phosphate ions have been found to bind also to monomeric ATP-actin, yet with a slightly higher affinity than to monomeric ADP-actin (binding constant 50 M - 1).
Introduction Since the discovery that actin hydrolyzes ATP during polymerization [1], many efforts have been made to better understand and quantitatively analyze this reaction. ATP hydrolysis has been found to cause actin filaments to treadmill due to the different critical monomer concentrations of the two ends [2-6]. ATP hydrolysis has been reported to lag behind assembly of monomers with filament ends [7-9]. ADP-actin wi'th tightly bound phosphate has been detected following ATP hydrolysis by chemically cross-linked actin dimers [10]. More recently, Rickard and Sheterline [11] reported that the critical monomer concentration of ADP-actin is decreased by millimolar phosphate concentrations. They proposed that ADPactin with a weakly bound phosphate ion may be Correspondence: A. Wegner, Institute of PhysiologicalChemistry, Ruhr-University Bochum, P.O. Box 102148, D-4630, Bochum, F.R.G.
an intermediate of the ATP hydrolysis reaction by actin, and that ATP-actin and ADP-actin with bound phosphate have a high affinity to filament ends whereas ADP-actin has a low binding affinity. In this paper we analyze quantitatively the equilibrium of binding of phosphate ions to ADP-actin and we investigate whether or not ATP and phosphate ions compete for binding to the surface of actin molecules. Materials and Methods Preparation of the proteins
Actin was prepared according to the method of Rees and Young [12]. The protein was applied to a Sephacryl S-200 column (2.5 × 90 cm). Part of the protein was modified with N-ethylmaleimide at cysteine-374 [13] and subsequently with 7-chloro4-nitro-2,1,3-benzoxadiazole at lysine-373 to produce a fluorescently labeled actir/ [14]. The concentration of actin was determined photometri-
0167-4838/87/$03.50 © 1987 ElsevierScience Publishers B.V. (BiomedicalDivision)
106
cally at 290 nm using an absorption coefficient of 24 900 M - 1. cm- 1 [2]. The gelsolin-actin complex was prepared from human platelet concentrate. Platelets were purified by differential centrifugation [15] with the modification that 20 nM prostaglandin E 1 was included in the suspension buffer to inhibit aggregation of the platelets [16]. Following the first resuspension, the purification was continued according to the method of Lind et al. [17]. The platelet extract was applied to an Agarose affinity column to which DNAase I (deoxyribonuclease I, EC 3.1.21.1) was covalently bound (1.5 x 12 cm, Affi-Gel 10, purchased from Bio-Rad). The column was equilibrated with monomeric actin to produce immobilized actin [18]. The gelsolin-actin complex was eluted from the affinity column by using 5 mM EGTA buffer [17]. The concentration of the gelsolin-actin complex was determined by the method of Lowry et al. [19] and Bradford [20]. For calculation of the molar concentration the molecular masses of gelsolin and actin were assumed to be 90 kDa and 42.3 kDa, respectively [21,22].
Preparation of the nucleotides 1,N6-Ethenoadenosine 5'-triphosphate (~ATP) was synthetized according to the method of Secrist et al. [23] with the modification that the crude product was apphed to a DEAE-Sephadex A-25 column (2.5 x 40 cm) and eluted with a linear NH4HCO 3 gradient (0.15-0.33 M) [24]. The cATP was analyzed as described previously [24]. ADP was separated from traces of ATP by chromatography on a DEAE-Sephadex A-25 column (2.5 x 40 cm). ADP was eluted with a linear NH4HCO 3 gradient (0.15-0.33 M). According to HPLC chromatography (RP 18 column, 1 mM tetrabutylammonium dihydrogenphosphate/3.3 % acetonitrile/65 mM KH2POa-H3PO4 (pH 2.2)), ADP contained less than 0.1% ATP. ADP-actin was prepared by exchange of ATP for ADP on a Sephadex G-25 Superfine column (2.5 x 50 cm) equilibrated with 500/aM A D P / 1 0 #M MgC12/5 mM triethanolamine-HC1 (pH 7.5)/200 mg/1 NaN 3.
Fluorescence Actin polymerization was followed by the
2.2-2.5-fold greater fluorescence intensity of polymeric actin compared to that of monomeric actin [14]. 5% of fluorescently labeled actin was copolymerized with unmodified actin. This low proportion of labeled actin does not significantly alter the polymerization rate or extent of assembly of unmodified actin [5]. The excitation wavelength was 436 nm, and the fluorescence intensity was measured at 530 nm. The changes of the fluorescence intensities were evaluated in terms of concentration of monomeric or polymeric actin. The fluorescence intensity of monomeric or polymeric actin was calibrated by measuring the fluorescence intensities of dilution series of monomeric or polymeric actin. The exchange of actin-bound nucleotides was measured by the increase of the fluorescence intensity on binding of cATP to monomeric actin [25,26]. The excitation wavelength was 360 nm and the emitted light was measured at 410 nm. The changes of the fluorescence intensities were evaluated in terms of free or actin-bound ~ATP. The fluorescence intensity of free or actin-bound ~ATP was calibrated by measuring the fluorescence intensities of dilution series of free and actin-bound ~ATP. Addition of phosphate (40 mM) did not significantly change the fluorescence intensity of the fluorescence label of actin or of cATP.
Experimental procedure The critical monomer concentration of ADPactin in the presence of various phosphate concentrations was measured both after depolymerization of polymeric ADP-actin (3 #M) and after polymerization of monomeric actin (2 #M) onto actin filaments (1 ~M). Samples were prepared by mixing a 20 mM MgC12 solution, a 1 M KCI solution, a 25 mM EGTA solution, a 100 mM KPOn solution, a 5 mM ADP solution, ADP-buffer (500 /~M ADP/10 ~M MgC12/5 mM triethanolamine-HCl (pH 7.5)/200 mg/1 NAN3), 25 /~M polymeric ADP-actin polymerized by the addition of 1 mM MgC12 and 100 mM KC1, and 25 /~M monomeric ADP-actin solution if necessary. The solutions were mixed in such a ratio that the final composition of the samples was 1 mM MgC12, 100 mM KC1, 0.5 mM EGTA. 500 /~M ADP, various concentrations of phosphate (0-45
107
mM), 5 mM triethanolamine-HC1 (pH 7.5), 200 mg/1 NAN3, polymeric ADP-actin and monomeric ADP-actin if necessary. Fluorescence intensities of the samples were measured at 10 min intervals until a constant final fluorescence intensity was reached (4 h). The critical monomer concentration of the pointed ends of actin filaments in the presence of ATP was determined similarly to the measurements on ADP-actin with the following alterations. ADP was replaced by ATP. Gelsolincapped actin filaments were used instead of polymeric ADP-actin. EGTA was replaced by 0.2 mM CaC12, which is necessary for tight binding of gelsolin to the barbed ends of actin filaments [27]. The exchange of actin-bound nucleotides was followed by titration of monomeric ADP- or ATP-actin (1 ~tM) with cATP. Samples were prepared by combining the solutions used for measurements of the critical monomer concentrations of ADP-actin with the following modifications. The concentration of ADP or ATP was in the range of 10-25/~M, and various CATP concentrations (0-250 /~M) were added. EATP was dissolved in 10/~M MgC12/5 mM triethanolamineHC1 (pH 7.5)/200 mg/1 NaN 3. First, the fluorescence intensity of (ATP was measured and then monomeric ADP- or ATP-actin was added. The exchange of ADP or ATP for cATP was followed by the increase in the fluorescence intensity. All experiments Were performed at 25 o C. Results
Binding of phosphate ions to monomeric and polymeric ADP-actin The effect of various concentrations of phosphate on the critical monomer concentration of ADP-actin is depicted in Fig. 1. Similar monomer concentrations were reached both after depolymerization of polymeric actin and after polymerization of monomeric actin onto actin filaments (Fig. 1). The monomer concentrations measured after depolymerization tended to be slightly lower than those measured after polymerization of monomers onto actin filaments. In agreement with Rickard and Sheterline [11], phosphate was found to decrease the critical monomer concentration. Half-maximal decrease occurred at about 13.5 mM
IK
~
2"
¢-I I
< + I
a_
1
0 .<
" ] ' ~ t ~ ~
l
0
i
i
I
........
i
|
5O
[P] /mM Fig. 1. Effect of various concentrations of phosphate on the critical monomer concentration of ADP-actin. [P], phosphate concentration; [ADP-G], concentration of monomeric ADPactin; [ADP-G-P], concentration of monomeric ADP-actin with bound phosphate. 1", monomer concentrations reached after polymerization of monomers onto filaments; Z, monomer concentration reached after depolymerization of actin filaments. - - , one-parameter fit of the equilibrium constant for binding of phosphate to monomeric ADP-actin K l ( K 1 =10 M - l ) . • . . . . . , two-parameter fit of K 1 ( K l = 8 M -1) and of the equilibrium constant for binding of ADP-actin with bound phosphate to a filament end K 4 ( K 4 = 5.5.10 6 M - ] ).
phosphate. The data summarized in Fig. 1 can be interpreted by the simple reaction scheme of binding of phosphate to ADP-actin depicted in Fig. 2. The four equilibrium constants defined in Fig. 2 are correlated with the concentrations of free phosphate [Pi], of monomeric ADP-actin [ADP-G], of monomeric ADP-actin with bound phosphate [ADP-G-P] and to the probability, ap, that the terminal filament subunit binds phosphate, in the following way: K1
[ADP-G-PI [ADP-G]- [Pi]
K1
(1 --
K3
[ADP-G]
K4
[ADP-G-P]
ap ap)"[Pi]
(1)
(2)
1-ap
(3)
ap
(4)
108
ADP~ +
p KI ~, ~ A O P ~ P P +
...."~'"..i
!...'"'..i p K2
ADP~
~"
~ ADP~P
Fig. 2. Reaction scheme of binding of phosphate to actin and of the assembly of monomeric actin with filament ends. P, phosphate.
The four equilibrium constants are not independent. K 2 c a n be expressed in terms of K~, K 3 and K4: K4
K2 = K33 "K1
(5)
The total monomer concentration, [ADP-G] + [ADP-G-P], can be calculated by using Eqns. 1-5: K1"[Pi]+ 1 [ADP-G] + [ADP-G-P] = K3 + K4"Ka"[Pi]
(6)
As the phosphate ions occur in great excess over actin monomers and filament subunits, the free phosphate concentration [Pi] is approximately equal to the total phosphate concentration. The equilibrium constant for binding of phosphate-free monomeric ADP-actin to actin filaments, K3, can be easily determined. In the absence of phosphate ( a p = 0 ) the monomer concentration ADP-G is equal to K31 (Eqn. 3). Since, according to Fig. 1 1.5 ~tM monomers coexist with polymeric actin in the absence of phosphate, K 3 turns out to be 6.7.105 M -1 (1/1.5 # M - l ) . It has been proposed [11] that ADP-actin with bound phosphate is similar to ATP-actin with respect to the polymerization reaction. In that case the critical monomer concentration of ADP-actin with bound phosphate would be expected to be similar to that of ATP-actin. The critical monomer concentration of ATP-
actin has been found to be 0.12 /~M under the same experimental conditions (Fig. 4). If this value was pertinent to ADP-actin with bound phosphate, K 4 would be 8 . 1 0 6 M -1 (1/0.12 /~M -1, a p = 1, Eqn. 4). The equilibrium constant for binding of phosphate to monomeric ADP-actin K 1 was fitted for K 3 = 6.7.105 M -1 and K 4 = 8 • 1 0 6 M -1. A curve calculated for K 1 = 10 M -1 is depicted in Fig. 1 as a continuous line. This calculated curve is in reasonable agreement with the experimental data. Generally, at great phosphate concentrations, the calculated monomer concentrations are lower than the measured monomer concentrations, suggesting that the critical monomer concentration of ADP-actin with bound phosphate is slightly higher than that of ATP-actin. We therefore determined K 1 and K 4 also by a twoparameter fit. The fitted line is depicted in Fig. 1 as a dotted line. According to this fit, the equilibrium constant for binding of phosphate to monomeric ADP-actin K 1 has been found to be 8 M-1 and the equilibrium constant for binding of monomeric ADP-actin bearing phosphate to a filament end K 4 has turned out to be 5.5.10 6 M -1 The critical monomer concentration of ADP-actin with bound phosphate is 0.18 /~M (1/K4). Both fits yield similar values of K 1 (10 and 8 M -1) and K l ( 8 . 1 0 6 and 5.5.106 M - l ) . The equilibrium constant of binding of phosphate ions to actin filaments K z can be calculated to be in the range of 100-a (Eqn. 5). Effect of phosphate on the critical monomer concentrations of the two ends of actin filaments in the presence of A TP The effect of various concentrations of phosphate on the critical monomer concentration of the pointed ends of actin filaments in the presence of ATP is depicted in Fig. 3. Actin filaments capped at the barbed ends were produced by polymerization of actin onto gelsolin-actin complex [16]. The monomer concentrations reached both after depolymerization of polymeric actin and after polymerization of monomeric ATP-actin onto the pointed ends of gelsolin-capped actin filaments are depicted in Fig. 3. A decrease in the critical monomer concentration of the pointed ends in the presence of phosphate has already been observed by Rickard and Sheterline [11]. In con-
109
::k
1.0
o.+ II+ I t~t x
+
t t:+I o i
so [P] !mM Fig. 3. Effect of various concentrations of phosphate on the critical monomer concentration of the pointed ends of gelsolin-capped actin filaments in the presence of ATP. [P], phosphate concentration; ~p, concentration of monomeric actin. T, monomer concentration reached after polymerization of monomers onto filaments; ±, monomer concentration reached after depolymerizationof actin filaments.
trast to the results of Rickard and Sheterline, who reported that under similar experimental conditions the critical monomer concentration of the pointed ends decreases half-maximally in the presence of 1 mM phosphate, we found that 15 mM phosphate is necessary for the same effect (Fig. 3). These results cannot be analyzed as an equilibrium because of the continuous ATP hydrolysis occurring during assembly and disassembly of the pointed ends [28,16]. In agreement with the results of Rickard and Sheterline [11] we found that phosphate has no significant effect on the critical monomer concentration of uncapped actin filaments in the presence of ATP (Fig. 4). As the association and dissociation reactions at the barbed ends are at least 10-fold faster than at the pointed ends [3,4,27], the critical monomer concentration is mainly determined by the barbed ends [3,5,29]. Thus, the determined critical monomer concentrations are approximately equal to the critical monomer concentration of the barbed end. One can conclude that in the presence of ATP, phosphate affects the pointed ends but not the barbed ends of actin filaments.
Effect of phosphate on the exchange of actin-bound nucleotide 3--
-0.1 -0.2
%0 ~~ w
0
w
.,~
gl
i
0.2
!
0.4
C/ilJM Fig. 4. Determination of the critical m o n o m e r concentration of actin in the presence of ATP. Various concentrations of monomeric actin (Cli) were added to 0.2 /~M polymeric actin. Subsequently the change in the monomer concentration ( A q ) was determined. Phosphate concentrations: e, zero; ©, 20 raM. 71, critical m o n o m e r concentration in the absence of phosphate.
As ATP-actin and ADP-actin with bound phosphate are similar with respect to the polymerization reaction, it is tempting to investigate whether ATP and phosphate ions compete for binding to actin. It is conceivable that phosphate binds near the binding site of the ~,-phosphate of the actinbound ATP. If ADP is bound to actin, the "~-phosphate-binding site of ATP would be free for binding of phosphate ions. On exchange of ADP for ATP, the phosphate ions would be displaced by the y-phosphate of ATP. We investigated this problem by analyzing the effect of phosphate on the exchange of actin-bound nucleotides. The exchange can not be measured directly, because no reliable method is available. This problem was circumvented by Waechter and Engel [26] who used cATP for observation of the exchange of actin-bound nucleotide. First the exchange of actin-bound ADP for ~ATP was determined in the absence of phosphate. A titration curve of monomeric ADP-actin with eATP is depicted in Fig. 5.
110
nucleotide concentrations: &
1.0
[ c ATP-G] [ ~ATP-G] + [ADP-G]
I--
[ c ATP-G] [G] [ ~ATP] [ADP] "Ks
0.5 ¸
(7)
[ c ATP] 1+ [ ~ - ' K s
/ go
/ s .o
0" ,o 0
2
l+
5
[~-ATP] / [ADP] Fig. 5. Titration of ADP-actin with cATP. [cATP], concentration of cATP; [ADP], concentration of ADP, which was kept constant at 20.1/~M. [~ATP-G], concentration of actin monomers with bound ~ATP; [G], total concentration of monomeric actin. - - , fit of the equilibrium constant of exchange of actin-bound ADP for cATP K s (K 5 = 1.4).
Actin-bound ADP (ADP-G) was displaced by EATP to yield actin-bound EATP (cATP-G):
[G] being the total concentration of actin monomers. The nucleotide concentrations were applied in great excess over bound nucleotides so that the free nucleotide concentrations were practically equal to the total nucleotide concentrations. Best agreement of titration curves calculated according to Eqn. 7 with the experimental data was obtained when K 5 was assumed to be 1.4 (Fig. 5). Titration of monomeric ATP-actin with c-ATP is depicted in Fig. 6. Actin-bound ATP (ATP-G) was displaced by ~ATP to yield actin-bound ~ATP (cATP-G): K6
ATP-G + cATP ~ cATP + ATP
g 5
ADP-G + c A T P ~ c A T P - G + A D P
The proportion of actin with bound c-ATP can be expressed in terms of K 5 and the ratio of the
1.0-
L~ I
12. I--
+
&
1.0
0.5
I
I---
jo
o_
f
/°
I
,O /o • ,o
0.5
/o
e
l
u
i
i
l
0
I
2
3
4
5
[(-ATP]I[ADP]
I
/
[~-ATP ] ! [ATP] Fig. 6. Titration of ATP-actin with cATP. [cATP], concentration of ~ATP; [ATP], concentration of ATP, which was kept constant at 12.8/~M. [cATP-G], concentration of actin monomers with bound cATP; [G], total concentration of monomeric actin. - - , fit of the equilibrium constant of exchange of actin-bound ATP for cATP K 6 ( K 6 = 0.6).
Fig. 7. Titration of ADP-actin with cATP in the presence of 40 mM phosphate. [cATP], concentration of ~ATP; [ADP], concentration of ADP which was kept constant at 20.1 ~tM. [EATP-G], concentration of monomeric cATP-actin; [cATPG-P], concentration of monomeric ~-ATP-actin with bound phosphate; [G], total concentration of monomeric actin. Middle continuous line, fit of the equilibrium constant for binding of phosphate to cATP-actin K 7 ( K 7 = 40 M - I ) . Upper and lower continuous line, test of the sensitivity of the fit to the choice of g 7 (upper line, K 7 = 1 0 0 M - I ; lower line, K 7 = 1 0 M-l).
111
~"
7 and 8 titration curves of ADP- or ATP-actin with cATP are depicted. The curves were quantitatively evaluated according to the reaction scheme displayed in Fig. 9. The proportion of actin with bound cATP can be expressed as a function of the ratio of the nucleotide concentrations, the phosphate concentration and the equilibrium constants K1, K 5 and K 7 to yield an equation for evaluation of titration of ADP-actin with eATP:
1.0-
& & L ÷
0.5-
A I O"
f l
0
!
w
|
!
10
20
[E-ATP] / [ATP] Fig. 8. Titration of ATP-actin with cATP in the presence of 40 mM phosphate. [cATP], concentration of cATP; [ATP], concentration of ATP which was kept constant at 12.8 #M. [~ATP-G], concentration of monomeric cATP-actin; [cATPG-P], concentration of monomeric cATP-actin with bound phosphate; [G], total concentration of monomeric actin. Middle continuous line, fit of the equilibrium constant for binding of phosphate to ATP-actin K 8 (K 8 = 50 M - l ) . Upper and lower continuous line, test of the sensitivity of the fit to the choice of K s (upper line, K s = 1 0 M - l ; lower line, Ks=100 M-l).
The equilibrium constant K 6 was fitted analogously to K 5. Best agreement of the calculated titration curves with the experimental data was obtained for K 6 = 016 (Fig. 6). The overall equilibrium constant for exchange of ATP for ADP can be calculated to be K s / K 6 = 2.3. The exchange of the nucleotide was followed also in the presence of 40 mM phosphate. In Figs.
[ ~ATP-G] + [ c ATP-G-P] [ADP-G] + [ADP-G-P] + [cATP-G] + [ ~ATP-G-P] [ c ATP-G] + [ c ATP-G-P]
[GI [ ~ATP] [ADP] 1 K l [ P i ] + l + [,ATP] K 5 Kv-[Pi] + 1 [ADP]
As the nucleotides and phosphate were present in excess over actin monomers, the free nucleotide and free phosphate concentrations were approximately equal to the total concentrations. The equilibrium constants K 1 and K 5 are known from determination of the critical monomer concentrations (Fig. 1) and from titration of ADP-actin with c-ATP (Fig. 5). The unknown equilibrium constant for binding of phosphate to ~ATP-actin K 7 was fitted to the experimental data. The best fit was achieved for K 7 = 40 M -1 (Fig. 7). The sensitivity of the fit to the choice of K v can be estimated from Fig. 7 where also titration curves
K5 £-ATP ADP
K6 ATP (-ATP
~-ATP
ATP c-ATP
ADP
~-ATP ADP A D P - ~ P ~' " c-ATP.~P ~-ATP
ADP
(8)
ATP ~-ATP ,' ~ " ATP.~P ATP ~-ATP
Fig. 9. Reaction scheme of binding of phosphate to actin and of exchange of actin-bound nucleotides. P, phosphate ions.
112
calculated for other values of K 7 are displayed. Phosphate ions tend to bind to eATP-actin more strongly (K 7 = 40 M -1) than to ADP-actin (K1 = 10 M-l). This result suggests that phosphate ions and y-phosphate of cATP do not compete for binding to actin. Titration of ATP-actin with cATP is depicted in Fig. 8. The equilibrium constant for binding of phosphate ions to ATP-actin K 8 was determined analogously with K 7. Best agreement of calculated with measured curves was obtained for K s = 50 M -t. The significance of the fit of K 8 can be judged from curves calculated for other values of K s (Fig. 8). It appears that ATP-actin binds phosphate slightly more strongly than ADP-actin. Competitive binding of phosphate ions and yphosphate of ATP can be excluded. Discussion
In this paper we determined the equilibrium constants for binding of phosphate ions to monomeric and polymeric ADP-actin. The decrease of the critical monomer concentration in the presence of phosphate was explained by the greater affinity of phosphate to polymeric ADP-actin than to monomeric ADP-actin (Eqn. 5). Our measurements on ADP-actin confirm the observations made by Rickard and Sheterline [11], who investigated the effect of phosphate on the critical monomer concentration of ADP-actin. However, our determinations of the critical monomer concentrations of the pointed ends in the presence of ATP disagree with the results of Rickard and Sheterline [11]. We found that 15 mM phosphate are necessary for a half-maximal decrease of the critical monomer concentration of the pointed end, whereas Rickard and Sheterline reported that 1 mM phosphate has the same effect. Both experiments were performed under almost identical conditions. We used gelsolin-actin complex for capATP
ATP
l-q
0
I
ATP ~
ATP
Oii;i!!: ii:!. i ~
2
ping of the barbed ends, whereas Rickard and Sheterline used cytochalasin. Cytochalasin may not only cap the barbed ends, but may also have other effects on actin [30]. According to most reliable nuclear magnetic resonance measurements, the physiologically occurring free phosphate concentrations are in the range of 5 mM [31]. If the in vitro experiments reported in this paper are pertinent to actin in a living cell, one can conclude that the physiological free phosphate concentration is not sufficient for abolition of the difference of the critical monomer concentrations of the two ends, or for inhibition of treadmilling of actin in vivo (see Fig. 3). Mockrin and Korn [10] have found that, following ATP hydrolysis by chemically cross-linked actin dimers, phosphate remains tightly bound to actin. They postulated that during actin polymerization an intermediate with tightly bound phosphate is formed. This rather stable intermediate is certainly not identical with the labile actin-phosphate complex investigated in this paper. Rickard and Sheterline [11] proposed that ADP-actin with weakly bound phosphate, is a later intermediate occurring after polymerization and ATP hydrolysis by actin. In that case, the release of the weakly bound phosphate would be the reaction by which the affinity for binding of actin to a filament end is affected. ATP-actin and ADP-actin with bound phosphate would have a high affinity and ADP-actin would have a low affinity of binding to filament ends. During the last years, the polymerization reaction of actin has been elucidated by the efforts of many laboratories. To date, six reaction steps have been characterized or postulated (Fig. 10): (1) activation of monomers by metal-ion binding [32-35]; (2) binding of activated ATP-actin to filament ends to form ATP caps [36,7-9]; (3) conformational change of bound actin molecules [37]; (4) hydrolysis of the actin-bound ATP to
3
ADP .~
/+
ADP
iiiii,i;: iiiii i: ~
P
5
.
P
p ADP ~
.
6
Fig. 10. Reaction steps occurring during actin polymerization: (1) monomer activation, (2) binding to a filament end, (3) conformational change, (4) hydrolysis of ATP and formation of tightly bound phosphate, (5) dissociation of the tight bond to yield weakly bound phosphate, (6) dissociation of the phosphate ion.
113
yield ADP-actin subunits with tightly bound phosphate [10]; (5) dissociation of the bond between actin and phosphate to yield weakly bound phosphate; (6) dissociation of the weakly bound phosphate from ADP-actin [11]. Acknowledgements We thank E. Werres for excellent technical assistance. We are indebted to Dr Wolter (Blutbank Hagen i. W.) for the generous gift of human platelet concentrate and to Dr Norma Selve for providing us with gelsolin-actin complex. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 168). References 1 Straub, F.B. and Feuer, G. (1950) Biochim. Biophys. Acta 4, 455-470 2 Wegner, A. (1976) J. Mol. Biol. 108, 139-150 3 Pollard, T.D. and Mooseker, M.S. (1981) J. Cell Biol. 88, 654-659 4 Bonder, E.M., Fishkind, D.J. and Mooseker, M.S. (1983) Cell 34, 491-501 5 Wegner, A. (1982) J. Mol. Biol. 161,607-615 6 Wegner, A. and Isenberg, G. (1983) Proc. Natl. Acad. Sci. USA 80, 4922-4925 7 Pardee, J.D. and Spudich, J.A. (1982) J. Cell Biol. 93, 648-654 8 Pollard, T.D. and Weeds, A.G. (1984) FEBS Lett. 170, 94-98 9 Carlier, M.-F., Pantaloni, D. and Korn, E.D. (1984) J. Biol. Chem. 259, 9983-9986 10 Mockrin, S.C. and Korn, E.D. (1981) J. Biol. Chem. 256, 9228-8233 11 Rickard, J.E. and Sheterline, P. (1986) J. Mol. Biol. 191, 273-280 12 Rees, M.K. and Young, M. (1967) J. Biol. Chem. 242, 4449-4458
13 Faust, U., Fasold, H. and Ortanderl, F. (1974) Eur. J. Biochem. 43, 273-279 14 Detmers, P., Weber, A., Elzinga, M. and Stephens, R.E. (1981) J. Biol. Chem. 256, 99-105 15 Harris, G.L. and Crawford, N. (1973) Biochim. Biophys. Acta 291,701-719 16 Selve, N. and Wegner, A. (1986) J. Mol. Biol. 187, 627-631 17 Lind, S., Yin, H.L. and Stossel, T.P. (1982) J. Clin. Invest. 69, 1384-1387 18 Lazarides, E. and Lindberg, U. (1974) Proc. Natl. Acad. Sci. USA 71, 4742-4646 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R. (1951) J. Biol. Chem. 193, 265-275 20 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 21 Yin, H.L. and Stossel, T.P. (1979) Nature (London) 281, 583-586 22 Elzinga, M., Collins, J.H., Kuehl, W.M. and Adelstein, R.S. (1973) Proc. Natl. Acad. Sci. USA 70, 2687-2691 23 Secrist, J.A., Barrio, J.R., Leonard, N.L. and Weber, G. (1972) Biochemistry 11, 3499-3506 24 Wanger, M. and Wegner, A. (1983) FEBS Lett. 162, 112-116 25 Miki, M., Ohnuma, H. and Mihashi, K. (1974) FEBS Lett. 46, 17-19 26 Waechter, F. and Engel, J. (1975) Eur. J. Biochem. 57, 453-459 27 Selve, N. and Wegner, A. (1986) Eur. J. Biochem. 160, 379-387 28 Cou6, M. and Korn, E.D. (1986) J. Biol. Chem. 260, 15033-15041 29 Wanger, M. and Wegner, A. (1985) Biochemistry 24, 1035-1040 30 Brenner, S.L. and Korn, E.D. (1981) J. Biol. Chem. 256, 8663-8670 31 Burt, C.T., Glonek, T. and Bhr~ny, M. (1977) Science 195, 145-149 32 Rich, S.A. and Estes, J.A. (1976) J. Mol. Biol. 104, 777-792 33 Rouayrenc, J.-F. and Travers, F. (1981) Eur. J. Biochem. 116, 73-77 34 Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886 35 Cooper, J.A., Buhle, E.L., Walker, S.B., Tsong, T.Y. and Pollard, T.D. (1983) Biochemistry 22, 2193-2202 36 Oosawa, F. and Kasai, M. (1962) J. Mol. Biol. 4, 10-21 37 Keiser, T., Schiller, A. and Wegner, A. (1986) Biochemistry 25, 4899-4906