(Received for publication, July 8, 1994; and in revised form, October 10, 1994)
From the
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
10
to 10
M
)
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.
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
,
-bidentate chelate in the
-configuration(1, 2, 3, 4) ;
conversely, the metal-ADP is bound as a
monodentate. 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
(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 . However, experimental evidence thus far
does not support this model, since thymosin
, 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 T
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
end, or
profilin could accelerate P
release from an F-ADP-P
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.
The concentrations of G-actin and profilin were determined
spectrophotometrically using extinction coefficients of 0.617
mg cm
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
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 to a solution
of G-actin (
20 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
, 25
µM EGTA, and the desired concentrations of MgCl
and ATP. The concentration of MgCl
in buffer was at
most 10 µM in excess of ATP, to avoid formation of actin
oligomers(25) . MgADP-G-actin and Mg
ADP-G-actin were
prepared as described previously(26) .
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 Ca
ATP-G-actin (2-5
µM) in G buffer containing 10 µM
ATP and
CaCl
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 Mg
ATP-G-actin was studied
in a similar fashion as follows. A stock solution of Ca
ATP-G-actin
was prepared at 20-30 µM in 5 mM Tris, 0.2
mM DTT, 5 µM CaCl
, 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
, 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
(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
and 0.1 M KCl,
and the same amounts of MgCl
and KCl were added, from a
[4 M KCl, 40 mM MgCl
] stock
solution, to the Mg
ATP-G-actin-profilin sample immediately prior
to filling the drive syringe A. For each (profilin +
Mg
ATP-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.
Mg
ADP-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.
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
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 Ca
ATP
G-actin (6 µM) in 5 mM Tris-Cl
,
0.2 mM DTT, 20 µM CaCl
, 10 µM
ATP, and profilin at different concentrations. Syringe B
contained 120 µM BAPTA, 20 µM MgCl
, 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 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 (
)with values of rate constants k
and k
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
yielded a plot of simulated
values of 1/t
which was more steeply cooperative
than the latter (with k
=
10 s
) but did not fit the data either. A better fit
to the data was obtained by introducing a supplementary slow step (k
) in the recycling of profilin, as described in Fig. SIII (subscript P represents profilin).
Fig. 2shows that the fit was improved by setting k < 2.5 s
. The slow step (k
) 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 Ca
ATP-G-actin (6 µM) were
measured at different profilin concentrations. The experiment was
carried out with Ca
ATP-G-actin and profilin present in syringe A
in G buffer containing 10 µM
ATP and 20 µM CaCl
. Syringe B contained 120 µM BAPTA,
20 µM MgCl
, 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
, 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 Ca
ATP-G-actin in 5 mM Tris, 0.2 mM DTT, 10 µM
ATP, 5-150 µM CaCl
, 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
, [Ca
] = 0.2
µM; b, 10 µM CaCl
,
[Ca
] = 0.5 µM; c, 20 µM CaCl
,
[Ca
] = 1 µM; d, 75 µM CaCl
,
[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-Ca
ATP-G-actin complex.
Syringe A contained 1.6 µM Ca
ATP-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 Ca
ATP-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/k
versus [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 Ca
ATP from the profilin-actin-Ca
ATP
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 Ca
ATP
is also increased
100-fold by profilin.
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
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
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.
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
(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
, 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).
Under
physiological ionic conditions (Mg-actin, 0.1 mM EGTA, 1
mM MgCl, 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
M
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 Mg
ADP-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
) 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.
[H]ADP-G-actin was prepared by polymerization
of [
H]ATP-G-actin 1:1 complex with 1 mM MgCl
and resuspension in G buffer containing 2.5
µM ADP, no CaCl
, 100 µM MgCl
, 25 µM EGTA, 1 µM Ap
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 [
H]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
[
H]ATP-G-actin in the presence of as low as 5
µM CaCl
or MgCl
in G buffer, no
appreciable dissociation of [
H]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-[H]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
, 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 (
) and
[
H]ADP radioactivity measurement
(
).
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. Ca
ATP-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
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
(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-Ca
ATP and PA
complexes, free
ATP, and Ca
at equilibrium could
be derived from the measured fluorescence decrease in each sample. The
concentration of free Ca
ATP was then calculated using a value of 5
µM for the equilibrium dissociation constant of
Ca
ATP(29) . The binding constant of Ca
ATP to the
profilin-actin complex was K =
([PA]
[Ca
ATP])/([PA-Ca
ATP]).
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 Ca
ATP for G-actin is lowered to
2.10
M
, 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.
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
-10
M
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
-10
M
, 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
M
) 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 . The
relative amounts of actin sequestered as profilin-actin (PA) and
thymosin
-actin (TA) can be calculated as follows.
Typically in a cell containing 100 µM T
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
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 5.10
M 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.
Note Added in Proof-The kinetics of metal/nucleotide exchange on profilin-actin were not affected by poly-L-proline.