The binding of ADF⁄ cofilin to F-actin induces large conformational changes in the structure of actin fila-ments, including changes in their mean twist [15], and the weakening of longitudi
Trang 1actin filaments
Andras Muhlrad1, Dmitry Pavlov2, Y Michael Peyser1and Emil Reisler2
1 Institute of Dental Sciences, School of Dental Medicine, Hebrew University of Jerusalem, Israel
2 Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, USA
Actin dynamics, the polymerization and
depolymeriza-tion of actin filaments and formadepolymeriza-tion of ordered actin
assemblies, is critical to many events of cell motility,
including the movement of whole cells, cell division,
vesicular transport and exo- and endocytosis The
essential processes of actin dynamics are closely
regula-ted in the cell by a large number of actin binding
pro-teins and small molecules The large family of actin
depolymerizing factor (ADF)⁄ cofilin (AC) proteins [1]
has a central role in regulating actin dynamics These
small proteins, which are ubiquitous in all eukaryotic
cells, increase the depolymerization and nucleation of
actin filaments and accelerate their treadmilling [2]
AC proteins accelerate the turnover of actin filaments
by severing them [3–6], thereby increasing the number
of the free pointed and barbed ends, or by increasing
monomer dissociation from the pointed end of
fila-ments [7], and⁄ or by both processes [8–10] The action
of these proteins on actin is promoted in most cases by
increasing pH [4], and is regulated by phosphatidyl inositides [11,12] and their phosphorylation (except for yeast cofilin) by kinases [13,14]
The binding of ADF⁄ cofilin to F-actin induces large conformational changes in the structure of actin fila-ments, including changes in their mean twist [15], and the weakening of longitudinal contacts between proto-mers along the long-pitch helix [16,17] and lateral contacts between the two strands [18,19] According to electron microscopy evidence, the weakening of longitudinal contacts in F-actin is due to conformational changes in subdomain 2 of actin, including the disordering of the DNase I binding loop (D-loop) [20] Solution studies also showed cofilin induced conformational changes in actin, particularly in subdomain 2 and its D-loop The fluorescence intensity of probes [tetramethyl rhodamine cadaverine (TRC) and dansyl diethylamine] attached to Gln41 on the D-loop decreased dramatically upon bind-ing of cofilin to F-actin [17,21] Moreover, subtilisin
Keywords
actin; cofilin; collisional quenching;
fluorescence; limited proteolysis
Correspondence
A Muhlrad, Institute of Dental Sciences,
School of Dental Medicine, Hebrew
University of Jerusalem, PO Box 12272,
Jerusalem 91120, Israel
Fax: +972 2 675 8561
Tel: +972 2 675 7587
E-mail: muhlrad@cc.huji.ac.il
(Received 3 January 2006, accepted 8
February 2006)
doi:10.1111/j.1742-4658.2006.05169.x
Inorganic phosphate (Pi) and cofilin⁄ actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling Here,
we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments This inhibition is also significant at physiological concen-trations of Pi, and more pronounced at low pH Cofilin prevents conforma-tional changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers These changes transform the nucleotide cleft of F-actin to G-actin-like
Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility
Abbreviations
AC, ADF ⁄ cofilin; ADF, actin depolymerizing factor; D-loop, DNase I binding loop; K sv , Ksv¼ [(F 0 ⁄ F) )1]Æquencher M )1, where F
0 and F are fluorescence values in the presence and absence of quencher respectively; Pi, inorganic phosphate; TRC, tetramethyl rhodamine cadaverine.
Trang 2cleavage of the D-loop between Met47 and Gly48 and
the tryptic cut after Arg62 and Lys68 in the 60–69 loop
of subdomain 2 became accelerated greatly upon
addi-tion of cofilin [21], confirming significant changes in the
subdomain 2 structure
The structure and dynamics of F-actin are also
affected by the presence of phosphate (Pi) in the
nuc-leotide binding cleft [22,23] In ATP-containing
solu-tions, under physiological condisolu-tions, G-actin contains
tightly bound ATP in the nucleotide binding cleft The
bound ATP hydrolyzes to ADP and Pi subsequent to
the polymerization of G-actin The formed Pi slowly
dissociates from the nucleotide cleft of the F-actin
pro-tomers The rate of dissociation is limited by an
isome-rization step, which is accompanied by conformational
changes The structure of actin filament is highly
dynamic, as there is continuous net association of
monomers at the barbed end and a net dissociation at
the pointed end, resulting in filament treadmilling
When ATP is present in the medium, there is always
ATP or ADP–Pi in the cleft of actin protomers located
in the vicinity of the barbed end of the filament,
pro-ducing an ATP or ADP–Pi cap at this end Because of
the presence of this cap at the barbed end the critical
concentration for polymerization of F-actin is low
F-ADP-actin can also be polymerized from ADP–
G-actin in the absence of ATP In this case all the
proto-mers contain ADP and there is no cap at the barbed
end ADP–F-actin is less stable and has a high critical
concentration for polymerization (reviewed in [24]) Pi
from the medium can bind stoichiometrically to the
nucleotide cleft of ADP–F-actin protomers and ADP–
G-actin if the Pi concentration in the solution is high
enough The Kd for Pi in ADP–F-actin protomers is
1.5 mm at pH 7.0 [23], while in G-ADP-actin the Kdfor
Pi is an order of magnitude higher [25] Because Kd
increases with increasing pH [23], it was concluded that
the actin bound Pi species is H2PO4 [23] Pi lowers
sig-nificantly the critical concentration for polymerization
of ADP-actin [26] by decreasing the rate of protomer
dissociation at both filament ends, and in particular at
the barbed end [23] On the other hand, Pi has a
negli-gible effect on the critical concentration for
polymeriza-tion of F-actin in the presence of ATP, because of the
protective ATP or ADP–Pi cap at the barbed end of the
filament However, Pi stabilizes F-actin structure both
in the absence (ADP–F-actin) and presence of ATP
(F-actin with ATP or ADP–Pi cap at the barbed end) in
the medium This is manifested in the ‘straightening’ of
the actin filament [22], the stabilization of longitudinal
bonds between protomers [27–29], the prevention of
breakage and destruction of filaments upon exposure to
SDS and potassium iodide (KI) [30], and the decreased
tryptic susceptibility of the 60–69 loop in subdomain 2 [31]
Phosphate and its BeFx analog {BeFx represents BeF3 ÆH2O or BeF2(OH)–Æ(H2O) beryllium fluoride complexes [32]}, like other actin filament stabilizing factors (e.g., phalloidin, tropomyosin, etc.), are antag-onistic to ADF⁄ cofilin, which destabilizes filaments Carlier et al [7] reported that plant ADF does not bind to BeFx containing F-actin and hypothesized that the binding of ADF to ATP or ADP–Pi containing F-actin protomers is also prevented According to sev-eral reports [3,33,34], Pi inhibits the severing of actin filaments by the AC-protein Acanthamoeba actophorin, and slows, whilst BeFx completely prevents, the bind-ing of actophorin to actin filaments In addition, acto-phorin was reported to promote the dissociation of Pi from freshly polymerized F-actin [34] However, no attempt was made to study the effect of physiological phosphate concentrations on cofilin binding to F-actin
at various pH values relevant to the regulation of
AC proteins
In view of the important and antagonistic effects of ADF⁄ cofilin and phosphate on actin dynamics, we examined here the binding of cofilin to F-actin and its effect on F-actin structure in the presence of various concentrations of Pi at pH 8.0 and 6.5 We also studied the effect of cofilin on the nucleotide cleft of F-actin We used yeast cofilin because in its presence the critical con-centration for actin polymerization is relatively low (0.7 lm) even at pH 7.8 [7] Thus, at high enough F-actin concentrations, its depolymerization does not significantly influence data analysis To monitor cofilin binding, we used fluorescence and proteolysis methods TRC-labeled F-actin shows a large fluorescence inten-sity decrease upon cofilin binding [21], while subdo-main 2 cleavage by subtilisin and trypsin is greatly accelerated by cofilin, independently of the increased fil-ament treadmilling [21] We found here that the rate of cofilin binding is strongly inhibited by Pi even at physio-logical Pi concentrations Cofilin greatly reduces the affinity of Pi to the nucleotide cleft because of conform-ational changes, which render the cleft of F-actin pro-tomers G-actin like The mutual regulation of cofilin and Pi binding to F-actin is pertinent to the regulation
of actin dynamics in the cell
Results
Fluorescence measurements of the effect of Pi
on the rate of cofilin binding to TRC–F-actin
A convenient way to study the binding of cofilin to F-actin is using actin in which Gln41 is labeled with
Trang 3TRC The fluorescence intensity of TRC–F-actin is
decreased > 70% upon binding of cofilin, while the
fluorescence intensity of G-actin changes little upon
addition of cofilin [21] The binding of 5 lm cofilin to
4 lm TRC–F-actin in the presence of 2 mm and
30 mm Pi at pH 8.0 and 6.5 was measured by
monitor-ing fluorescence changes in a stopped-flow fluorometer
(Fig 1) To saturate F-actin, we used 30 mm Pi
because of its low affinity to F-actin [23,25] To check the effect of physiological concentrations of Pi [35,36],
we also assayed cofilin binding in the presence of
2 mm Pi The ionic strength of all reaction mixtures was adjusted with KCl In the absence of Pi, the TRC fluorescence intensity decreased fast upon addition of cofilin (Fig 1) The rate of cofilin binding depended
on pH in the absence of Pi; it was about four-fold fas-ter at pH 6.5 than at pH 8.0 (Table 1) Both 30 and
2 mm Pi significantly inhibited the rate but not the extent of cofilin binding, however, the inhibition was greater at the higher Pi concentration (Fig 1) In the presence of Pi, unlike in its absence, the rate of cofilin binding was pH independent between pH 6.5 and 8.0 (Table 1) We also checked the effect of phosphate on the fluorescence emission spectrum of TRC–F-actin (data not shown) We found that except for a very small red shift, Pi essentially did not affect the spec-trum of the TRC moiety on the D-loop of F-actin The incubation time (1–24 h) of TRC–F-actin with Pi had no effect on cofilin binding (data not shown)
Effect of cofilin on Pi binding to F-actin and on the conformation of the nucleotide binding cleft Although Pi inhibited the rate of cofilin binding to F-actin, we could not detect any displacement of cofilin
by even 30 mm Pi, using either TRC–F-actin fluores-cence or subtilisin digestion assays (data not shown) This indicates very low affinity of Pi to cofilin-occupied F-actin It is plausible that cofilin induced changes in the nucleotide binding cleft are responsible for a reduction
of Pi affinity to F-actin To test whether such changes indeed occur in F-actin, we examined the fluorescence emission of etheno-ADP (e-ADP) on actin, with and without the bound cofilin Figure 2A shows a 54% fluorescence increase upon binding of cofilin to F-actin, confirming nucleotide cleft perturbation Addition of cofilin to G-actin increased slightly (6.3%) the
fluores-0.1
0.2
0.3
0.4
0.5
Time (sec)
A
30 m M Pi
2 m M Pi
0 m M Pi
Time (sec)
B
0
0.1
0.2
0.3
0.4
0.5
30 m M Pi
2 m M Pi
0 m M Pi
Fig 1 Stopped-flow fluorescence measurements of the effect of
30 m M Pi on the binding of cofilin to TRC–F-actin at pH 8.0 and 6.5.
TRC–F-actin, 4 l M , was incubated with 30 m M NaPi or KPi in
pH 8.0 or pH 6.5 F-buffer, respectively, for 1 h on ice The ionic
strength was equalized in all solutions with NaCl or KCl at pH 8.0
and 6.5, respectively Cofilin (5.0 l M ) was added and the time
course of fluorescence intensity change was monitored in a
stopped-flow instrument at 20 C, as described in Experimental
procedures (A), pH 8.0; (B), pH 6.5.
Table 1 Effect of Pi on the rate constants of cofilin binding to TRC–F-actin at pH 8.0 and 6.5 Data were obtained by fitting the binding curves in Fig 1 to mono-exponential expression All rates were normalized to the rate determined in the absence of Pi at the same pH.
pH
Pi concentration (m M )
Rate constants of cofilin binding (s)1)
Normalized rates (%)
Trang 4cence of e-ATP in the nucleotide cleft (data not shown).
We also studied the effect of cofilin on the nitromethane
quenching of e-ADP in F-actin (Fig 2B) The KSV
values obtained in the absence and presence of coflilin
were 3.32 and 1.41, respectively, which indicates that
the cofilin induced perturbation in the nucleotide
bind-ing cleft decreases the accessibility of the F-actin bound
nucleotide to collisional quenchers
Monitoring the Pi stabilization of the D-loop in subfragment 2 by subtilisin digestion
It has been shown that BeFx Pi analogs protect sub-domain 2 of F-actin from digestion by trypsin and subtilisin and Pi inhibits the tryptic cleavage of the 60–69 loop of subdomain 2 [31] Here, we show that
Pi also inhibits strongly the subtilisin cleavage of the D-loop in F-actin at pH 8.0 (Fig 3) and 6.5 (data not shown) This inhibition decreased with decreasing Pi concentration, but could be observed even at 2 mm Pi The inhibitory effect of Pi on the subtilisin digestion indicates stabilization of the D-loop
Monitoring the binding of cofilin to F-actin by limited proteolysis
The effect of Pi on the binding of cofilin to F-actin was also studied by subtilisin cleavage of the D-loop
in subdomain 2 We showed previously that the digestion of subdomain 2 of F-actin by trypsin and subtilisin was greatly accelerated by cofilin [21] We used the effects of cofilin and Pi on actin proteolysis
to assay cofilin binding to F-actin in the presence
of 30 mm Pi at pH 6.5 and 8.0 To this end, TRC– F-actin (10 lm) was digested with subtilisin in the presence and absence of 12 lm cofilin and 30 mm Pi (Fig 4) The digestion was started 30 s after the
0
100000
200000
300000
A
Wavelength (nm)
8 µM cofilin
no cofilin
B
0.95
1.00
1.05
1.10
1.15
1.20
Nitromethane (m M )
/
Fig 2 Effect of cofilin on the fluorescence emission spectrum and
quenching of e-ADP in the nucleotide cleft of F-actin The
fluores-cence spectrum (A) and quenching by nitromethane (B) of 8 l M
e-ADP–F-actin in 2 m M MgCl2, 20 m M PIPES, pH 6.5, were
moni-tored as described in Experimental procedures n, no cofilin; m,
8 l M cofilin.
Fig 3 Inhibition of subtilisin digestion of F-actin by Pi at pH 8.0 TRC–F-actin (10 l M ) was incubated with 0–30 m M Pi at pH 8.0 for
10 min, and then digested with 100 lgÆmL)1subtilisin for 60, 120 and 180 s The ionic strength was equalized in all samples with KCl The samples were run on SDS ⁄ PAGE and analyzed by densi-tometry n, no Pi; m, 2 m M Pi; , 5 m M Pi; r, 10 m M Pi; d, 20 m M
Pi; h, 30 m M Pi.
Trang 5addition of cofilin The ionic strength was equalized
in all samples by adding KCl or NaCl Cofilin
greatly accelerated the rate of subtilisin cleavage
both in the presence and absence of Pi at pH 6.5
and pH 8.0 At pH 8.0 the digestion in the presence
of cofilin was equally fast with and without Pi, while
at pH 6.5 the acceleration of the cleavage was
lar-ger in the absence of Pi than in its presence We
also studied the effect of incubation time of
Pi–F-actin with cofilin on the rate of subtilisin
diges-tion at pH 8.0 and 6.5 (Fig 5) At pH 8.0, after
3 min incubation with cofilin, the cleavage rate of
Pi–F-actin became as fast as that of F-actin without
Pi, and 20 s incubation with cofilin was enough to
almost completely activate the subtilisin digestion
(Fig 5A) Similar results were obtained by trypsin
digestion of Pi–F-actin in the presence of cofilin at
pH 8.0 (data not shown) On the other hand, at
pH 6.5 the subtilisin cleavage of Pi–F-actin was still
inhibited after 20 s, and even 180 s, incubation
with cofilin (Fig 5B) The rate of cofilin-activated
subtilisin cut of Pi–F-actin is faster at pH 8.0 than
at pH 6.5, while the binding rates of cofilin at
these pH values, as monitored by TRC fluorescence,
are equal
Discussion
Cofilin and phosphate are important regulators of actin dynamics in the cell Their effects on the struc-ture of actin filaments are antagonistic to each other Cofilin, and AC proteins in general, disorder the
Fig 4 Effect of cofilin on the subtilisin digestion of Pi-TRC–F-actin
at pH 8.0 and 6.5 TRC–F-actin (10 l M ) was incubated with 30 m M
NaPi or KPi in pH 8.0 or pH 6.5 F-buffer, respectively, for 1 h on
ice The ionic strength was equalized in all solutions with NaCl or
KCl at pH 8.0 and 6.5, respectively After 30 s incubation with
12 l M cofilin, the actin was digested with 25 lgÆmL)1subtilisin for
20, 50, 80 s at 22 C The samples were run on SDS ⁄ PAGE and
analyzed by densitometry Closed symbols, pH 6.5; open symbols,
pH 8.0; circle, Pi only, no cofilin; downward triangle, no addition;
diamond, Pi and cofilin; sqaure, cofilin only, no Pi.
Fig 5 Effect of incubation time with cofilin on the rate of subtilisin digestion of Pi–F-actin TRC–F-actin (10 l M ) was incubated with
30 m M NaPi or KPi in a pH 8.0 or pH 6.5 F-buffer, respectively, for
1 h on ice The ionic strength was equalized in all solution with NaCl or KCl at pH 8.0 and 6.5, respectively After 20 or 180 s incu-bation with 12 l M cofilin, actin was digested with 25 lgÆmL)1 sub-tilisin for 20, 50, 80 s at 22 C in the pH 8.0 or pH 6.5 F-buffer, respectively The samples were run on SDS ⁄ PAGE and analyzed by densitometry (A), pH 8.0; (B) pH 6.5 ., Pi only, no cofilin; n, no addition; r, Pi and 20 s incubation with cofilin; d, Pi and 3 min incubation with cofilin; m, 20 s incubation with cofilin only, no Pi.
Trang 6filament structure by changing the conformation of
subdomain 2, while phosphate has a stabilizing effect
on F-actin structure The antagonistic effects of Pi and
AC proteins on F-actin are also indicated by increased
dissociation of Pi from actin filaments in the presence
of Acanthamoeba actophorin and inhibited binding of
this AC protein at high, nonphysiological Pi
concen-tration [34] Here, we found that the rate but not the
extent of yeast cofilin binding to F-actin is inhibited
even at physiological concentrations of Pi at pH values
8.0 and 6.5, which promote and inhibit AC protein
induced actin depolymerization, respectively [4]
In agreement with Ressad et al [37], we observed
that in the absence of Pi the binding of cofilin is faster
at low pH than at high pH This is in contrast to
cofi-lin’s depolymerizing effect, which is stronger at high
pH However, according to our observations, the rate
of cofilin binding to F-actin in the presence of
2–30 mm Pi is the same at pH 6.5 and 8.0 The
influ-ence of Pi on the relative rates of cofilin binding at
these two pH values can be explained by the finding
that the Pi species that binds to F-actin is H2PO4 [23]
At pH 8.0 the main species of Pi is HPO42–, while at
pH 6.5 the H2PO4 species is dominant Thus, at equal
concentrations more Pi is bound to F-actin at pH 6.5
than at pH 8.0, yielding apparently greater inhibition
of cofilin binding at pH 6.5 than at pH 8.0 (relative to
the rate observed in the absence of Pi) However,
another explanation of the stronger inhibition of
cofi-lin binding at lower pH can be also suggested
Accord-ing to the molecular dynamics modelAccord-ing of Wriggers &
Schulten [38], Pi leaves the nucleotide cleft of F-actin
through a back door mechanism In this mechanism,
His73, which is methylated with pKa¼ 6.56, has a
central role, because its positively charged, protonated
form inhibits the release of the negatively charged Pi
Because the protonation of histidine decreases with
increasing pH, it follows that the affinity of Pi to
F-actin is lower at high pH The pH dependence of the
Pi inhibition of cofilin binding may contribute to the
pH regulation of the actin depolymerizing activity of
cofilin, which is important in view of the localized pH
fluctuations in the cell
Subtilisin digestions of F-actin revealed that after
30 s incubation with cofilin at pH 8.0 the rate and
the extent of actin cleavage was the same in the
presence and absence of 30 mm Pi On the other
hand, at pH 6.5 the rate of F-actin cleavage was
inhibited by Pi even after 3 min incubation with
cofi-lin, suggesting incomplete displacement of Pi from
actin by cofilin The presence of residual Pi is not
detected via TRC fluorescence data, because these
are unchanged by Pi (data not shown) and monitor
only cofilin binding to F-actin The difference in subtilisin digestion of F-actin at the two pH values probably derives from the stronger binding of Pi at
pH 6.5 than at pH 8.0 (see above) It is also possible that the effect of Pi (in the nucleotide cleft) on F-actin structure is cooperative, as is the case for the BeFx analog of Pi [31], and that the Pi coopera-tivity is higher at pH 6.5 than at pH 8.0 It is more difficult to detect any significant effect of Pi on the preformed F-actin–cofilin complexes High concentra-tions of Pi (30 mm) cannot displace cofilin from F-actin, probably because of further weakening of the intrinsically weak binding of Pi to F-actin This may be due to cofilin induced allosteric changes in the nucleotide binding cleft of actin The cofilin induced increase in the fluorescence and a decrease
in quenching of e-ADP (Fig 2) also indicate nucleo-tide cleft perturbation in F-actin The latter indicates that in the presence of cofilin the bound nucleotide
in F-actin becomes less accessible and probably more buried in the F-actin structure Notably, cofilin bind-ing has an opposite effect on ADP and Pi in the nucleotide binding cleft, i.e., it buries ADP and faci-litates Pi release from the cleft We may speculate that the Pi release is promoted by opening of the
‘back door’ [38], while ADP becomes less accessible through the closing of the ‘main door’ on the top of the protomer or by some other mechanism Similarly
in G-actin, cofilin decreases the accessibility of the bound nucleotide, and inhibits nucleotide exchange [40] perhaps by closing the ‘main door’ of the nuc-leotide binding cleft Taken together, cofilin binding transforms the nucleotide binding cleft of F-actin protomers to a more G-actin-like state, as indicated
by the higher fluorescence and less accessibility to collisional quenchers of the bound e-ADP, which is characteristic of G-actin [40] Moreover, the affinity
of Pi to G-actin is an order of magnitude lower than to F-actin [25], similarly to cofilin containing F-actin Although these results per se do not show weaker Pi binding to F-actin in the presence of cofi-lin, they document cofilin induced changes in the nucleotide cleft, which may be linked to lower Pi affinity to F-actin The lowering of Pi affinity by cofilin has a physiological significance in the forma-tion of lamellipodia of moving cells because it facili-tates the binding and branching activity of Arp2⁄ 3, which can bind only to ADP–F-actin protomers on F-actin [41]
Pi, which stabilizes F-actin structure and decreases the critical concentration for actin polymerization, and cofilin, which has the opposite effect, are antagonistic
to each other This may be physiologically significant
Trang 7because Pi concentration in the cell can approach
2 mm [35,36], which particularly at low pH decreases
the rate of cofilin binding to F-actin On the other
hand, cofilin accelerates Pi dissociation from F-actin
[34] and prevents conformational changes that
accom-pany Pi binding Thus, Pi, cofilin, and pH can
intri-cately regulate actin dynamics with a profound impact
on the actin based cell motility
Experimental procedures
Reagents
TRC and e-ATP were obtained from Molecular Probes
(Eugene, OR) ATP, trypsin, soybean trypsin inhibitor,
sub-tilisin (Carlsberg), phenylmethylsulfonyl fluoride were
pur-chased from Sigma Chemical Co (St Louis, MO) Bacterial
transglutaminase was a generous gift from K Seguro
(Ajimoto Co., Inc., Kawasaki, Japan)
Proteins
G-actin was prepared from back and leg muscles of rabbit
by the method of Spudich & Watt [42] and stored in
G-buf-fer containing 5.0 mm Tris⁄ HCl, 0.2 mm CaCl2, 0.2 mm
ATP, 0.5 mm dithiotreitol, pH 8.0 F-actin was prepared
from G-actin by polymerizing it with 2.0 mm MgCl2 Yeast
cofilin was prepared as described previously [17] The
con-centrations of cofilin and unlabeled skeletal muscle a-actin
were determined spectrophotometrically by using the
extinc-tion coefficients E1%280¼ 9.2 and E1%290¼ 11.5 cm)1,
respectively (The optical density of actin was measured in
the presence of 0.5 m NaOH, which shifts the maximum of
absorbance from 280 nm to 290 nm) Molecular masses
were assumed to be 42 and 15.9 kDa for skeletal actin and
yeast cofilin, respectively
Proteolysis
Labeled or unlabeled F-actin (10 lm) was digested in the
presence and absence of cofilin at pH 8.0 (in 20.0 mm
Tris-HCl, 2.0 mm MgCl2, 0.2 mm ATP, 0.5 mm dithiotreitol)
and pH 6.5 (in 20.0 mm PIPES, 2.0 mm MgCl2, 0.2 mm
ATP, 0.5 mm dithiotreitol) by 25 lgÆmL)1 subtilisin or
800 lgÆmL)1 trypsin, respectively The products of
diges-tions were analyzed by SDS⁄ PAGE Protein bands on SDS
gels were analyzed by densitometry
Chemical modification
Actin labeled with TRC at Gln41 (TRC–actin) was
pre-pared by incubating 50 lm skeletal G-actin with 100 lm
TRC and 0.18 mgÆmL)1 bacterial transglutaminase in
G-buffer pH 8.0, at 22C for 2 h Reagent excess was
removed on PD-10 filtration column (Amersham Pharmacia Biotech Inc., Piscataway, NJ) equilibrated with G-buffer
Preparation of e-ADP–F-actin
ATP in skeletal muscle G-actin was substituted with e-ATP as follows G-actin was passed through a desalting column (Amersham, PD10) of Sephadex G-25 equilibrated with ATP-free G-buffer The eluted actin was supplemen-ted with 20-fold molar excess of e-ATP and was incubasupplemen-ted for 1 h on ice Excess e-ATP was removed from G-actin
by passing it through another PD10 column Actin was polymerized by addition of 2.0 mm MgCl2 and during the polymerization the actin-bound e-ATP was hydrolyzed to e-ADP
Fluorescence measurements
Fluorescence emission spectra were recorded in a PTI spectrofluorometer (Photon Technology Industries, South Brunswick, NJ), in G-buffer for G-actin, and in G-buffer containing 2.0 mm MgCl2for F-actin The excitation wave-length for TRC and e-ADP was set at 544 and 350 nm, respectively For quenching of e-ADP and time course of TRC fluorescence change the emission wavelength was set
at 420 and 583 nm, respectively The time course of TRC fluorescence change was monitored in an Applied Photo-physics (Leatherhead, Surrey, UK) SX-18 MV stopped-flow apparatus supplied with excitation and emission monochro-mators
Acknowledgements
This work was supported by USPHS grant AR 20231 and NSF grant MCB 0316269 (to E.R.)
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