Báo cáo Y học: Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin pdf

Changes in the intensity of fluorescence of F-actin whilst under pressure suggested that sADP-—F-actin was initially depoly- merized to sADP-G-actin; subsequently there was quick exchang

Fluorescence study of the high pressure-induced denaturation

of skeletal muscle actin

Yoshihide Ikeuchi', Atsusi Suzuki?, Takayoshi Oota”, Kazuaki Hagiwara’, Ryuichi Tatsumi’, Tatsumi Ito’

and Claude Balny”

‘Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan;

? Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Japan, 3INSERM Unité 128,

IFR 24, CNRS, Montpellier, France

Ikkai & Ooi [Ikkai, T & Ooi, T (1966) Biochemistry 5, 1551—

1560] made a thorough study of the effect of pressure on

G- and F-actins However, all of the measurements in their

study were made after the release of pressure In the present

experiment in situ observations were attempted by using

sATP to obtain further detailed kinetic and thermodynamic

information about the behaviour of actin under pressure

The dissociation rate constants of nucleotides from actin

molecules (the decay curve of the intensity of fluorescence of

sATP-G-actin or sADP-F-actin) followed first-order

kinetics The volume changes for the denaturation of G-actin

and F-actin were estimated to be —72 mLmol! and

—67 mL-‘mol' in the presence of ATP, respectively Changes

in the intensity of fluorescence of F-actin whilst under

pressure suggested that sADP-—F-actin was initially depoly-

merized to sADP-G-actin; subsequently there was quick

exchange of the sADP for free cATP, and then polymer- ization occurred again with the liberation of phosphate from sATP bound to G-actin in the presence of excess ATP In the higher pressure range (> 250 MPa), the partial collapse of

the three-dimensional structure of actin, which had been

depolymerized under pressure, proceeded immediately after release of the nucleotide, so that it lost the ability to exchange bound ADP with external free ATP and so was denatured irreversibly An experiment monitoring sATP fluorescence

also demonstrated that, in the absence of Mg**-ATP, the

dissociation of actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure

Keywords: actin; denaturation; dissociation; fluorescence;

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 and 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 for polymerization are present in the upper

region of the actin molecule, designated as the ‘pointed end’

and also in the bottom region known as a ‘bared end’ (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]

Correspondence to Y Ikeuchi, Department of Bioscience and

Biotechnology, Faculty of Agriculture, Kyushu University,

6-10-1, Hakozaki, higashi-ku, Fukuoka, 812-8581, Japan

Tel./Fax: +81 92 642 2950, E-mail: ikeuchiy@agr.kyushu-u.ac.jp

Abbreviations HMM, heavy meromyosin; NaPP;,

sodium pyrophospate

(Received 9 July 2001, revised 17 October 2001, accepted 7 November

2001)

Pressure exerts a great influence on the properties of

proteins by rearrangement and/or destruction of 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 fibres and actin—myosin interaction [8,9] In addition, Garica et al [10] and Crenshaw et al [11] reported the effect of hydrostatic pressure on the equilibrium of actin

polymerization

The direct effect of pressure on G- and F-actins was first investigated by Ikkai & Ooi [12], and they reported the following results: (a) actin is irreversibly denatured

> 150 MPa without ATP, but > 250 MPa with ATP The amount of protein denatured by pressure is dependent

on the initial protein concentration; (b) ATP protects actin from pressure-induced denaturation; (c) a reversible F ~ G

transformation occurs with release of ADP and P; in the

presence of ATP under pressure; (d) a volume change for the F-actin > G-actin transformation is estimated to be

—82 mL-‘mol of monomer from the pressure denaturation curve although it is considered questionable whether the value may be indicative of the im vivo AV of assembly

However, it must be borne in mind that all of the

measurements reported from that study were obtained only after release of pressure Therefore it is most important to make measurements under pressure in order to get accurate detailed thermodynamic information on the pressure- induced denaturation of actin

The aim of the present study was to complete a study of

F > Gtransition and denaturation of actin under pressure

Use of a Hitachi F2000 fluorospectrophotometer 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 muscle were

obtained from acetone dried powder according to the

procedure of Pardee & Spudich [13] Unless used immedi-

ately, G-actin with ATP was stored at —20°C after

lyophilization Myosin was extracted with Guba—Straub

solution from rabbit skeletal muscle according to the

method of Perry [14] and heavy meromyosin (HMM) was

obtained by limited trypsin digestion of myosin [15] 1:N°-

ethenoadenosine 5’-triphosphate (gATP) was synthesized

from ATP (Sigma Co.) according to the method of Secrist

et al [16] eATP-labelled G-actin was prepared as described

by Waechter & Engel [17] The stoichiometry of the

binding of sATP was determined according to the proce-

dure of Miki et al [18] sATP-G-actin was converted into

sADP-F-actin by adding 50 mm KCl (polymerization),

and then dialysed against a large volume of cold 50 mm

KCl, 0.2 mm dithiothreitol, 1 mm NaN; and 10 mm Tris/

HCl (pH 7.5)

Tris/HCl buffer was selected because of its negligible

effect of pressure on pH values Protein concentration was

measured using the extinction coefficient at 280 nm for a

1% solution of 6.47 for HMM [19] and at 290 nm for a 1%

solution of 6.6 for ATP-G-actin [20]

High pressure apparatus

High pressure devices used for this study consisted of a

thermostated high pressure vessel equipped with sapphire

windows and a pump capable of raising pressure to

400 MPa (Teramecs Co., Ltd, Kyoto, Japan) The vessel

was placed in the light beam of a Hitachi F2000 spectro-

fluorometer A quartz cuvette containing sample solutions

was placed inside the vessel

140r- 120E

Fig 1 Fluorescence spectra of G-actin under 100L

various pressure conditions |, 0.1 MPa; 2,

100 MPa; 3, 200 MPa; 4, 300 MPa; 5,

400 MPa; 6, return from 400 MPa to

0.1 MPa (dotted line) Inset: the pressure

dependence of the centre of spectral mass of

© © T

G-actin intrinsic fluorescence (@), Com-

pression; (A), decompression Excitation

N © I

wavelength, 295 nm; emission range,

300-400 nm; temperature, 20 °C Protein

concentration, 0.5 mgmL™! in 2 mm

Tris/HCl pH 7.5, 0.2 mm ATP, 0.2 mm

dithiothreitol, 0.2 mm CaCh, | mm NaN3

ao © I

Fluorescence spectroscopy

Fluorescence measurements were made in a Hitachi F2000 fluorospectrophotometer, inside which the high-pressure vessel was placed Temperature was maintained by circu- lating water from a temperature-controlled bath The fluorescence spectra were quantified by specifying the centre

of spectral mass [21] The excitation wavelength for the intrinsic fluorescence spectrum was 295 nm which excites tryptophan residues in the actin molecule

To determine the kinetics of the pressure-induced dena- turation of sATP G-actin (or sADP-—F-actin), samples were kept at elevated pressures, and the changes in the fluores- cence intensity under pressure were monitored The excita- tion wavelength was set to 360 nm and emission was

recorded at 410 nm [17,22] The relative fluorescence

intensity was plotted as function of pressure time as shown below We fitted the data to the first-order reaction scheme using data fitting program (KALEIDAGRAPH, Abelbeck Software) to evaluate the apparent denaturation rate constant (A) The value of volume change was obtained by plotting Ink vs pressure [7]

RESULTS AND DISCUSSION

In situ pressure-induced changes in spectrum and the centre of spectral mass of the intrinsic fluorescence

of ATP-G-actin Following pressure increase, a red shift in the spectra with a decrease in the intrinsic fluorescence intensity of G-actin was observed (Fig 1, inset) Fig | shows the changes in the centre of spectral mass of intrinsic fluorescence spectrum of G-actin with ATP (0.5 mgmL™', pH 7.5) in a pressure range from 0.1 MPa to 400 MPa at a fixed temperature of

20 °C The transition of the curve of the centre of spectral mass occurred between roughly 250 and 350 MPa and the curve reached plateau near 400 MPa However, the decom- pression curve did not correspond with the curve observed upon pressure elevation, indicating that G-actin was irreversibly denatured even in the presence of ATP under pressures as high as 400 MPa although ATP was thought to

play a role in stabilizing actin structure [6]

> compression

Nt

decompression \*

Pressure (MPa)

350 400

300

Wavelength (nm)

300r

ƒ NH

@® h YN " z "

£ 150L bn OH

°o

2

Le

50-

Wavelength (nm)

Fig 2 Variation in fluorescence spectra of sATP-G-actin and

eADP-F-actin at 0.1 MPa or 250 MPa 1, G-actin with eATP at

0.1 MPa; 2, F-actin with sADP at 0.1 MPa; 3, G-actin with sATP at

250 MPa; 4, F-actin with cADP at 250 MPa; 5, buffer with eATP

at 0.1 MPa; 6, buffer with eATP at 250 MPa Excitation wavelength,

360 nm; emission range, 380-580 nm; temperature, 20 °C G-actin

solution contained 2 mgmL7! G-actin, 2mm Tris/HCl pH 7.5,

0.2mm cATP, 0.2 mm dithiothreitol, 0.2 mm CaCl, 1 mm NaN3

F-actin solution contained 2mgmL7! F-actin, 10 mm Tris/HCl

pH 7.5, 50 mm KCl, 0.2 mm eATP, 0.2 mm dithiothreitol, 0.2 mm

CaCl, | mm NaN3 Inset shows the chemical structure of eATP [16]

/n situ pressure-induced changes in the fluorescence

spectra of sATP-G-actin and sADP-F-actin

We attempted i situ observation of the behaviour of actin

under pressure by using sATP which emits strong fluores-

cence at 410 nm when it binds to actin The chemical

structure of cATP is illustrated in inset of Fig 2 [16] The

fluorescence emission spectra of sATP-G-actin, sADP-F-

actin and the eATP buffer are displayed in Fig 2, which

shows that the intensity of fluorescence at 410 nm of sATP-

G-actin was higher than that of sADP-—F-actin Both actins

and sATP buffer showed an increase in intensity of

fluorescence when exposed to a pressure of 250 MPa

However, the increase of intensity of fluorescence of sATP

buffer itself was much smaller than that of sATP bound to

G-actin Therefore, the increase of fluorescence seems to be

due mainly to the conformational change of actin under

pressure

/n situ pressure-induced changes in the intensity

of fluorescence of epsilon nucleotides bound

to G- and F-actins

Fig 3 shows changes in the relative intensity of fluorescence

of cATP-G-actin and eADP-—F-actin in the presence of

sATP as the pressure was raised from 0.1 MPa to 400 MPa

The Y-axis is calibrated in 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 further increase in pressure, it decreased gradually in a

relatively lower pressure range and steeply in a higher

pressure range At 400 MPa it dropped almost to the same

level as the ceATP buffer Thus, the decrease in intensity of

230 MPa 250 MPa 675 mPa

ore

'0

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Sc

ö

®

hm

5

=0.5F

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2

Ss

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Time (sec)

Fig 3 Change in the relative fluorescence 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, 410 nm; temperature, 20 °C Protein concentration, 2 mgmL!

in 2mm Tris/HCl pH 8.0, 0.2mm ceATP, 0.2 mm dithiothreitol, 0.2 mm CaCh, | mm NaN3 The pressure was maintained for 3 min after reaching the indicated pressure as indicated by the arrows

fluorescence evidently corresponded to the dissociation of sADP bound to F-actin For G-actin a pattern similar to that of F-actin was obtained except that the intensity had 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 fluorescence 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 not change while the pressure was maintained at

100 MPa 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

100 MPa

200 MPa

Time (sec)

Fig 4 Time courses of change in the relative fluorescence intensity of eATP-G-actin under various pressures The experimental conditions were the same as in Fig 3 Filled arrowheads show the point at which the designated pressure was reached and open arrowheads show the start of decompression.

2 F ° " ẽ

2 0.6F ° 5

© r n

& a ‘

Pressure time (sec)

Fig 5 Logarithm of the relative fluorescence intensity of sATP-G-actin

as a function of pressure time at various pressures The solid lines show

the best curve fit of a first order kinetics The experimental conditions

were the same as in Fig 3 The | to 9 represent the pressure intensities

at intervals of 25 MPa from 200 MPa up to 400 MPa Each fluores-

cence intensity was expressed relative to the value at the start of decline

in fluorescence intensity

is fully reversible, which was also confirmed by measure-

ment of the fluorescence spectrum (data not shown) On the

other hand, the relative intensity of fluorescence decreased

slowly at 200 MPa and rapidly at 300 MPa (the protein was

held at these constant pressures) and, in this instance, it did

not return to the initial level after release of the pressure

To estimate the volume change of G-actin during

denaturation, the time dependence of the relative intensity

of fluorescence of eATP-G-actin was investigated under

pressures ranging from 200 MPa to 400 MPa at 25 MPa

intervals (Fig 5) The decrease in the intensity when

pressure was kept constant actually reflects the dissociation

of sATP from G-actin As shown in Fig 5, change in the

relative intensity of fluorescence obeyed first-order kinetics

Assuming that the dissociation rate constant of sATP from

actin corresponds to its denaturation rate, the volume

change for the denaturation was estimated to be —-72 mL:

mol"! in the presence of ATP This is in the same range as

the value reported by Ikkai & Ooi [12] who estimated the

value from irreversible pressure-induced denaturation after

release of pressure and by Garcia et al [10] who calculated

the value from the pressure dissociation curve of actin

subunits

Fig 6 shows the time dependence of the relative intensity

of fluorescence of sADP-—F-actin in the presence of 0.2 mm

sATP and 50 mm KCI at several pressure values The

intensity of fluorescence continued to increase as the

pressure was elevated, and it increased for some time even

after the intended pressure was reached (i.e a thermal effect

due to compression) The extent of increase in intensity was

dependent on the pressure applied This may be attributable

to the increase in the amount of depolymerized actin

because sATP bound to G-actin generates stronger fluor-

escence than sADP-F-actin (see Fig 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 sADP-F-actin (.e the GF equilibrium)

wo ơ

Holding pressure

Nn ơ

n

(MPa)

Beh

Time (sec)

Fig 6 Time courses of change in the relative fluorescence intensity of

£ADP-E-actin under various pressures from 0.1 to 250 MPa Protein concentration, 2 mgmL™ in 10 mm Tris/HCl pH 7.5, 0.2 mm eATP,

50 mm KCl, 0.2 mm dithiothreitol, 0.2 mm CaCh, 1 mm NaN3

The intensity began to decrease as soon as the pressure reached 250 MPa (data not shown) When the time dependence of change in the intensity of sADP-—F-actin at several pressure values above 250 MPa was investigated, the decrease in intensity obeyed first-order kinetics as in the case

of G-actin [23] The volume change for the denaturation of

sADP-F-actin was —67 mL-mol”', 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 sATP bound to G-actin with free sATP or ATP in the solvent at 100 MPa where G-actin

is not denatured (Fig 4) In the presence of sATP, the fluorescence intensity showed no change under conditions

of contstant pressure, whereas in the presence of ATP its decrease with time was exponential Both actins exposed to

a pressure of 100 MPa for 5 min showed the same DNase I inhibition capacity (one of the biochemical properties of G- actin [24,25]) after release of pressure (data not shown) This implied that the decrease in the intensity of fluorescence in the presence of ATP was not attributable to the denatur- ation of G-actin Rather these data would represent the rapid exchange between the bound and the free nucleotides

at relatively low pressure such as 100 MPa

sADP bound to F-actin is not easily exchanged with free nucleotides at the normal atmospheric pressure unless external force is applied [2] Hence, to determine whether sADP bound to F-actin is capable of exchanging nucleo- tides under pressure, a similar experiment as in the case of sATP-G-actin was conducted (Fig 7, inset) The result indicated that ADP bound to F-actin could be replaced by the free ATP in the pressure range at which the irreversible denaturation does not take place (see Fig 6)

F-actin, in contrast with G-actin, is not denatured even in

the presence of EDTA EDTA will deprive G-actin of divalent cation leading to a quick irreversible denaturation

- N

100 MPa

+ATP

>

@

— c E

®

g a

a

2

—

SS

®

50 100 150 200 250 300 350

Time (sec)

Fig 7 Exchange of sATP bound to G-actin by free sATP or ATP in the

solvent under pressure at 100 MPa The samples were diluted to a final

concentration of 2 mgmL*! with a solution containing sATP (solid

line} or ATP (dotted line) immediately before monitoring of the fluo-

rescence intensity Protein concentration, 2 mgmL™ in 2 mm Tris/

HCl pH 7.5, 0.2 mm eATP or ATP, 0.2 mm dithiothreitol, 0.2 mm

CaCl, 1mm NaN Inset represents exchange of eADP bound to

F-actin by free seATP or ATP under pressure The experimental con-

ditions were the same as in the case of G-actin except that F-actin was

subjected to 200 MPa pressure

[4] Subsequently fluorescence measurements of eA DP-F-

actin were made in the presence and absence of EDTA and

ATP to confirm the dissociation—association equilibrium of

actin under pressure Fig 8 shows the time dependence of

fluorescence intensity of sADP-—F-actin at 0.1 MPa (see

inset) or 100 MPa No change in the intensity was observed

even upon maintaining pressure constant at 100 MPa

regardless of whether EDTA was present or not This result

could be interpreted as follows: eADP-—F-actin was first

depolymerized to seADP-G-actin, quickly exchanged its

sADP for external free sATP, and then polymerized again

accompanying the liberation of phosphate from eATP

bound to G-actin That is to say, the cycling F ~ G > F

transformation (FG 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 that the partial collapse of

the three-dimensional structure 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 the release

of sADP bound to depolymerized G-actin, leading to

random aggregation after release of pressure because neutral

salt (50 mm KCl) was present in the solution (see below) [4]

Effect of pressurization on the behaviour

of the actin-HMM complex

Ikkai & Ooi [26] found that, in the absent of ATP, turbid

solutions of actomyosin became transparent with increasing

pressure (< 250 MPa) This phenomenon was not inter-

preted as being due to the dissociation of actin and myosin

under pressure Then im situ observations were made by

monitoring the fluorescence of an eADP bound actin—

HMM (the products of myosin digested by trypsin) complex

to clarify whether or not the dissociation of the actin-HMM

complex occurs under pressure (Figs 10 and 11)

—_ +>

> Tr 100 MPa

8 L

S 1.2

oO

L2 nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnng

Time (sec)

Fig 8 Effect of EDTA on the release of ADP bound to F-actin with or without free eATP at 0.1 MPa (inset) and 100 MPa Protein concen- tration, 2 mg-mL™! in 10 mm Tris/HCl pH 7.5, 0.2 mm eATP, 50 mm KCl, 0.2 mm dithiothreitol, | mm EDTA, | mm NaN3 The other experimental conditions were the same as in Fig 3 Solid line, without EDTA; dotted line, with EDTA

When eATP, but no Mg” ,was present in the solution, in which conditions actin did not detach from the actin-HMM complex, little change in the fluorescence occurred up to

250 MPa (solid line in Fig 10) This suggested that HMM prevented F-actin from its depolymerization and subse- quent denaturation On an increase in pressure, the intensity began to decrease, which means that denaturation of actin was occurring (see Fig 5), but its rate was relatively slow compared that of F-actin alone (dotted line in Fig 10) As shown in Fig 10, the behaviour of actin in the actin-HMM complex was quite different from that of F-actin alone, indicating that the actin—HMM complex did not dissociate under relatively low pressure (P < 250 MPa) That was deduced because if the dissociation of actin from the complex (subsequent to depolymerization) happened under pressure, then the intensity of fluorescence would have been increased accompanying an increase of free sADP-—G-actin

as the pressure was elevated (Figs 2 and 6)

Ì 250 MPa

© a

Time (sec)

Fig 9 Effect of EDTA on the release of sADP bound to F-actin with and without free ATP at 250 MPa The experimental conditions were the same as in Fig 8 1, With eATP; 2, without eATP; 3, with EDTA and sATP; 4, with EDTA, without sATP; 5, buffer.

300r

200 MPa

Ez200E ” wy 350 MPa

Oo

°

© 100Ƒ

=

LL

ed

Time (sec)

Fig 10 Change in the fluorescence intensity of F-actin or acto-HMM

complex in the presence of sATP as pressure was elevated from 0.1 to

400 MPa Dotted line, F-actin; solid line, acto-HMM complex Pro-

tein concentration, 3.4 mgmL~' HMM (10 pm) and/or 0.42 mgmL"!

F-actin (10 pm) in 10 mm Tris/HCl pH 7.5, 50 mm KCI, 2 mm eATP,

0.2 mm dithiothreitol, 0.2 mm CaCh, 1 mm NaN3 The pressure was

kept for approximately 30-60 s after reaching the indicated pressure as

shown by the arrows

The effect of Mg””-sodium pyrophospate (NaPP;) on

the behaviour of actin in the actin-HMM complex (1: 1

molar ratio where actin filament was saturated by HMM

molecules) under pressure was investigated (Fig 11) It

should be noted that in this case sATP is not present in the

solution and Mg” * -NaPP; is capable of dissociating actin—

HMM complex without its hydrolysis When F-actin

without HMM was pressurized, it began to denature at

low pressure (150 MPa), as compared to the result shown

in Fig 3, because of a lack of sATP (line | in Fig 11) This

suggests that ATP had a protective effect against denatur-

ation when F-actin was under pressure as pointed out by

Bombardier ef al [6] and Ikkai & Ooi [12] When

pyrophosphate without Mg?* was added to the actin—

HMM solution, the change in fluorescence intensity was

small up to 200 MPa, as shown in Fig 10, because the

actin-HMM complex did not dissociate under such con-

ditions (line 2 in Fig 11) On the other hand, in the

presence of Mg’ '-NaPP;, where the actin-HMM complex

can be dissociated, and in the absence of sATP in the

external solution, the fluorescence 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, the decay in the intensity of fluorescence

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 have been due to the depolymerizing effect of

a small amount of HMM, which stimulated fragmentation

of F-actin, as reported by Ikeuchi e¢ al [27] Interestingly,

higher pressures (> 350 MPa), the intensities of fluores-

cence of HMM alone and 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 high pressure

150r 150 MPa

{ 200 MPa

250 MPa

300 MPa

100

350 MPa

400 MPa

200 300 400 500 600 700 80

Time (sec)

0 100

Fig 11 Change in the fluorescence intensity of F-actin, acto- HMM complex and HMM with and without Mg”*-NaPP, as pressure was elevated from 0.1 to 400 MPa 1, F-actin alone (10 um) with | mm MgCl and 2 mm NaPP,; (dotted line); 2, acto-HMM complex (actin/ HMM ratio 1 : 1) with 2 mm NaPP;; 3, acto-HMM complex (actin/ HMM ratio | : 1) with 1 mm MgCl and 2 mm NaPP;; 4, acto- HMM complex (actin/HMM ratio 10: 1) with 1mm MgCl and 2 mm NaPP;; 5, HMM alone (10 pm) with 1 mm MgCl and 2 mm NaPP; (dotted line) The other experimental conditions were the same as in Fig 10 except that cATP was not present in the solution The pressure was kept for approximately 2 min after reaching the indicated pressure shown by the arrows

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 the actin-HMM complex could be dissociated by pressure even without ATP although whether or not depolymerization of actin pro- ceeded prior to the dissociation of the complex was obscure However, our present data did not support this idea as stated above (Fig 10) The different interpretation regard- ing the dissociation of acto-HMM under pressure could be explained by the difference in the HMM/F-actin molar ratio used Namely, Ikkai & Ooi [26] measured the turbidity of acto-HMM solution under conditions at which the binding between F-actin and HMM was not saturated (F-actin: HMM = 5:1) unlike our conditions (F-actin: HMM = 1: 1) Therefore, the changes in the turbidity reported by them were presumed to be attributable mainly to the depolymerization of F-actin which was unbound to HMM If this is true, it may be understandable

to interpret the phenomenon as the dissociation of acto- HMM However, such a change in the turbidity (ie dissociation of acto-HMM) is probably not observed when the binding between F-actin and HMM is fully saturated (our condition) Although we do not have a satisfactory explanation for the nondissociation of acto-HMM under pressure as yet, our interpretation is that the association of actin and HMM, which are in the rigor complex, is so strong

as to resist high pressure (P < 250 MPa) Of course, further studies with respect to this point are needed

depolymerised actin

» 250 MPa < P (` ——TBT—>

Fig 12 Schematic interpretation of the

(eth neutral selÙ 250 MPa > P of ADP under pressure In brief: 1, below 250 MPa,

(with or without \) denatured once depolymerized actin is repolymerized

depolymerised actin

On the other hand, the behaviour of the actin-HMM

complex in the presence of Mg~°-NaPP, (i.e under

dissociation conditions) was different from that in the

absence of ATP That is, the actin-HMM_ complex

evidently dissociated into actin and HMM because the

fluorescence intensity rapidly decreased prior to reaching

200 MPa (ines 3, 4 in Fig 11) Ikkai & Ooi [26] have

reported that the 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 significant

dissociation of the complex In any event HMM protects

denaturation of F-actin up to 200 MPa in the absence of

ATP (compare line | and line 2 in Fig 11), whereas high

pressure under conditions that favour actin-HMM complex

dissociation (or in the presence of Mg**-NaPP, or

Mg’ -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 actin molecule (i.e the decay curve of the fluorescence

intensity of sATP-G-actin) obeyed good first order kinetics

(Fig 5) The volume change for the denaturation, calculat-

ed from their rate constants, was close to that obtained by

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 free ATP (Figs 7-9) The present results mostly

verified their data and speculations (i.e the value of volume

change, protecting effect of ATP on denaturation, repoly-

merization and so on), but we emphasize that our in situ

experiments show more direct and clearer evidence for those

facts than the ex situ experiment by Ikkai & Ooi [12] On the

other hand, information obtained from the fluorescence

measurements of the acto-HMM system (Fig 10) was

contradictory to the idea of Ikkai & Ooi [26] that the acto-

HMM complex in the absence of Mg” -ATP dissociates

into actin and HMM under pressure The reason for the

discrepancy was mentioned above

Apart from the fluorescence experiments, we attempted

spectroscopic measurement such as NMR and also bio-

chemical assays of actin after pressure release Although

details of the data are discussed elsewhere [23], the disap-

pearance of a characteristic 'H NMR signal at 2.06 p.p.m.,

which is considered to originate from the methyl proton of

methionine in the vicinity of the DNasel binding site in actin

[28], and the loss in biochemical activity (DNase I inhibition

Ph

random aggregation after release of pressure

release of ADP If EDTA is present, this step is accelerated; 3, after release of pressure, ran- dom aggregation of denatured actin occurs

capacity) were almost identical The DNasel binding site is located on the surface of the actin molecule [1] Taking these facts into account, we have inferred that the rapid collapse of the three-dimensional structure around the upper region known as the ‘pointed end’ (e.g burying into the inside of the molecule) is caused following the dissociation of the bound nucleotide (ATP) The 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 Scientific Research from the Ministry of Education, Science, Sports and Culture

of Japan (No 10460118) We thanks Dr Goodenough, University of Reading, UK, for reading this manuscript

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