Fluorescence study of the high pressure-induced denaturation
of skeletal muscle actin
Yoshihide Ikeuchi
1
, Atsusi Suzuki
2
, Takayoshi Oota
2
, Kazuaki Hagiwara
2
, Ryuichi Tatsumi
1
, Tatsumi Ito
1
and Claude Balny
3
1
Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan;
2
Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Japan;
3
INSERM Unite
Â
128,
IFR 24, CNRS, Montpellier, France
Ikkai & Ooi [ Ikkai, T. & O oi, T . (1966) Biochemistry 5, 1551±
1560] made a thorough study of the eect of pressure on
G- and F-actins. However, all of the measurements in their
study were made after the release of p ressure. In the present
experiment in situ observations were attempted by using
eATP to obtain f urther detailed kinetic and thermodynamic

Keywords: a ctin; denaturation; dissociation; ¯uorescence;
heavy meromyosin; high pressure.
Actin, the major protein in muscle, is composed of two
domains that are separated by a cleft in which one molecule
of ATP or ADP and one divalent cation are present [1].
Actin undergoes transformation from a monomeric form
(G-actin) to a long, helical polymer (F-actin). This conver-
sion of G- to F-actin can be induced by the addition of
neutral salt a nd is coupled with dephosphorylation of ATP
into ADP and inorganic phosphate. Generally, the G ® F
transformation can be repeated by cycling the experimental
salt concentration in the presence of ATP [2]. The sites
responsible f or polymerizatio n a re present i n t he upper
region of the actin molecule, designated as the Ôpointed endÕ
and also i n the bottom region known as a Ôbared endÕ (i.e.
polymerization is due to end-to-end interaction) [3]. Actin
becomes unstable if it loses bound nucleotides and divalent
cations [4]. This results in irreversible denaturation. There-
fore, ATP is considered to contribute to the promotion of
polymerization and the stabilization of the actin structure
[5,6].
Pressure exerts a great in¯uence on t he properties of
proteins by rearrangement and/or destruction o f noncova-
lent bonds such as hydrogen bonds, hydrophobic and
electrostatic interactions, which normally stabilize the
tertiary structure of proteins [7]. There are some reports
describing the effect of hydrostatic pressure on intact muscle
®bres and a ctin±myosin interaction [8,9]. In addition,
Garica et al.[10]andCrenshawet al. [11] reported the
effect of hydrostatic pressure on the equilibrium of actin

,
sodium pyrophospate.
(Received 9 July 2001, revised 17 October 2001, a ccepted 7 November
2001)
Eur. J. Biochem. 269, 364±371 (2002) Ó FEBS 2002
The aim of the presen t study was to complete a study of
F ® G transition and denaturation of actin under pressure.
Use of a Hitachi F2000 ¯uorospectrophotometer equipped
with a pressure pump and vessel allowed in situ observation
of actin behaviour under pressure.
MATERIALS AND METHODS
Protein preparations
Actin preparations from rabbit skeletal m uscle w ere
obtained from a ceto ne dried powder according to the
procedure o f Pardee & Spudich [13]. Unless used i mmedi-
ately, G-actin with ATP was stored at )20 °Cafter
lyophilization. Myosin was extracted with Guba±Straub
solution from rabbit s keletal m uscle according to the
method of Perry [14] and heavy meromyosin (HMM) was
obtained by limited trypsin digestion of myosin [15]. 1:N
6
-
ethenoadenosine 5 ¢-triphosphate ( eATP) was synthesized
from ATP (Sigma Co.) according to t he method of Secrist
et al. [16]. eATP-labelled G-actin was prepared as described
by Waechter & Engel [17]. T he stoichiometry of t he
binding of eATP was determined according to the proce-
dure of Miki et al.[18].eATP-G-actin was converted into
eADP±F-actin by adding 50 m
M

lating water from a temperature-controlled bath. The
¯uorescence spectra were quanti®ed by specifying the centre
of spectral mass [21]. The excitation wavelength for the
intrinsic ¯uorescence spectrum was 295 nm which excites
tryptophan residues in the actin molecule.
To determine the kinetics of the p ressure-induced dena-
turation of eATP G-actin (or eADP±F-actin), samples were
kept at elevated pressure s, and the changes in the ¯uores-
cence intensity under pressure were monitored. The excita-
tion wavelength was s et to 360 nm and em ission was
recorded at 410 nm [17,22]. The relative ¯uorescence
intensity was plotted as function of pressure time as shown
below. We ®tted the data to the ®rst-order reaction scheme
usingdata®ttingprogram(
KALEIDAGRAPH
,Abelbeck
Software) to evaluate the apparent denaturation rate
constant (k). The value of volume change was obtained by
plotting lnk vs. pressure [7].
RESULTS AND DISCUSSION
In situ
pressure-induced changes in spectrum and the
centre of spectral mass of the intrinsic ¯uorescence
of ATP-G-actin
Following pressure increase, a red shift in the spectra with a
decrease in the intrinsic ¯uorescence intensity of G-actin was
observed (Fig. 1, inset). Fig. 1 shows the changes in the
centre of spectral mass of intrinsic ¯uorescence spectrum of
G-actinwithATP(0.5mgámL
)1

140
250 300 350 400 450
Fluorescence intensity
Wavelength (nm)
1
2
4
5
3
6
Fig. 1. Fluorescence spectra of G-actin under
various pressure conditions. 1, 0.1 MPa; 2,
100 MPa; 3, 200 MPa; 4, 300 MPa; 5,
400 MPa; 6, return from 400 MPa to
0.1 M Pa (dotted line). In set: the pressure
dependence of the centre o f s pectral mass of
G-actin intrinsic ¯uoresce nce. (d), Com-
pression; (m), decompression. Excitation
wavelength, 295 nm; emission range,
300±400 nm; temperature, 20 °C. Protein
concentrat ion, 0 .5 mgámL
)1
in 2 m
M
Tris/HCl pH 7.5, 0.2 m
M
ATP, 0.2 m
M
dithiothreitol, 0.2 m
M

Fig. 3 shows changes in the relative intensity of ¯uorescence
of eATP-G-actin a nd eADP±F-ac tin i n the presence of
eATP as the pressure was raised from 0.1 MPa to 400 MPa.
The Y-axis is calibrated i n values relative to the intensity at
0.1 MPa. In F-actin the relative intensity increased with a
rise in pressure to around 230 MPa, then reached a plateau.
On a f urther increase in pressure, it decreased gradually in a
relatively lower pressure range and steeply in a higher
pressure range. At 400 MPa it d ropped a lmost t o the same
level as the eATP buffer. Thus, the decrease in intensity of
¯uorescence evidently corresponded to the dissociation of
eADP bound to F-actin. For G-actin a pattern similar to
that of F-actin was obtained except that the intensity h ad
already begun to decrease at the time the pressure reached
230 MPa. This indicates that F-actin is somewhat more
resistant to pressure than is G-actin.
The time course of change in the relative intensity of
¯uorescence of eATP-G-actin under pressures of 100, 200
and 300 MPa is illustrated in Fig. 4. At 100 MPa, the
intensity increased slightly upon pressure elevation, but it
did n ot change while the pressure was maintained at
100 M Pa. After release of pressure, the intensity immedi-
ately returned to its original level. This indicates that the
conformational change of G-actin pressurized at 100 MPa
350 400 450 500 550 600
Wavelength (nm)
1
2
3
4

300
250
200
150
100
50
0
Fluorescence intensity
ε
Fig. 2. Variation in ¯ uorescence spectra of eATP-G-actin and
eADP±F-actin at 0.1 MPa or 250 MPa. 1, G-ac tin with eATP at
0.1 M Pa; 2, F-actin with eADP at 0.1 MPa; 3, G- ac tin with eATP at
250MPa;4,F-actinwitheADP at 250 MPa; 5, buer w ith eATP
at0.1MPa;6,buerwitheATP at 250 MPa. E xcitation wavelen gth,
360 nm; emission range, 380±580 nm; temperature, 20 °C. G-actin
solution contained 2 mgámL
)1
G-actin, 2 m
M
Tris/HCl pH 7.5,
0.2 m
M
eATP, 0.2 m
M
dithiothreitol, 0.2 m
M
CaCl
2
,1m
M

230 MPa
250 MPa
275 MPa
300 MPa
350 MPa
400 MPa
Fig. 3. Change in the relative ¯uorescence intensity of G-actin and
F-actin as pressure was elevated from 0.1 to 400 MPa. Solid line,
G-actin; dotted line, F-actin. Excitation wavelength, 360 nm; emission
range, 4 10 nm; temperature, 20 °C. Protein concentration, 2 mgámL
)1
in 2 m
M
Tris/HCl pH 8.0, 0.2 m
M
eATP, 0.2 m
M
dithiothreitol,
0.2 m
M
CaCl
2
,1m
M
NaN
3
. The pressure was maintained for  3min
after reaching the indicated pressure as indicated by the arrows.
Fig. 4. Time courses of change in the relative ¯uorescence intensity of
eATP-G-actin under various pressures. The experimental conditions

of ¯uorescence of eADP±F-actin in t he presence of 0.2 m
M
eATP a nd 50 m
M
KCl at several pressure values. The
intensity of ¯uorescence continued to increase as the
pressure was elevated, and it i ncreased for some time even
after the inten ded pressure was r eached (i.e. a thermal effect
due to compression). The extent of increase in intensity was
dependent on the pressure applied. This may be a ttributable
to the i ncrease in t he amount of depolymerized actin
because eATP bound to G-actin generates stronger ¯uor-
escence than eADP±F-actin (see F ig. 2). No notable
alterations in the intensity were observed while pressures
ranging from 0.1 to 240 MPa were maintained. This
suggests a rapid reassociation of depolymerized actin
subunits into eADP±F-actin (i.e. the G«F equilibrium).
The intensity began to decrease as soon as the pressure
reached 250 MPa ( data not shown). When the time
dependence of change in t he intensity of eADP±F-actin at
several pressure values above 250 MPa was investigated, the
decrease in intensity obeyed ®rst-order kinetics as in the case
of G-actin [23]. The volume change for the denaturation of
eADP±F-actin was )67 mLámol
)1
, which was close to that
of G-actin (see Fig. 5).
Effect of pressurization on the exchangeability
of nucleotides bound to actin with free nucleotides
Fig. 7 shows the exchange of eATP bound to G-actin with

1
0 50 100 150 200 250 300 350
Relative fluorescence intensity
Pressure time (sec)
1
2
3
4
5
6
7
8
9
Fig. 5. Logarithm of the relative ¯uorescence intensity of eATP-G-actin
as a function of pressure time at various pressures. The solid lines sh ow
the best curve ®t of a ®rst ord er k inetics. Th e experimental conditions
were the same as in Fig. 3. The 1 to 9 represent the pressure intensities
at intervals of 25 MPa from 200 MPa up to 400 MPa. Each ¯uores-
cence intensity was expressed relative to the value at the start of decline
in ¯uorescence intensity.
0
0.5
1
1.5
2
2.5
3
3.5
0 50 100 150 200 250 300 350 400
Time (sec)

,1m
M
NaN
3
.
Ó FEBS 2002 Pressure-induced denaturation of actin (Eur. J. Biochem. 269) 367
[4]. Subsequently ¯uorescence measurements of eADP±F-
actin were made in the presence and absence of EDTA and
ATP to con®rm the dissociation±association equilibrium of
actin u nder pressure. Fig. 8 shows the time dependence o f
¯uorescence intensity of eADP±F-actin at 0.1 MPa ( see
inset) or 100 MPa. No change in the intensity was observed
even upon maintaining p ressure constant at 100 MPa
regardless of whether E DTA was present or not. T his result
could be interpreted as follows: eADP±F-actin was ®rst
depolymerized to eADP±G-actin, quickly exchanged its
eADP for external free eATP, and then polymerized again
accompanying the liberation of phosphate from eATP
bound to G-actin. That is to say, the c ycling F ® G ® F
transformation (F«G equilibrium under a certain pressure)
is thought to occur without denaturation in the pressure
range used (see Fig. 12). In a higher pressure range, above
250 MPa (Fig. 9), it was inferred t hat t he partial collapse of
the three-dimensional s tructure of actin, depolymerized
under pressure, proceeds immediately after release of the
nucleotide, so that it loses the exchangeability of bound
ADP with external free ATP. EDTA promoted t he release
of eADP bound to depolymerized G-actin, leading t o
random aggregation after release of pressure because n eutral
salt ( 50 m

pressure, then the intensity of ¯uorescence would h ave been
increased accompanying an increase of free eADP± G-actin
as the pressure was e levated (Figs 2 and 6).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 200 400 600 800 1000
Time (sec)
100 MPa
Relative fluorescence intensity
0.8
0.9
1
1.1
1.2
0 200 400 600 800 1000
Time (sec)
0.1 MPa
Relative fluorescence
intensity
Fig. 8. Eect of EDTA on t he release of eADP bound to F-actin with or
without free eATP at 0.1 MPa (inset) and 100 MPa. Protein concen-
tration, 2 mgámL
)1
in 10 m

Fig.9. EectofEDTAonthereleaseofeADP bound to F-actin with
and w ithout free eATP at 250 MPa. T he experimental conditions were
the same as in F ig. 8. 1, W ith eATP; 2 , without eATP ; 3, with EDTA
and eAT P; 4, with EDTA, without eATP; 5, buer.
Fig. 7. Exchange of eATP bound to G -actin by free eATP or A TP in the
solvent under pressure at 100 MPa. Thesamplesweredilutedtoa®nal
concentration of 2 mgámL
)1
with a solution containing eATP (solid
line) or A TP (dotted line) immediately before m onitoring of the ¯uo-
rescence in tensit y. Prote in co ncentration, 2 mgámL
)1
in 2 m
M
Tris/
HClpH7.5,0.2m
M
eATP or ATP, 0.2 m
M
dithiothreitol, 0.2 m
M
CaCl
2
,1m
M
NaN
3
. Inset represents exchange of eADP bound to
F-actin by free eATP or ATP under pressure. The experimental con-
ditions w ere the same as in the case of G-actin except that F-actin was

presence of Mg
2+
-NaPP
i
, where the actin±HMM complex
can be dissociate d, and in the absence of eATP i n the
external solution, the ¯uorescence intensity began to
decrease prior to reaching 200 MPa ( line 3 in Fig. 11).
When the molar ratio of actin to HMM was reduced from
1 : 1 to 1 : 10,
2
the decay in the intensity of ¯uorescence
proceeded immediately after reaching 100 MPa (line 4 in
Fig. 11), indicating the rapid depolymerization of F-actin
and subsequent its denaturation. This result was unexpect-
ed but might h ave been due to the d epolymerizing effect of
a small amount of HMM, which s timulated fragmentation
of F-actin, as reported by Ikeuchi et al. [27]. Interestingly,
higher pressures (> 350 MPa), the intensities of ¯uores-
cence of HMM alone an d the actin±HMM complex with a
large amount of HMM increased (lines 2, 3 and 5 in
Fig. 11). This reason is not clear, but might arise from the
large conformational change of the HMM molecule itself
under h igh pressure.
In order to explain a decrease in the turbidity of the
actomyosin system under pressure Ikkai & Ooi [26] had
proposed another possibility. This was that t he actin±HMM
complex c ould b e dissociated by pressure even without ATP
although whether or not depolymerization of actin pro-
ceeded prior to the dissociation of t he complex was obscure.

200 MPa
250 MPa
300 MPa
275 MPa
375 MPa
400 MPa
350 MPa
Fluorescence intensity
Fig. 10. Change in the ¯uorescence intensity of F-actin or acto-HMM
complex in the presence of eATP as pressure was elevated from 0.1 to
400 MPa. Dotted line, F-actin; solid line, acto-HMM complex. Pro-
tein concentration, 3.4 mgámL
)1
HMM (10 l
M
)and/or0.42 mgámL
)1
F-actin (10 l
M
)in10m
M
Tris/HCl pH 7. 5, 50 m
M
KCl, 2 m
M
eATP,
0.2 m
M
dithiothreitol, 0.2 m
M

-NaPP
i
as pressure was
elevated from 0.1 to 400 MPa. 1, F-actin alone (10 l
M
)with1m
M
MgCl
2
and 2 m
M
NaPP
i
(dotted line); 2, acto-HMM complex (actin/
HMM ratio 1 : 1) with 2 m
M
NaPP
i
; 3, acto-HMM comp lex (actin/
HMM ratio 1 : 1) with 1 m
M
MgCl
2
and 2 m
M
NaPP
i
;4,acto-HMM
complex (actin/HMM ratio 10 : 1) with 1 m
M

¯uorescence intensity rapidly decreased prior to reaching
200 MPa (lines 3, 4 in F ig. 11). I kkai & Ooi [ 26] have
reported that t he dissociation of the actin±HMM complex
was quite possible in the presence of ATP under pressure
because of the reduction of Mg-activated ATPase and
pressure > 150 MPa was required to induce a signi®cant
dissociation of the complex. In any event HMM protects
denaturation of F-actin up to 200 MPa in the absence of
ATP (compare line 1 and line 2 in Fig. 11), whereas high
pressure under conditions that favour actin±HMM complex
dissociation (or in t he presence of Mg
2+
-NaPP
i
or
Mg
2+
-ATP) promotes the denaturation of actin following
the dissociation of actin±HMM complex (lines 3, 4 in
Fig. 11).
In conclusion, the dissociation rates of nucleotides from
the a ctin molecule (i.e. the decay curve of the ¯uo rescence
intensity o f eATP-G-actin) obeyed good ®rst order kinetics
(Fig. 5 ). The volume change for the d enaturation, calculat-
ed from their rate constants, was close to that obtained b y
Ikkai & Ooi [12] who estimated it after release of pressure.
In addition the denaturation of G-actin under pressure is
coupled with loss in the exchangeability of bound ATP
against f ree ATP ( Figs 7±9). T he present r esults mostly
veri®ed their data and speculations (i.e. the value of volume

nucleotide (ATP). T he scheme of the pressure-induced
denaturation process of actin in the presence of ATP is
shown in Fig. 12 on a basis of present observations.
ACKNOWLEDGEMENTS
This study was supported in part by a Grant-in-Aid for Scienti®c
Research from the Ministry of Education, Science, Sports and C ulture
of Japan (No. 10460118). We thanks Dr Goodenough, University of
Reading, UK, for reading this manuscript.
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250 MPa < P
250 MPa > P
(with or without
EDTA)
depolymerised
actin
repolymerisation
(with neutral salt)
rapid exchange
ADP
ATP
Pi
rapid release
of ADP
depolymerised actin
denatured
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random aggregation
after release of pressure
[1]
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