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  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 information about the behaviour of actin under pressure. The dissociation rate constants of nucleotides from actin molecules ( the decay curve of the intensity o f ¯uorescence of eATP -G-actin or eADP±F-actin) followed ®rst-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be )72 mLámol )1 and )67 mLámol )1 in the p resence of ATP, respectively. Ch anges in the intensity of ¯uorescence of F-actin whilst under pressure suggested that eADP±F-actin was initially depoly- merize d to eADP±G-actin; subsequently there was quick 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 t he 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 ¯uorescence also demonstrated that, in the absence of Mg 2+ -ATP, the dissociation of 1 actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure. 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 polymerization. The direct effect of pressure on G- and F -actins was ®rst investigated by Ikkai & Ooi [12], and they reported the following results: (a) a ctin 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 i in the presence of ATP under pressure; (d) a volume change for the F-actin ® G-actin transformation is estimated to be )82 mLámol )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. 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 p ressure- 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; NaPP i , 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 KCl (polymerization), and then dialysed against a large volume of cold 50 m M KCl, 0.2 m M dithiothreitol, 1 m M NaN 3 and 10 m M Tris/ HCl (pH 7.5). Tris/HCl buffe r was selected because of i ts negligible effect of pressure on pH values. Protein concentration was measured using the extinction coef®cient 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- ¯uorometer. A quartz cuvette containing sample solutions was placed inside the vessel. Fluorescence spectroscopy Fluorescence measurements w ere made in a Hitachi F2000 ¯uorospectrophotometer, inside which the high-pressure vessel was placed. Temperature was maintained by circu- 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 , p H 7.5) in a pressure range from 0.1 MPa to 400 MPa at a ®xed 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 a s high as 400 MPa although ATP was thought to play a role in s tabilizing actin structure [6]. 288 290 292 294 296 298 300 0 100 200 300 400 Center of Mass /100, cm -1 Pressure (MPa) Compression decompression 0 20 40 60 80 100 120 140 250 300 350 400 450 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 CaCl 2 ,1m M NaN 3 . Ó FEBS 2002 Pressure-induced denaturation of actin (Eur. J. Biochem. 269) 365 In situ pressure-induced changes in the ¯uorescence spectra of eATP-G-actin and eADP±F-actin We attempted in situ observation of the behaviour of actin under p ressure by using eATP w hich emits strong ¯uores- cence at 410 nm when i t b inds to actin. The chemical structure of eATP is illustrated in i nset of Fig. 2 [16]. The ¯uorescence emission spectra of eATP-G-actin, eADP±F- actin and the eATP buffer are displayed in Fig. 2, which shows that the intensity of ¯uorescence 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 ¯uorescence when exposed to a p ressure of 250 MPa. However, the increase of intensity of ¯uorescence of eATP buffer itself was much smaller than that of eATP bo und to G-actin. Therefore, the increase of ¯uorescence seems to b e due mainly to the conformational change of actin under pressure. In situ pressure-induced changes in the intensity of ¯uorescence of epsilon nucleotides bound to G- and F-actins 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 5 6 + N N N N O H H OH H OH CH 2 H O POPOPHO NH OOO O - O - O - 1, N 6 -ethenoadenosine 5'-triphosphate ( -ATP) 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 NaN 3 . F-actin solution contained 2 mgámL )1 F-actin, 10 m M Tris/HCl pH 7.5, 50 m M KCl, 0.2 m M eATP, 0.2 m M dithiothreitol, 0.2 m M CaCl 2 ,1m M NaN 3 . Inset sh ows th e che mical stru cture of eATP [16]. 0 0.5 1 1.5 0 500 1000 1500 Relative fluorescence intensity Time (sec) 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 were the same as in Fig. 3. Filled arrowh eads show the point at which the designated p ressure was reached and open a rrowhead s s how the start of decompression. 366 Y. Ikeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 is fully reversible, which was also con®rmed by measure- ment of the ¯uorescence spectrum (data not shown). O n t he other h and, the relative intensity of ¯uorescence d ecreased 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 ¯uorescence o f eATP-G-actin was investigated under pressures ranging from 200 M Pa to 400 M Pa at 25 MPa intervals (Fig. 5). The decrease in the intensity when pressure was kept c onstant actually re¯ects the dissociation of eATP from G-actin. As s hown in Fig. 5 , c hange i n t he relative intensity of ¯uorescence obeyed ®rst-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 mLá mol )1 in the p resence 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]whocalculated the value from the pressure dis sociation curve o f a ctin subunits. Fig. 6 s hows the time dependen ce of the relative intensity 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 free eATP or ATP in the s olvent at 100 MPa where G-actin is not denatured (Fig. 4). In the presence of eATP, the ¯uorescence 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 re lease of pressure (data not shown). T his implied that the decrease in the intensity of ¯uorescence 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 p ressure such a s 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 t he 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). F-actin, in contrast with G-actin, is not denatured even in the presence o f E DTA. ED TA will deprive G-actin of divalent cation leading to a quick irreversible denaturation 0 0.2 0.4 0.6 0.8 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) 0.1 50 100 125 150 180 200 230 240 250 Holding pressure Relative fluorescence intensity (MPa) Fig. 6. Time courses of change in the relative ¯uorescence intensity of eADP±F-actin under various pressures from 0.1 to 250 MPa. Protein concentration, 2 mgámL )1 in 10 m M Tris/HCl pH 7.5, 0.2 m M eATP, 50 m M KCl, 0.2 m M dithiothreitol, 0.2 m M CaCl 2 ,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 M KCl) was present in the s olution ( see b elow) [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 in situ observations were made by monitoring the ¯uorescence 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 pressu re (Figs 10 and 11). When eATP, but no Mg 2+ ,was present in t he solution, in which conditions actin did not d etach from th e actin±HMM complex, little change in the ¯uorescence occurred up to 250 MPa (solid line in Fig. 10). This suggested that HMM prevented F -actin from its dep olymerization a nd subse- quent denaturation. On an increase in pressure, the intensity began to decrease, which means that denaturation o f 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 F ig. 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 disso ciate under relatively low pressure (P < 250 MPa). That was deduced because if t he dissociation of actin from the complex (subsequent to depolymerization) happened under 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 M Tris/HCl pH 7.5, 0.2 m M eAT P, 50 m M KCl, 0.2 m M dithiothreitol, 1 m M EDTA, 1 m M NaN 3 . The other experimental conditions were th e same as in Fig. 3. Solid line, with ou t EDTA; dotted line, with EDTA. 0 0.5 1 1.5 2 0 200 400 600 800 1000 Time (sec) 250 MPa Relative fluorescence intensity 1 2 3 4 5 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 subjected to 200 MPa pressure. 368 Y. Ikeuchi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The effect of Mg 2+ -sodium pyrophospate (NaPP i )on the behaviour of actin in the actin±HMM complex (1 : 1 molar ratio where actin ®lament was saturated by HMM molecules) under pressure was investigated (Fig. 11). I t should be noted that in this case eATP is not present in the solution and Mg 2+ -NaPP i 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 A TP had a protective effect against denatur- ation when F-actin was under pressure as pointed out by Bombardier et al.[6]andIkkai&Ooi[12].When pyrophosphate withou t M g 2+ was add ed to the actin± HMM solution, the change in ¯uorescence 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 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. 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 t he difference in the HMM/F-actin molar r atio used. Namely, Ikkai & Ooi [26] measured the turbidity of acto-HMM solution under conditions at which t he binding between F -actin a nd HMM w as 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 a ttributable mainly to the depolymerization o f F -actin which was unbound to HMM. If this is true, i t 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, ou r 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). O f course, further studies with respect to this point are needed. 0 100 200 300 0 100 200 300 400 500 Time (sec) 100 MPa 150 MPa 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 CaCl 2 ,1m M NaN 3 . The pressure was kept for approximately 30±60 s after reaching the indicated pressure as shown by the arrows. 0 50 100 150 0 100 200 300 400 500 600 700 800 Time (sec) 250 MPa 200 MPa 300 MPa 400 MPa 350 MPa 150 MPa 1 2 3 4 5 Fluorescence intensity Fig. 11. Change in the ¯uorescence intensity of F-actin, acto-HMM complex and HMM with and without Mg 2+ -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 MgCl 2 and 2 m M NaPP i ;5,HMMalone(10l M )with1m M MgCl 2 and 2 m M NaPP i (dotted l ine). The other exp erimental conditions were the same as i n Fig. 10 except that eATP was not p resent in the solution. The pressure was kept for approximate ly 2 min after reaching the indicated p ressure shown by the arrows. Ó FEBS 2002 Pressure-induced denaturation of actin (Eur. J. Biochem. 269) 369 On the other hand, the behaviour of the actin±HMM complex in the presence of Mg 2+ -NaPP i (i.e. under dissociation conditions) was different from that in the absence of ATP. That is, the actin±HMM complex evidently d issociated into actin and HMM because the ¯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 change, protecting e ffect of A TP on denaturation, repoly- merization and so on), but we emphasize that our in situ experiments show more direct and clearer evidence 3 for those facts t han the ex situ experiment by Ikkai & Ooi [12]. On the other hand, information obtained from t he ¯uorescence measurements of the a cto-HMM system ( Fig. 10) was contradictory to the idea of Ikkai & Ooi [26] that the acto- HMM complex in the absence of Mg 2+ -ATP dissociates into actin and HMM under pressure. The reason for the discrepancy was mentioned a bove. Apart from the ¯uorescence 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 1 H NMR signal at 2.06 p.p.m., which is c onsidered to originate from the methyl proton of methionine in the vicinity of the DNaseI binding site in actin [28], and the loss i n b iochemical activity (DNase I i nhibition capacity) were almost identical. The DNaseI binding site is located o n the surface of t he 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 Ôpointed endÕ (e.g. burying into the i nside of the molecule) i s caused following the dissociation o f the bound 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. REFERENCES 1. Kabsh,W.,Mannherz,H.G.,Such,D.,Pai,E.F.&Holmes,K.C. (1990) Atomic structure of the actin: Dnase I complex. Nature 347, 37±44. 2. Oosawa,F.&Kasai,M.(1971)Actin.InSubunits in Biological Systems (Timashe, S.N. & Fasman, G.D., eds) part A , pp. 261± 322. Dekker, N ew York. 3. Pollard, T.D. (1986) A ctin and actin-binding proteins. A critical evaluation of mechanisms and function. Annu. Rev. Biochem. 55, 987±1035. 4. Lewis, M.S., Maruyama, K., Carroll, W.R., Kominz, D.R. & Laki, K. (1963) Physical propeties and polymerization reactions of native and inactivated G-actin. B i och em ist ry 2, 34±39. 5. Carlier, M F. (1989) Role of nucleotide hydrolysis in the dynamics of actin ®laments and microtubules. Int. Rev. 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Heintz, D., Kany, H. & Kalbitzer, H.R. (1996) Mobility of t he N-terminal segment of rabbit skeletal muscle F-actin detected by 1 Hand 19 F nuclear magnetic resonance spectroscopy. Bio- chemistry 35, 12686±12693. Ó FEBS 2002 Pressure-induced denaturation of actin (Eur. J. Biochem. 269) 371 . Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin Yoshihide Ikeuchi 1 , Atsusi Suzuki 2 , Takayoshi Oota 2 ,. 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