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Effect of high hydrostatic pressure on chicken myosin subfragment-1
International Journal of Biological Macromolecules 30 (2002) 227 /232 www.elsevier.com/locate/ijbiomac
Effect of high hydrostatic pressure on chicken myosin subfragment-1 Tomohito Iwasaki *, Kastuhiro Yamamoto Department of Food Science, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan Received 27 August 2001; received in revised form 4 March 2002; accepted 8 April 2002
Abstract Hydrostatic pressure-induced structural changes in subfragment-1 (S1) of myosin molecule were studied. ATP-induced emission spectra of S1 were used to detect global structural change of S1 by pressure treatment. The fluorescence intensity of unpressurized S1 increased by addition of ATP. The increment of fluorescence of pressurized S1 up to 150 MPa was almost the same as control, whereas it became smaller above 200 MPa. ATP binding ability of S1 examined using 1, N6-ethenoadenosine 5?-diphosphate (oADP) indicated that the binding of o-ADP to S1 decreased in the range of 250 /300 MPa. S1 pressurized below 250 MPa and unpressurized S1 similarly bound to F-actin, although binding of S1 pressurized above 250 MPa decreased. Electron microscopic observation revealed arrowhead structure in control acto-S1, while disordered arrowhead structure was observed in acto-S1 prepared from pressurized S1 at 300 MPa. S1 pressurized below 250 MPa retained the same actin activated ATPase activity as the control, whereas the activity decreased to 60% at 300 MPa. Pressure treated S1 was easily cleaved by tryptic digestion into three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which were the same as those in unpressurized one. It is concluded that pressure-induced global structural changes of S1 begin to occur about 150 MPa, and the local structural changes in ATPase and actin binding sites followed with elevating pressure to 250 /300 MPa. # 2002 Published by Elsevier Science B.V. Keywords: Subfragment-1; Myosin; Pressure
1. Introduction Application of high hydrostatic pressure on protein induces aggregation [1,2] gelation [3], or subunits dissociation [4 /8]. These pressure-induced change in proteins are thought to be useful for controlling enzyme activity as well as analysis of interaction among protein molecules or subunits. Pressure-induced aggregation and gelation of proteins are caused by disruption and reformation of noncovalent bonds. Myosin is a major protein of myofibril and it consists of two heads and a tail. ATPase and actin binding sites are located in the heads, and the tails self-associate to form thick filament at low ionic strength. These are some reports on pressure-induced denaturation of myosin [9 /11]. Yamamoto et al. [10] reported that
Abbreviations: PMSF, phenylmethylsulfonylflouride; TPCKtrypsin, L-1 tosylamido-2 phenylethyl chloromethyl ketone treated trypsin; o-ADP, 1, N6-ethenoadenosine 5?-diphosphate. * Corresponding author. Tel./fax: /81-11-388-4834 E-mail address: [email protected] (T. Iwasaki).
Ca2- and K -EDTA-activated myosin ATPase activities decreased from 100 MPa. Hydrophobicity of myosin increased from 100 MPa and it reached plateau value at 300 MPa, and the helix content of myosin decreased above 300 MPa [10]. Turbidity of pressurized myosin solution markedly increased within 5 min of pressure treatment at 210 MPa, indicating formation of aggregates. The aggregates were formed by head to head interaction and the shape of aggregate looked like daisywheel [9]. The size of pressure-induced myosin aggregates was dependent on the magnitude of applied pressure and the duration of the treatment. Oligomers containing over 10 myosin molecules were made by pressure treatment at 210 MPa for 30 min [9]. ATPase activity of myosin was apparently related to the size of pressure-induced oligomer. These findings also indicate that the head (subfragment-1, S1) is the most pressuresensitive portion in myosin molecule. S1 has ATPase and actin binding sites, and it consists of three domains, which are 27, 50 and 20 kDa fragments. The nucleotide binding site is located in the junction of the 27 and 50 kDa fragments [12,13], and the actin binding site is located in the 50 and 20 kDa
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fragments junction [14,15]. The detail of pressureinduced changes in actin binding and ATP binding sites of S1 are not well understood. It is important to elucidate alteration of these portions in the myosin molecule for comprehensive understanding of pressureinduced denaturation of myosin. As mentioned above, myosin head (S1) tends to aggregate by pressure treatment. Since the analysis of local structural change in S1 seems to be difficult using pressurized S1 solution containing large aggregates, we removed large aggregates from pressurized S1 by centrifugation and used it in this study.
2. Materials and methods 2.1. Preparation of proteins Myosin was prepared from fresh chicken pectoral muscle according to Offer et al. [16]. S1 was prepared by the method of Weeds and Pope [17] with some modification. Myosin in 0.12 M NaCl, 20 mM Na phosphate (pH 7.0) and 1 mM EDTA was digested with 1/400 (w/w) of a-chymotrypsin at 23 8C for 7 min. Digestion was terminated by addition of 0.1 M phenylmethylsulfonylflouride (PMSF) to a final concentration of 0.5 mM, then the protein solution was dialyzed against 40 mM NaCl and 5 mM Na phosphate (pH 6.5) at 4 8C with three changes of dialysate. S1 was further purified by ammonium sulfate fractionation and Whatman DE-52 column chromatography with a NaCl gradient from 0 to 0.2 M. The unretarded fractions were pooled and precipitated by ammonium sulfate at 58% saturation. Actin was prepared from rabbit skeletal muscle according to Spudich and Watt [18]. 2.2. Application of hydrostatic pressure Two milliliters of S1 (1.7 /25 mM) solution containing 0.05 /0.2 M NaCl and 10 mM Tris /HCl (pH 7.0) was put into a plastic tube, then the tube was firmly sealed with a plug without trapping of any air. The sample tube was placed in a pressure vessel, having a diameter of 30 mm and a length of 100 mm, filled with water. Hydrostatic pressure was generated by pumping water into the vessel with a hydraulic machine. Application of pressure was performed at 20 8C for 10 min. After pressure release, the pressurized S1 solution was ultracentrifuged at 194 000/g for 60 min. S1 in the supernatant was used in the present study. 2.3. Fluorescence measurements Fluorescence spectra were recorded with a Hitachi F2000 spectrofluorometer. The tryptophan fluorescence of S1 was measured in 0.2 M NaCl and 20 mM Na
phosphate (pH 7.0) at 20 8C with excitation wavelength of 295 nm according to Werber et al. [19]. After the emission spectra were recorded, ATP was added to give a final concentration of 1 mM, and the spectra were recorded again. Fluorescence intensity at the peak of the spectrum (334 nm) was used to calculate the increment of ATP-induced fluorescence (DF ) which was expressed as percentages of the fluorescence intensity obtained the unpressurized S1 by the following equation; DF (%) [(FATP FATP ) pressurized]=[(FATP FATP ) unpressurized]100 here, FATP and F ATP are the fluorescence intensities at 334 nm before and after addition of ATP, respectively. The fluorescence of 1, N6-ethenoadenosine 5?-diphosphate (o-ADP) was measured at 20 8C with the excitation wavelength of 320 nm and the emission wavelength of 410 nm. The interaction of o-ADP with S1 was estimated using quenching of the fluorescence of the free nucleotide induced by acrylamide as described by Ando et al. [20]. o-ADP was added to 4 mM S1 in 200 mM acrylamide, 2 mM MgCl2 5 mM KCl, and 40 mM Tris /HCl (pH 7.7). In control experiment, the fluorescence of o-ADP was measured under the same conditions without S1. The ATP binding ability of pressurized S1 was estimated from the difference between fluorescences of pressurized and unpressurized S1. 2.4. Binding experiments The binding of pressurized S1 to F-actin was measured as described Chalovich and Eisenberg [21], and Yamamoto and Moos [22]. Centrifugation was performed at 135 000 /g for 10 min in a Beckman Airfuge with a type A-100 rotor. The supernatant, in which unbound S1 remained, was collected and the concentration was measured by UV absorbance. 2.5. ActoS1 ATPase measurements Actin activated S1 ATPase activity was measured with a pH-stat at pH 7.0 and 25 8C in a reaction mixture containing 0.02 M NaCl, 0.5 mM ATP and 2 mM MgC12. The protein concentrations of S1 and Factin were 0.38 and 1.6 mM, respectively. Titration was performed with 20 mM KOH. 2.6. Electron microscopy A drop of acto /S1 (0.1 /0.2 mg/ml) was applied to a carbon-coated grid which had been glow-discharged just before use. The grid was negatively stained with 2% aqueous uranyl acetate. The specimens were observed in a Hitachi H-800 electron microscope at an accelerating
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voltage of 75 kV. Images were recorded at a magnification of 40 000. 2.7. SDS-PAGE of tryptic digested fragment Pressurized S1 (8.33 mM) was digested with 1/400 (w/ w) of L-1 tosylamido-2 phenylethyl chloromethyl ketone treated trypsin (TPCK-trypsin) in 0.1 M NaCl and 10 mM Tris /HCl (pH 7.0) at 20 8C for 5 /30 min. Digestion was terminated by heat treatment at 100 8C. Electrophoresis of the digests was performed on a 7.5% polyacrylamide gel [22].
Fig. 2. The ATP-induced fluorescence increment of S1 after pressure treatment. Relative fluorescence intensity at 334 nm was calculated as described under Section 2.
3. Results 3.1. The ATP-induced tryptophan fluorescence lt is known that the structure of S1 is changed by binding of ATP [19]. Fig. 1 shows the fluorescence spectrum of unpressurized S1 (a) and pressurized S1 at 300 MPa (b) before and after addition of ATP. The fluorescence intensity of unpressurized S1 with ATP was higher than that without ATP. On the other hand, there was no difference in fluorescence spectrum of pressurized S1 before and after addition of ATP. In order to estimate pressure effect on S1 structure, we calculated the ATP-induced fluorescence increment (DF ) with varying pressure (Fig. 2). The ATP-induced fluorescence increments did not change from 0.1 to 150 MPa. However, DF linearly decreased above 200 MPa. The differential spectra of ATP-induced fluorescence intensity showed negative value, since the fluorescence intensity of S1 after addition of ATP was lower than that of S1 before addition of ATP at 300 MPa. These results suggest pressure-induced global structural change in S1.
Fig. 1. Emission spectra of S1 before and after addition of ATP. One micromole of S1 in 0.2 M NaCl and 10 mM Tris-HCl (pH 7.0) was pressurized at 300 MPa, then centrifuged at 194 000/g for 60 min. The supernatant was collected, and ATP was added to 1 mM. (a) unpressurized S1 (0.1 MPa); (b) pressurized S1. Excitation wavelength was 295 nm. Solid and dotted lines indicate the spectra before and after addition of ATP, respectively.
We investigated pressure effect on the ATP binding site of S1 by fluorescence quenching. Ando et al. [20] developed a method of measuring nucleotide binding to S1. They found that the fluorescence of free o-nucleotides was quenched by acrylamide, while the quenching was prevented when the o-nucleotides bound to S1. We titrated pressure treated S1 with o-ADP in the presence of acrylamide using this method (Fig. 3). The intensity of the acrylamide nonquenchable fluorescence, i.e. the amount of o-ADP bound to S1, decreased with elevating pressure. The amount of o-ADP bound to S1 did not change up to 200 MPa, while it decreased above 250 MPa. The intensity of ATP-induced fluorescence decreased above 200 MPa (Fig. 2), while binding of o-ADP to S1 decreased above 250 MPa (Fig. 3). These results may explain the difference between global and local structural change in S1.
Fig. 3. Effect of pressure treatment on the binding of o-ADP to S1. oADP was added for give a final concentration of 8 mM, then pressurized 1 mM S1 in 0.1 M NaCl, 200 mM acrylamide, 2 mM MgC12, 20 mM Na phosphate (pH 7.0), and its fluorescence was recorded as described in Section 2. The bound o-ADP was calculated from the fluorescence intensity at 410 nm before and after addition oADP.
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3.4. Changes in actin activated ATPase activity of pressurized S1
Fig. 4. Effect of pressure treatment of S1 on the binding to F-actin. Factin was incubated with pressurized S1 in 50 mM NaCl, 5 mM Na phosphate (pH 7.0), and 1 mM MgCl2 for 20 min at room temperature, then centrifuged at 135 000/g for 10 min. Protein concentration of the supernatant (unbound S1) was measured by UV absorbance at 280 nm. (m) 5 mM S1; (k) 2 mM S1. Actin concentration was 5 mM.
3.2. Pressure effect on actin binding site of S1 The effect of pressure on binding ability of S1 to Factin was studied (Fig. 4). S1 binding to F-actin causes formation of acto-S1, which can be sedimented by ultracentrifuge. Bound S1 was calculated by subtracting unbound S1 from total S1. The binding did not change up to 200 MPa, and it decreased at 300 MPa though S1 still retains 90% of actin-binding ability at this pressure. These findings indicate structural change in actin binding site of S1 by pressure at 300 MPa.
We measured actin activated S1 ATPase activity to investigate the interaction between F-actin and pressurized S1. Fig. 6 shows pressure effect of S1 on actinactivated S1 ATPase activity. The activity remained at almost the same level as an unpressurized S1 at least up to 250 MPa, but the activity was remarkably decreased at 300 MPa. The diminution of ATPase activity of pressurized S1 is consistent with decreasing actin binding ability of pressurized one shown in Fig. 4, though pressure treated S1 at 300 MPa still retains ATPase activity of about 0.43 mmol/min/mg. Since the conformational change of S1 was induced by pressure, the interaction between pressurized S1 and F-actin was weak. 3.5. Tryptic digestion of pressure treated S1 S1 contains three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which are easily produced by tryptic digestion of S1. The ATPase site is located in the 27 and 50 kDa fragments, and the actin binding site is in the 50 and 20 kDa fragments. Fig. 7 shows the SDS-PAGE pattern of tryptic fragments of S1
3.3. Morphology of acto-S1 complex Fig. 5 shows morphology of acto-S1. A typical arrowhead structure was observed in the control actoS1 complex which was prepared from unpressurized S1 and F-actin. When the pressurized S1 at 300 MPa was mixed with F-actin, the formed acto-S1 showed disordered arrowhead structure. The morphological observations support overall conformational alteration of S1 by pressure treatment.
Fig. 6. Effect of pressure treatment of S1 on acto-S1 ATPase activity. Acto-S1 ATPase activity was assayed in 20 mM NaCl, 0.5 mM ATP, and 2 mM MgCl2 with 0.38 mM S1 and 1.6 mM actin at pH 7.0 and 25 8C.
Fig. 5. Transmission electron micrographs of acto-S1. (a) control; (b) acto-S1 formed from pressurized S1 at 300 MPa for 10 min and unpressurized F-actin. Scale bar indicates 0.2 mm.
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Fig. 7. SDS-PAGE of tryptic digested S1. Pressure-treated S1 in 0.1 M NaCl and 10 mM Tris /HCl (pH 7.0) was digested with 1/400 (w/w) of trypsin at 20 8C for 5 /30 min. The reaction was terminated by heat treatment at 100 8C. Polyacrylamide gel concentration was 7.5%. Control denotes undigested S1. HC and LC denote S1 heavy chain and light chain, respectively. (a) unpressurized S1, (b), (c) and (d); pressurized S1 at 100, 200 and 300 MPa, respectively.
after pressure treatment. These tryptic fragments of S1 obtained from 100 to 300 MPa were the same as that of unpressurized one. Setton and Muhlrad [23] showed disappearance of tryptic 50 kDa fragment accompanying loss ATPase activity in the mild heat treated S1. The band of 50 kDa fragment in a gel was observed in pressurized S1 regardless of degree of pressure, although the Mg2-ATPase activity of S1 was decreased after pressure treatment at 300 MPa (Fig. 6).
4. Discussion The tryptophan fluorescence intensities of myosin and its proteolytic fragments are useful to investigate structural change induced by binding of nucleotides [19,24/27], though it is difficult to interpret S1 fluorescence data in terms of local sites structure[28]. S1 has five Trp residues in its heavy chain [29]. ATP-induced fluorescence increase is caused by only one or two tryptophan residues [19,30], which are located in the 50 kDa tryptic fragment [31]. Trp510 especially plays an important role in the fluorescence change of S1 [30,32]. Sugimoto et al. [33] showed that the global conformational change of skeletal muscle S1 in the presence of MgATP was caused by a hinge-like bending movement between the catalytic and regulatory domains, and the global change of S1 was correlated with closing of ATP binding pocket by cross-linking of 50 and 20 kDa fragments in S1. This finding indicates that the ATPinduced fluorescence spectral change can be used to detect the alteration of global structure of S1. From these points of view, the fluorescence increment shown in Fig. 1 is possibly caused by global conformational change of S1, which is induced by addition of ATP. The ATP-induced fluorescence increment markedly decreased above 200 MPa. The reduction of ATP-induced
fluorescence increment shown in Fig. 2 suggests that the nucleotide binding ability of pressurized S1 is smaller than that of unpressurized one. Nucleotide binding ability did not change from 0.1 to 200 MPa. The binding was decreased at 250 /300 MPa (Fig. 3). On the other hand, the change in actin binding was also similar to nucleotide binding of S1, which was remarkably decreased at 250/300 MPa (Fig. 4) These findings indicate that the local structural changes in ATP and actin binding sites follow global structural change. A previous report [10] showed that the inactivation of ATPase activity was caused by structural change of active site of head, and the following hydrophobic interaction among heads led to the masking of the active sites, resulting in further loss of ATPase activity [10]. Ca2- and K -EDTA-ATPase activities of pressurized myosin decreased from 100 MPa, and completely inactivated at 300 MPa [10,11]. In the present observation, the ATPase activity decreased from 250 MPa, and the remaining ATPase activity after application of pressure at 300 MPa was approximately 60% (Fig. 6). The discrepancy between the present study and the previous studies [10,11] can be explained by the difference of size of oligomer in the pressurized sample. When myosin was pressurized at relatively low pressure, such as 70 MPa, single headed species were formed through intramolecular head to head interaction [9]. The hydrophobic interaction among myosins caused formation of oligomers at relatively high pressure such as 140 MPa [9]. Oligomers containing over 10 myosin molecules appeared by pressure treatment above 300 MPa, and the formation of oligomer apparently accompanied decrease of ATPase activity [10]. S1 also aggregated by application of pressure [9]. In the previous studies [10,11], ATPase activity was measured using pressurized myosin containing large aggregates. It is probable that the ATPase activity of pressure-induced myosin aggre-
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gates is lower than that of small oligomers such as dimer or trimer. S1 used in this study did not contain large oligomers, so that it retained relatively high ATPase activity even after high pressure treatment such as 300 MPa (Fig. 6). Tryptic digestion of S1 produces 27, 50 and 20 kDa fragments [34]. Setton and Muhlrad [23] and Burke et al. [35] found that S1 lost its ATPase activity at 35 8C. The loss of ATPase activity was accompanied by increase of tryptic susceptibility of the 50 kDa domain of S1. Tryptic fragments of pressurized S1 analyzed by SDSPAGE were essentially the same as those in unpressurized one at least up to 300 MPa, suggesting that the pressure denaturation of S1 was not concomitant with unfolding of 50 kDa fragment. The present results are summarized as follows: (1) pressure treatment at 150 MPa induces global deformation of S1 without losing its biological activities such as ATPase and actin binding, (2) ATPase and actin binding sites lose those intrinsic structures with elevating pressure above 250 MPa. The actin activated Mg2-ATPase activity also decreased above 250 MPa, (3) pressureinduced conformational change in 50 kDa fragment, which is involved in ATPase, is different from that induced by heat.
References [1] Aoki K, Hiramatsu K, Tanaka M, Kaneshina S. Biochim Biophys Acta 1968;160:368. [2] Schmid G, Ludemann H-D, Jaenicke R. Eur J Biochem 1979;97:407. [3] Bridgman PW. J Biol Chem 1914;19:511. [4] Ruan K, Weber G. Biochemistry 1988;27:3295. [5] Ruan K, Weber G. Biochemistry 1989;28:2114.
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Ruan K, Weber G. Biochemistry 1993;32:6295. Foguel D, Weber G. J Biol Chem 1995;270:28759. Cioni P, Strambini GB. J Mol Biol 1996;263:789. Yamamoto K, Hayashi S, Yasui T. Biosci Biotech Biochem 1993;7:383. Yamamoto K, Yoshida Y, Morita J, Yasui T. J Biochem 1994;116:215. Ikeuchi Y, Tanji H, Kim K, Suzuki A. J Agric Food Chem 1992;40:1756. Mahmood R, Yount RG. J Biol Chem 1984;259:12956. Nakamaye KL, Wells JA, Bridenbaugh RL, Okamoto Y, Yount RG. Biochemistry 1985;24:5226. Mornet D, Pantel P, Audemard E, Kassab R. Biochem Biophys Res Commun 1979;89:925. Yamamoto K, Sekine T. J Biochem 1979;86:1869. Offer G, Moos C, Starr R. J Mol Biol 1973;74:653. Weeds AG, Pope B. J Mol Biol 1977;111:129. Spudich JA, Watt S. J Biol Chem 1971;246:4866. Werber MM, Szent-Gyo¨rgyi AG, Fasman G. Biochemistry 1972;11:2872. Ando T, Duke JA, Tonomura Y, Morales MF. Biochem Biophys Res Commun 1982;109:1. Chalovich JM, Eisenberg E. J Biol Chem 1982;257:2432. Yamamoto K, Moos C. J Biol Chem 1983;258:8395. Setton A, Muhlrad A. Arch Biochem Biophys 1984;235:411. Johnson KA, Taylor EW. Biochemistry 1978;17:3432. Trentham DR, Eccleston JF, Bagshaw CR. Q Rev Biophys 1976;9:217. Kishi K, Noda H. J Biochem 1983;93:717. Bachouchi N, Garrigos M, Morel JE. J Mol Biol 1986;191:247. Rayment I, Smith C. Annu Rev Physiol 1996;58:671. Tong SW, Elzinga M. J Biol Chem 1990;265:4893. Papp SJ, Highsmith S. Biochem Biophys Acta 1993;1020:169. Werber MM, Peyser M, Muhlrad A. Biochemistry 1987;26: 2903. Hiratsuka T. J Biol Chem 1992;267:14949. Sugimoto Y, Tokunaga M, Takezawa Y, Ikebe M, Wakabayashi K. Biophys J 1995;68:29S. Balint M, Wolf I, Tarcsafalvi A, Gergely J, Sreter FA. Arch Biochem Biophys 1978;190:793. Burke M, Zaager S, Bliss J. Biochemistry 1987;26:1492.
Effect of high hydrostatic pressure on chicken myosin subfragment-1 Tomohito Iwasaki *, Kastuhiro Yamamoto Department of Food Science, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan Received 27 August 2001; received in revised form 4 March 2002; accepted 8 April 2002
Abstract Hydrostatic pressure-induced structural changes in subfragment-1 (S1) of myosin molecule were studied. ATP-induced emission spectra of S1 were used to detect global structural change of S1 by pressure treatment. The fluorescence intensity of unpressurized S1 increased by addition of ATP. The increment of fluorescence of pressurized S1 up to 150 MPa was almost the same as control, whereas it became smaller above 200 MPa. ATP binding ability of S1 examined using 1, N6-ethenoadenosine 5?-diphosphate (oADP) indicated that the binding of o-ADP to S1 decreased in the range of 250 /300 MPa. S1 pressurized below 250 MPa and unpressurized S1 similarly bound to F-actin, although binding of S1 pressurized above 250 MPa decreased. Electron microscopic observation revealed arrowhead structure in control acto-S1, while disordered arrowhead structure was observed in acto-S1 prepared from pressurized S1 at 300 MPa. S1 pressurized below 250 MPa retained the same actin activated ATPase activity as the control, whereas the activity decreased to 60% at 300 MPa. Pressure treated S1 was easily cleaved by tryptic digestion into three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which were the same as those in unpressurized one. It is concluded that pressure-induced global structural changes of S1 begin to occur about 150 MPa, and the local structural changes in ATPase and actin binding sites followed with elevating pressure to 250 /300 MPa. # 2002 Published by Elsevier Science B.V. Keywords: Subfragment-1; Myosin; Pressure
1. Introduction Application of high hydrostatic pressure on protein induces aggregation [1,2] gelation [3], or subunits dissociation [4 /8]. These pressure-induced change in proteins are thought to be useful for controlling enzyme activity as well as analysis of interaction among protein molecules or subunits. Pressure-induced aggregation and gelation of proteins are caused by disruption and reformation of noncovalent bonds. Myosin is a major protein of myofibril and it consists of two heads and a tail. ATPase and actin binding sites are located in the heads, and the tails self-associate to form thick filament at low ionic strength. These are some reports on pressure-induced denaturation of myosin [9 /11]. Yamamoto et al. [10] reported that
Abbreviations: PMSF, phenylmethylsulfonylflouride; TPCKtrypsin, L-1 tosylamido-2 phenylethyl chloromethyl ketone treated trypsin; o-ADP, 1, N6-ethenoadenosine 5?-diphosphate. * Corresponding author. Tel./fax: /81-11-388-4834 E-mail address: [email protected] (T. Iwasaki).
Ca2- and K -EDTA-activated myosin ATPase activities decreased from 100 MPa. Hydrophobicity of myosin increased from 100 MPa and it reached plateau value at 300 MPa, and the helix content of myosin decreased above 300 MPa [10]. Turbidity of pressurized myosin solution markedly increased within 5 min of pressure treatment at 210 MPa, indicating formation of aggregates. The aggregates were formed by head to head interaction and the shape of aggregate looked like daisywheel [9]. The size of pressure-induced myosin aggregates was dependent on the magnitude of applied pressure and the duration of the treatment. Oligomers containing over 10 myosin molecules were made by pressure treatment at 210 MPa for 30 min [9]. ATPase activity of myosin was apparently related to the size of pressure-induced oligomer. These findings also indicate that the head (subfragment-1, S1) is the most pressuresensitive portion in myosin molecule. S1 has ATPase and actin binding sites, and it consists of three domains, which are 27, 50 and 20 kDa fragments. The nucleotide binding site is located in the junction of the 27 and 50 kDa fragments [12,13], and the actin binding site is located in the 50 and 20 kDa
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fragments junction [14,15]. The detail of pressureinduced changes in actin binding and ATP binding sites of S1 are not well understood. It is important to elucidate alteration of these portions in the myosin molecule for comprehensive understanding of pressureinduced denaturation of myosin. As mentioned above, myosin head (S1) tends to aggregate by pressure treatment. Since the analysis of local structural change in S1 seems to be difficult using pressurized S1 solution containing large aggregates, we removed large aggregates from pressurized S1 by centrifugation and used it in this study.
2. Materials and methods 2.1. Preparation of proteins Myosin was prepared from fresh chicken pectoral muscle according to Offer et al. [16]. S1 was prepared by the method of Weeds and Pope [17] with some modification. Myosin in 0.12 M NaCl, 20 mM Na phosphate (pH 7.0) and 1 mM EDTA was digested with 1/400 (w/w) of a-chymotrypsin at 23 8C for 7 min. Digestion was terminated by addition of 0.1 M phenylmethylsulfonylflouride (PMSF) to a final concentration of 0.5 mM, then the protein solution was dialyzed against 40 mM NaCl and 5 mM Na phosphate (pH 6.5) at 4 8C with three changes of dialysate. S1 was further purified by ammonium sulfate fractionation and Whatman DE-52 column chromatography with a NaCl gradient from 0 to 0.2 M. The unretarded fractions were pooled and precipitated by ammonium sulfate at 58% saturation. Actin was prepared from rabbit skeletal muscle according to Spudich and Watt [18]. 2.2. Application of hydrostatic pressure Two milliliters of S1 (1.7 /25 mM) solution containing 0.05 /0.2 M NaCl and 10 mM Tris /HCl (pH 7.0) was put into a plastic tube, then the tube was firmly sealed with a plug without trapping of any air. The sample tube was placed in a pressure vessel, having a diameter of 30 mm and a length of 100 mm, filled with water. Hydrostatic pressure was generated by pumping water into the vessel with a hydraulic machine. Application of pressure was performed at 20 8C for 10 min. After pressure release, the pressurized S1 solution was ultracentrifuged at 194 000/g for 60 min. S1 in the supernatant was used in the present study. 2.3. Fluorescence measurements Fluorescence spectra were recorded with a Hitachi F2000 spectrofluorometer. The tryptophan fluorescence of S1 was measured in 0.2 M NaCl and 20 mM Na
phosphate (pH 7.0) at 20 8C with excitation wavelength of 295 nm according to Werber et al. [19]. After the emission spectra were recorded, ATP was added to give a final concentration of 1 mM, and the spectra were recorded again. Fluorescence intensity at the peak of the spectrum (334 nm) was used to calculate the increment of ATP-induced fluorescence (DF ) which was expressed as percentages of the fluorescence intensity obtained the unpressurized S1 by the following equation; DF (%) [(FATP FATP ) pressurized]=[(FATP FATP ) unpressurized]100 here, FATP and F ATP are the fluorescence intensities at 334 nm before and after addition of ATP, respectively. The fluorescence of 1, N6-ethenoadenosine 5?-diphosphate (o-ADP) was measured at 20 8C with the excitation wavelength of 320 nm and the emission wavelength of 410 nm. The interaction of o-ADP with S1 was estimated using quenching of the fluorescence of the free nucleotide induced by acrylamide as described by Ando et al. [20]. o-ADP was added to 4 mM S1 in 200 mM acrylamide, 2 mM MgCl2 5 mM KCl, and 40 mM Tris /HCl (pH 7.7). In control experiment, the fluorescence of o-ADP was measured under the same conditions without S1. The ATP binding ability of pressurized S1 was estimated from the difference between fluorescences of pressurized and unpressurized S1. 2.4. Binding experiments The binding of pressurized S1 to F-actin was measured as described Chalovich and Eisenberg [21], and Yamamoto and Moos [22]. Centrifugation was performed at 135 000 /g for 10 min in a Beckman Airfuge with a type A-100 rotor. The supernatant, in which unbound S1 remained, was collected and the concentration was measured by UV absorbance. 2.5. ActoS1 ATPase measurements Actin activated S1 ATPase activity was measured with a pH-stat at pH 7.0 and 25 8C in a reaction mixture containing 0.02 M NaCl, 0.5 mM ATP and 2 mM MgC12. The protein concentrations of S1 and Factin were 0.38 and 1.6 mM, respectively. Titration was performed with 20 mM KOH. 2.6. Electron microscopy A drop of acto /S1 (0.1 /0.2 mg/ml) was applied to a carbon-coated grid which had been glow-discharged just before use. The grid was negatively stained with 2% aqueous uranyl acetate. The specimens were observed in a Hitachi H-800 electron microscope at an accelerating
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voltage of 75 kV. Images were recorded at a magnification of 40 000. 2.7. SDS-PAGE of tryptic digested fragment Pressurized S1 (8.33 mM) was digested with 1/400 (w/ w) of L-1 tosylamido-2 phenylethyl chloromethyl ketone treated trypsin (TPCK-trypsin) in 0.1 M NaCl and 10 mM Tris /HCl (pH 7.0) at 20 8C for 5 /30 min. Digestion was terminated by heat treatment at 100 8C. Electrophoresis of the digests was performed on a 7.5% polyacrylamide gel [22].
Fig. 2. The ATP-induced fluorescence increment of S1 after pressure treatment. Relative fluorescence intensity at 334 nm was calculated as described under Section 2.
3. Results 3.1. The ATP-induced tryptophan fluorescence lt is known that the structure of S1 is changed by binding of ATP [19]. Fig. 1 shows the fluorescence spectrum of unpressurized S1 (a) and pressurized S1 at 300 MPa (b) before and after addition of ATP. The fluorescence intensity of unpressurized S1 with ATP was higher than that without ATP. On the other hand, there was no difference in fluorescence spectrum of pressurized S1 before and after addition of ATP. In order to estimate pressure effect on S1 structure, we calculated the ATP-induced fluorescence increment (DF ) with varying pressure (Fig. 2). The ATP-induced fluorescence increments did not change from 0.1 to 150 MPa. However, DF linearly decreased above 200 MPa. The differential spectra of ATP-induced fluorescence intensity showed negative value, since the fluorescence intensity of S1 after addition of ATP was lower than that of S1 before addition of ATP at 300 MPa. These results suggest pressure-induced global structural change in S1.
Fig. 1. Emission spectra of S1 before and after addition of ATP. One micromole of S1 in 0.2 M NaCl and 10 mM Tris-HCl (pH 7.0) was pressurized at 300 MPa, then centrifuged at 194 000/g for 60 min. The supernatant was collected, and ATP was added to 1 mM. (a) unpressurized S1 (0.1 MPa); (b) pressurized S1. Excitation wavelength was 295 nm. Solid and dotted lines indicate the spectra before and after addition of ATP, respectively.
We investigated pressure effect on the ATP binding site of S1 by fluorescence quenching. Ando et al. [20] developed a method of measuring nucleotide binding to S1. They found that the fluorescence of free o-nucleotides was quenched by acrylamide, while the quenching was prevented when the o-nucleotides bound to S1. We titrated pressure treated S1 with o-ADP in the presence of acrylamide using this method (Fig. 3). The intensity of the acrylamide nonquenchable fluorescence, i.e. the amount of o-ADP bound to S1, decreased with elevating pressure. The amount of o-ADP bound to S1 did not change up to 200 MPa, while it decreased above 250 MPa. The intensity of ATP-induced fluorescence decreased above 200 MPa (Fig. 2), while binding of o-ADP to S1 decreased above 250 MPa (Fig. 3). These results may explain the difference between global and local structural change in S1.
Fig. 3. Effect of pressure treatment on the binding of o-ADP to S1. oADP was added for give a final concentration of 8 mM, then pressurized 1 mM S1 in 0.1 M NaCl, 200 mM acrylamide, 2 mM MgC12, 20 mM Na phosphate (pH 7.0), and its fluorescence was recorded as described in Section 2. The bound o-ADP was calculated from the fluorescence intensity at 410 nm before and after addition oADP.
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3.4. Changes in actin activated ATPase activity of pressurized S1
Fig. 4. Effect of pressure treatment of S1 on the binding to F-actin. Factin was incubated with pressurized S1 in 50 mM NaCl, 5 mM Na phosphate (pH 7.0), and 1 mM MgCl2 for 20 min at room temperature, then centrifuged at 135 000/g for 10 min. Protein concentration of the supernatant (unbound S1) was measured by UV absorbance at 280 nm. (m) 5 mM S1; (k) 2 mM S1. Actin concentration was 5 mM.
3.2. Pressure effect on actin binding site of S1 The effect of pressure on binding ability of S1 to Factin was studied (Fig. 4). S1 binding to F-actin causes formation of acto-S1, which can be sedimented by ultracentrifuge. Bound S1 was calculated by subtracting unbound S1 from total S1. The binding did not change up to 200 MPa, and it decreased at 300 MPa though S1 still retains 90% of actin-binding ability at this pressure. These findings indicate structural change in actin binding site of S1 by pressure at 300 MPa.
We measured actin activated S1 ATPase activity to investigate the interaction between F-actin and pressurized S1. Fig. 6 shows pressure effect of S1 on actinactivated S1 ATPase activity. The activity remained at almost the same level as an unpressurized S1 at least up to 250 MPa, but the activity was remarkably decreased at 300 MPa. The diminution of ATPase activity of pressurized S1 is consistent with decreasing actin binding ability of pressurized one shown in Fig. 4, though pressure treated S1 at 300 MPa still retains ATPase activity of about 0.43 mmol/min/mg. Since the conformational change of S1 was induced by pressure, the interaction between pressurized S1 and F-actin was weak. 3.5. Tryptic digestion of pressure treated S1 S1 contains three domains, i.e. 27 kDa (N-terminal), 50 and 20 kDa (C-terminal) fragments, which are easily produced by tryptic digestion of S1. The ATPase site is located in the 27 and 50 kDa fragments, and the actin binding site is in the 50 and 20 kDa fragments. Fig. 7 shows the SDS-PAGE pattern of tryptic fragments of S1
3.3. Morphology of acto-S1 complex Fig. 5 shows morphology of acto-S1. A typical arrowhead structure was observed in the control actoS1 complex which was prepared from unpressurized S1 and F-actin. When the pressurized S1 at 300 MPa was mixed with F-actin, the formed acto-S1 showed disordered arrowhead structure. The morphological observations support overall conformational alteration of S1 by pressure treatment.
Fig. 6. Effect of pressure treatment of S1 on acto-S1 ATPase activity. Acto-S1 ATPase activity was assayed in 20 mM NaCl, 0.5 mM ATP, and 2 mM MgCl2 with 0.38 mM S1 and 1.6 mM actin at pH 7.0 and 25 8C.
Fig. 5. Transmission electron micrographs of acto-S1. (a) control; (b) acto-S1 formed from pressurized S1 at 300 MPa for 10 min and unpressurized F-actin. Scale bar indicates 0.2 mm.
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Fig. 7. SDS-PAGE of tryptic digested S1. Pressure-treated S1 in 0.1 M NaCl and 10 mM Tris /HCl (pH 7.0) was digested with 1/400 (w/w) of trypsin at 20 8C for 5 /30 min. The reaction was terminated by heat treatment at 100 8C. Polyacrylamide gel concentration was 7.5%. Control denotes undigested S1. HC and LC denote S1 heavy chain and light chain, respectively. (a) unpressurized S1, (b), (c) and (d); pressurized S1 at 100, 200 and 300 MPa, respectively.
after pressure treatment. These tryptic fragments of S1 obtained from 100 to 300 MPa were the same as that of unpressurized one. Setton and Muhlrad [23] showed disappearance of tryptic 50 kDa fragment accompanying loss ATPase activity in the mild heat treated S1. The band of 50 kDa fragment in a gel was observed in pressurized S1 regardless of degree of pressure, although the Mg2-ATPase activity of S1 was decreased after pressure treatment at 300 MPa (Fig. 6).
4. Discussion The tryptophan fluorescence intensities of myosin and its proteolytic fragments are useful to investigate structural change induced by binding of nucleotides [19,24/27], though it is difficult to interpret S1 fluorescence data in terms of local sites structure[28]. S1 has five Trp residues in its heavy chain [29]. ATP-induced fluorescence increase is caused by only one or two tryptophan residues [19,30], which are located in the 50 kDa tryptic fragment [31]. Trp510 especially plays an important role in the fluorescence change of S1 [30,32]. Sugimoto et al. [33] showed that the global conformational change of skeletal muscle S1 in the presence of MgATP was caused by a hinge-like bending movement between the catalytic and regulatory domains, and the global change of S1 was correlated with closing of ATP binding pocket by cross-linking of 50 and 20 kDa fragments in S1. This finding indicates that the ATPinduced fluorescence spectral change can be used to detect the alteration of global structure of S1. From these points of view, the fluorescence increment shown in Fig. 1 is possibly caused by global conformational change of S1, which is induced by addition of ATP. The ATP-induced fluorescence increment markedly decreased above 200 MPa. The reduction of ATP-induced
fluorescence increment shown in Fig. 2 suggests that the nucleotide binding ability of pressurized S1 is smaller than that of unpressurized one. Nucleotide binding ability did not change from 0.1 to 200 MPa. The binding was decreased at 250 /300 MPa (Fig. 3). On the other hand, the change in actin binding was also similar to nucleotide binding of S1, which was remarkably decreased at 250/300 MPa (Fig. 4) These findings indicate that the local structural changes in ATP and actin binding sites follow global structural change. A previous report [10] showed that the inactivation of ATPase activity was caused by structural change of active site of head, and the following hydrophobic interaction among heads led to the masking of the active sites, resulting in further loss of ATPase activity [10]. Ca2- and K -EDTA-ATPase activities of pressurized myosin decreased from 100 MPa, and completely inactivated at 300 MPa [10,11]. In the present observation, the ATPase activity decreased from 250 MPa, and the remaining ATPase activity after application of pressure at 300 MPa was approximately 60% (Fig. 6). The discrepancy between the present study and the previous studies [10,11] can be explained by the difference of size of oligomer in the pressurized sample. When myosin was pressurized at relatively low pressure, such as 70 MPa, single headed species were formed through intramolecular head to head interaction [9]. The hydrophobic interaction among myosins caused formation of oligomers at relatively high pressure such as 140 MPa [9]. Oligomers containing over 10 myosin molecules appeared by pressure treatment above 300 MPa, and the formation of oligomer apparently accompanied decrease of ATPase activity [10]. S1 also aggregated by application of pressure [9]. In the previous studies [10,11], ATPase activity was measured using pressurized myosin containing large aggregates. It is probable that the ATPase activity of pressure-induced myosin aggre-
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gates is lower than that of small oligomers such as dimer or trimer. S1 used in this study did not contain large oligomers, so that it retained relatively high ATPase activity even after high pressure treatment such as 300 MPa (Fig. 6). Tryptic digestion of S1 produces 27, 50 and 20 kDa fragments [34]. Setton and Muhlrad [23] and Burke et al. [35] found that S1 lost its ATPase activity at 35 8C. The loss of ATPase activity was accompanied by increase of tryptic susceptibility of the 50 kDa domain of S1. Tryptic fragments of pressurized S1 analyzed by SDSPAGE were essentially the same as those in unpressurized one at least up to 300 MPa, suggesting that the pressure denaturation of S1 was not concomitant with unfolding of 50 kDa fragment. The present results are summarized as follows: (1) pressure treatment at 150 MPa induces global deformation of S1 without losing its biological activities such as ATPase and actin binding, (2) ATPase and actin binding sites lose those intrinsic structures with elevating pressure above 250 MPa. The actin activated Mg2-ATPase activity also decreased above 250 MPa, (3) pressureinduced conformational change in 50 kDa fragment, which is involved in ATPase, is different from that induced by heat.
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