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

Eur. J. Biochem. 269, 364–371 (2002) (cid:211) FEBS 2002

Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin

Yoshihide Ikeuchi1, Atsusi Suzuki2, Takayoshi Oota2, Kazuaki Hagiwara2, Ryuichi Tatsumi1, Tatsumi Ito1 and Claude Balny3

1Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan; 2Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Japan; 3INSERM Unite´ 128, IFR 24, CNRS, Montpellier, France

exchange of the eADP for free eATP, and then polymer- ization occurred again with the liberation of phosphate from eATP 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 eATP ﬂuorescence also demonstrated that, in the absence of Mg2+-ATP, the actin-heavy meromyosin (HMM) complex dissociation of into actin and HMM did not occur under high pressure.

Keywords: actin; denaturation; dissociation; ﬂuorescence; heavy meromyosin; high pressure.

Ikkai & Ooi [Ikkai, T. & Ooi, T. (1966) Biochemistry 5, 1551– 1560] made a thorough study of the e(cid:128)ect 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 eATP 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 eATP-G-actin or eADP–F-actin) followed ﬁrst-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be )72 mLÆmol)1 and )67 mLÆmol)1 in the presence of ATP, respectively. Changes in the intensity of ﬂuorescence of F-actin whilst under pressure suggested that eADP–F-actin was initially depoly- merized to eADP–G-actin; subsequently there was quick

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.

(a) actin is

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 ﬁ 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 (cid:212)pointed end(cid:213) and also in the bottom region known as a (cid:212)bared end(cid:213) (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].

The direct effect of pressure on G- and F-actins was ﬁrst investigated by Ikkai & Ooi [12], and they reported the following results: 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 ﬁ G transformation occurs with release of ADP and Pi in the presence of ATP under pressure; (d) a volume change for the F-actin ﬁ G-actin transformation is estimated to be )82 mLÆmol)1 of monomer from the pressure denaturation curve although it is considered questionable whether the value may be indicative of the in vivo DV of assembly. it must be borne in mind that all of the However, 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.

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; NaPPi, sodium pyrophospate. (Received 9 July 2001, revised 17 October 2001, accepted 7 November 2001)

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Fluorescence spectroscopy

The aim of the present study was to complete a study of F ﬁ G transition 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.

M A T E R I A L S A N D M E T H O D S

Protein preparations

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 eATP G-actin (or eADP–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 (k). The value of volume change was obtained by plotting lnk vs. pressure [7].

R E S U L T S A N D D I S C U S S I O N

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 (cid:176)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:N6- ethenoadenosine 5¢-triphosphate (eATP) 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 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 mM KCl (polymerization), and then dialysed against a large volume of cold 50 mM KCl, 0.2 mM dithiothreitol, 1 mM NaN3 and 10 mM Tris/ HCl (pH 7.5).

Insitupressure-induced changes in spectrum and the centre of spectral mass of the intrinsic ﬂuorescence of ATP-G-actin

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

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. 1 shows the changes in the centre of spectral mass of intrinsic ﬂuorescence spectrum of G-actin with ATP (0.5 mgÆmL)1, pH 7.5) in a pressure range from 0.1 MPa to 400 MPa at a ﬁxed temperature of 20 (cid:176)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 indicating that G-actin was upon pressure elevation, 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].

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.

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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 MPa (dotted line). Inset: the pressure dependence of the centre of spectral mass of G-actin intrinsic ﬂuorescence. (d), Com- pression; (m), decompression. Excitation wavelength, 295 nm; emission range, 300–400 nm; temperature, 20 (cid:176)C. Protein concentration, 0.5 mgÆmL)1 in 2 mM Tris/HCl pH 7.5, 0.2 mM ATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3.

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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 (cid:176)C. Protein concentration, 2 mgÆmL)1 in 2 mM Tris/HCl pH 8.0, 0.2 mM eATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3. The pressure was maintained for (cid:25) 3 min after reaching the indicated pressure as indicated by the arrows.

ﬂuorescence 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 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.

Insitupressure-induced changes in the ﬂuorescence spectra of eATP-G-actin and eADP–F-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

We attempted in situ observation of the behaviour of actin under pressure by using eATP which emits strong ﬂuores- cence at 410 nm when it binds to actin. The chemical structure of eATP is illustrated in inset of Fig. 2 [16]. The ﬂuorescence emission spectra of eATP-G-actin, eADP–F- actin and the eATP buffer are displayed in Fig. 2, which shows that the intensity of ﬂuorescence at 410 nm of eATP- G-actin was higher than that of eADP–F-actin. Both actins and eATP buffer showed an increase in intensity of ﬂuorescence when exposed to a pressure of 250 MPa. However, the increase of intensity of ﬂuorescence of eATP buffer itself was much smaller than that of eATP bound to G-actin. Therefore, the increase of ﬂuorescence seems to be due mainly to the conformational change of actin under pressure.

Insitupressure-induced changes in the intensity of ﬂuorescence of epsilon nucleotides bound to G- and F-actins

Fig. 2. Variation in ﬂuorescence spectra of eATP-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 eADP at 0.1 MPa; 3, G-actin with eATP at 250 MPa; 4, F-actin with eADP at 250 MPa; 5, bu(cid:128)er with eATP at 0.1 MPa; 6, bu(cid:128)er with eATP at 250 MPa. Excitation wavelength, 360 nm; emission range, 380–580 nm; temperature, 20 (cid:176)C. G-actin solution contained 2 mgÆmL)1 G-actin, 2 mM Tris/HCl pH 7.5, 0.2 mM eATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3. F-actin solution contained 2 mgÆmL)1 F-actin, 10 mM Tris/HCl pH 7.5, 50 mM KCl, 0.2 mM eATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3. Inset shows the chemical structure of eATP [16].

Fig. 3 shows changes in the relative intensity of ﬂuorescence of eATP-G-actin and eADP–F-actin in the presence of eATP 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 eATP buffer. Thus, the decrease in intensity of

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.

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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.

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 eADP–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 eADP–F-actin was )67 mLÆmol)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

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 eATP 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 eATP from actin corresponds to its denaturation rate, the volume change for the denaturation was estimated to be )72 mLÆ mol)1 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. 7 shows the exchange of eATP bound to G-actin with free eATP or ATP in the solvent at 100 MPa where G-actin is not denatured (Fig. 4). In the presence of eATP, 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.

eADP 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 eADP bound to F-actin is capable of exchanging nucleo- tides under pressure, a similar experiment as in the case of eATP-G-actin was conducted (Fig. 7, inset). The result indicated that eADP 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).

Fig. 6 shows the time dependence of the relative intensity of ﬂuorescence of eADP–F-actin in the presence of 0.2 mM eATP and 50 mM KCl 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 eATP bound to G-actin generates stronger ﬂuor- escence than eADP–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 eADP–F-actin (i.e. the G«F equilibrium).

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

Fig. 5. Logarithm of the relative ﬂuorescence intensity of eATP-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 1 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. Fig. 6. Time courses of change in the relative ﬂuorescence intensity of eADP–F-actin under various pressures from 0.1 to 250 MPa. Protein concentration, 2 mgÆmL)1 in 10 mM Tris/HCl pH 7.5, 0.2 mM eATP, 50 mM KCl, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3.

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Fig. 8. E(cid:128)ect of EDTA on the release of eADP bound to F-actin with or without free eATP at 0.1 MPa (inset) and 100 MPa. Protein concen- tration, 2 mgÆmL)1 in 10 mM Tris/HCl pH 7.5, 0.2 mM eATP, 50 mM KCl, 0.2 mM dithiothreitol, 1 mM EDTA, 1 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 Mg2+,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 eADP–G-actin as the pressure was elevated (Figs 2 and 6).

Fig. 7. Exchange of eATP bound to G-actin by free eATP or ATP in the solvent under pressure at 100 MPa. The samples were diluted to a ﬁnal concentration of 2 mgÆmL)1 with a solution containing eATP (solid line) or ATP (dotted line) immediately before monitoring of the ﬂuo- rescence intensity. Protein concentration, 2 mgÆmL)1 in 2 mM Tris/ HCl pH 7.5, 0.2 mM eATP or ATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3. Inset represents exchange of eADP bound to F-actin by free eATP 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.

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[4]. Subsequently ﬂuorescence measurements of eADP–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 eADP–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 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 cycling F ﬁ G ﬁ 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 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 eADP 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].

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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 in 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).

Fig. 9. E(cid:128)ect of EDTA on the release of eADP bound to F-actin with and without free eATP at 250 MPa. The experimental conditions were the same as in Fig. 8. 1, With eATP; 2, without eATP; 3, with EDTA and eATP; 4, with EDTA, without eATP; 5, bu(cid:128)er.

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Fig. 10. Change in the ﬂuorescence 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 mgÆmL)1 HMM (10 lM) and/or 0.42 mgÆmL)1 F-actin (10 lM) in 10 mM Tris/HCl pH 7.5, 50 mM KCl, 2 mM eATP, 0.2 mM dithiothreitol, 0.2 mM CaCl2, 1 mM NaN3. The pressure was kept for approximately 30–60 s after reaching the indicated pressure as shown by the arrows.

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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 (cid:136) 5 : 1) conditions (F-actin : HMM (cid:136) 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 (i.e. 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.

The effect of Mg2+-sodium pyrophospate (NaPPi) 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 eATP is not present in the solution and Mg2+-NaPPi 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 eATP (line 1 in Fig. 11). This suggests that ATP had a protective effect against denatur- ation when F-actin was under pressure as pointed out by [12]. When Bombardier et al. pyrophosphate without Mg2+ 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- in the ditions (line 2 in Fig. 11). On the other hand, presence of Mg2+-NaPPi, where the actin–HMM complex can be dissociated, and in the absence of eATP 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 et 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.

Fig. 11. Change in the ﬂuorescence intensity of F-actin, acto-HMM complex and HMM with and without Mg2+-NaPPi as pressure was elevated from 0.1 to 400 MPa. 1, F-actin alone (10 lM) with 1 mM MgCl2 and 2 mM NaPPi (dotted line); 2, acto-HMM complex (actin/ HMM ratio 1 : 1) with 2 mM NaPPi; 3, acto-HMM complex (actin/ HMM ratio 1 : 1) with 1 mM MgCl2 and 2 mM NaPPi; 4, acto-HMM complex (actin/HMM ratio 10 : 1) with 1 mM MgCl2 and 2 mM NaPPi; 5, HMM alone (10 lM) with 1 mM MgCl2 and 2 mM NaPPi (dotted line). The other experimental conditions were the same as in Fig. 10 except that eATP was not present in the solution. The pressure was kept for approximately 2 min after reaching the indicated pressure shown by the arrows.

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capacity) were almost identical. The DNaseI 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 (cid:212)pointed end(cid:213) (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.

Fig. 12. Schematic interpretation of the behaviour of F-actin in the presence of free ATP under pressure. In brief: 1, below 250 MPa, once depolymerized actin is repolymerized with or without EDTA if ATP is fully present; 2, above 250 MPa F-actin is ﬁrst depolymer- ized and is then denatured with the rapid release of ADP. If EDTA is present, this step is accelerated; 3, after release of pressure, ran- dom aggregation of denatured actin occurs.

A C K N O W L E D G E M E N T S

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.

R E F E R E N C E S

On the other hand, the behaviour of the actin–HMM complex in the presence of Mg2+-NaPPi (i.e. under dissociation conditions) was different from that in the absence of ATP. That the actin–HMM complex is, evidently dissociated into actin and HMM because the ﬂuorescence intensity rapidly decreased prior to reaching 200 MPa (lines 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 1 and line 2 in Fig. 11), whereas high pressure under conditions that favour actin–HMM complex dissociation (or in the presence of Mg2+-NaPPi or Mg2+-ATP) promotes the denaturation of actin following the dissociation of actin–HMM complex (lines 3, 4 in Fig. 11).

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In conclusion, the dissociation rates of nucleotides from the actin molecule (i.e. the decay curve of the ﬂuorescence intensity of eATP-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 Mg2+-ATP dissociates into actin and HMM under pressure. The reason for the discrepancy was mentioned above.

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