Báo cáo Y học: High pressure-induced changes of biological membrane Study on the membrane-bound Na+/K+-ATPase as a model system pdf

High pressure-induced changes of biological membrane Study on the membrane-bound Na + /K + -ATPase as a model system Michiko Kato 1 , Rikimaru Hayashi 1 , Takeo Tsuda 2 and Kazuya Taniguchi 2 1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Japan; 2 Biological Chemistry, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan In order to study the pressure-in duced changes of biological membrane , hydros tatic pr essure s of from 0.1 t o 400 MPa were applied to membrane-bound Na + /K + -ATPase f rom pig k idney as a model system o f protein and lipid membrane. The a ctivity showed at least a three-step change induced by pressures of 0.1±100 MPa, 100±220 MPa, and 220 MPa or higher. At p ressures o f 100 MPa or lower a decrease in t he ¯uidity of lipid bilayer a nd a reversible conformational change in transmembrane protein is induced, leading to the functional disorder o f membrane-associated ATPase activ- ity. A pressure of 100±220 M Pa causes a reversible phase transition in parts of the lipid bilayer from the liquid crys- talline to the gel phase and the dissociation of and/or con- formational c hanges in the protein subunits. T hese changes could cause a separation o f t he interface between a and b subunits and b etween protein a nd the lipid bilayer to c reate transmembrane tunnels at the interface. Tunnels would be ®lled with w ater from the aqueous environment a nd tak e up tritiated w ater. A pressure of 220 MPa or higher irreve rsibly destroys and fragments the gross membrane structure, due to protein unfolding and i nterface separation, which is am- pli®ed by the i ncreased pressure. T hese ®ndings p rovide an explanation for the high p ressure-induced membrane-dam- age t o subcellular organelles. Keywords: hydrostatic pressure; membrane; Na + /K + - ATPase; h ydrogen±tritium exchange. The high hydrostatic pressure treatment of microbial cells, as well as plant and animal tissues at 100±400 MPa solubilizes cellular components, such as metals, amino acids, a nd proteins [ 1±5], permeates extracellular c om- pounds, such as s alts, into cells and tissues [6,7], and lead to hemolysis [8], in t he case of animal tissue. After pressure treatment, electron m icroscopic observation o f yeast cells and b iochemical analysis of animal tissues have shown that membranes of nuclei [9], lysosomes [4,5], and vacuoles [9] undergo signi®cant disrupting, in addition to a small amount of damage to cell membranes and cell walls. In order to understand these morphological and biochemical phenomena, studies of functional and structural changes of biological membranes, as induced by high pressure (in situ observation), are in order. In the present study, a membrane-bound Na + /K + - ATPase from pig kidney was used for this purpo se. The enzyme consists of two subunits, an a subunit ( M r 94 000± 120 000) and a b subunit (M r 40 000±57 000). The former contains the catalytic center r equired for ATP hydrolysis [10]. I n terms of gross structure, approximately t wo-thirds o f the total enzyme protein (water soluble domain) protrudes from the lipid bilayer and is in contact with the aqueous environment, while the remainder (transmembrane seg- ment) is surrounded by the lipid bilayer [ 11,12]. In the overall reaction, the enzyme t ransports sodium out of the cell and potassium into the cell [10] according to the Post±Albers mechanism a s f ollows: t he Na + -bound enzyme (Na + -dependent ATPase) is phosphorylated by ATP in the presence of Mg 2+ and f orms ADP-sensitive phospho- enzyme followe d by releasing Na + to form the K + -sensitive phospho-enzyme. This is dephosphorylated to form the K + -bound enzym e (K + -activated phosphatase) and ®nally the K + -bound enzyme accepts Na + and releases K + . Na + /K + -ATPase undergoes conformational changes during the reaction [11,13±16]. To d etect such conforma- tional changes, t he enzyme is labeled with ¯uo rescence probes, such as N-(p-(2-ben zimidazolyl)phenyl)maleimide (BIPM) [17] and ¯uorescein 5 ¢-isothiocyanate (FITC) [ 18]. The former b inds to Cys964, which is located in the transmembrane s egment [17], and the latter b inds to Lys501, which is located in the water-soluble domain [19±21]. Moderately high pressures of u p to 200 MPa suppress Na + /K + -ATPase activity b y d ecreasing m embrane ¯uidity, which, in turn, hinders conformational transitions of the protein [22,23]. These pressures also appear to dissociate and/or unfold protein subunits of the enzyme [ 24]. In this study, p ressure-induced functional and structural changes o f the membrane-bound Na + /K + -ATPase from pig k idney have been studied in detail as a model system of Correspondence to M. Kato, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Fax: + 81 75 75 3 6128, Tel.: + 81 75 753 6495, E-mail: m k@kais.kyoto-u.ac.jp Abbreviations: FITC, ¯uorescein 5¢-isothiocyanate; BF/F, th e r atio of FITC ¯uorescence emitted at the excitation wavelength for BIPM (305 nm) to FITC ¯uorescence emitted at the excitation wavelength for FITC (470 nm); BIPM, N-(p-(2-benzimidazolyl)phenyl) maleimide; C 12 E 8 , octa-ethyleneglycol mono n-dodecyl ether; H±T exchange, hydrogen±tritium exchange; pNPP, p-nitrophenylphos- phate; soy-PtdCho, soybean phosphatidylcholine. (Received 2 July 2001, revised 28 September 2001, accepted 2 3 October 2001) Eur. J. Biochem. 269, 110±118 (2002) Ó FEBS 2002 the cell membrane consisting of lipid bilayer and trans- membrane proteins. EXPERIMENTAL PROCEDURES Materials ATP (Tris salt), pNPP (Tris salt), egg albumin and soy- PtdCho were obtained from Sigma. Rabbit muscle pyruvate kinase (a s alt-free lyophilized p owder of speci®c activity, 100±200 Uámg )1 ), bovine pancreatic trypsin (N-a-tosyl- L - phenylalanylchloromethane treated, speci®c activity, 7 000± 13 000 Uámg )1 for N-a-benzoyl- L -arginine ethylester), and NADH were purchased from Nacalai T esque (Kyoto, Japan). Chicken he art l actate dehydroge nase (lyophiliz ed powder of speci®c activity, 300 Uámg )1 or higher) and phosphoenolpyruvate were obtained from Wako Pure Chemicals ( Osaka, Japan). C 12 E 8 was purchased from Nikko Chemicals (Tokyo, Japan). Tritiated water (37 M BqámL )1 ) was purchased from Dupont. P ig kidney Na + /K + -ATPase (speci®c activity, 1240 lmolámg pro- tein )1 áh )1 ), BIPM- [17] and FITC- [18] labeled enzymes, and BIPM/FITC doubly l abeled enzyme were prepared by Taniguchi, Graduate School of Science, Hokkaido Univer- sity, according t o the method of Jùrgensen [11]. The buffer used in the high pressure experiments were 40 m M Tris/HCl, pH 7.4, to minimize the pressure-induced pH changes [25]. In situ spectroscopic measurements under high pressure Absorbance and ¯uorescence under high pressure were measured using a Shimadzu UV-2500PC spectrophotome- ter (Kyoto, Japan) and a Shimadzu RF-5300PC s pectro- ¯uorometer, respectively, equipped with a high pressure photometer cell (Type PCI-400SRF; Teramex Co., Kyoto, Japan), which contains three s apphire windows. The enzyme was added to the reaction mixture, which had been previously incubated at ambient pressure and at 37 °C, and a portion of the m ixture was placed in the high pressure photometer cell, which was maintained at 37 °Cby circulating water. Immediately a fter the cell was sealed, pressure was applied with a hand-type pump (Type TP-500, Teramex Co.) using water as the pressure medium. Typi- cally, after 90 s (required to a ttain the des ired pressure), spectroscopic changes were recorded. Activity measurements Three activities of N a + /K + -ATPase were determined a s follows. Na + /K + -ATPase activity was measured by mon- itoring the decrease in NADH absorbance at 340 nm (e 340  6.2 2 ´ 10 3 M )1 ácm )1 ) using a coupled enzyme assay method [26]. The assay mixture contained 40 m M Tris/HCl, pH 7.4, 160 m M NaCl, 16 m M KCl, 25 m M sucrose, 5 m M MgCl 2 ,0.1m M EDTA, 4 m M ATP-Tris, 1 m M phosphoenolpyruvate, 0.3 m M NADH, 5 lgámL )1 of p yru- vate k inase, and 2.5 lgámL )1 LDH i n a total v olume o f 2 m L. The reaction was initiated by an addition of 50 ng of Na + /K + -ATPase. Na + -dependent ATPase activity was also measured u sing the coupled enzyme assay meth- od as described above except that 4 m M ATP-Tris and 16 m M KCl was replaced with 100 l M ATP-Tris and 16 m M choline chloride, respectively. It was already shown that high concentration of Na + activates pyruvate kinase without K + [27]. The reaction was initiated by an addition of 1 lgenzyme.K + -activated phosphatase activity was determined as K + -dependent pNPPase activity by measur- ing the amount of p-nitrophenol (e 420  1.33 ´ 10 4 M )1 ácm )1 ) released from pNPP. The assay mixture contained 40 m M Tris/HCl, pH 7.4, 4 m M pNPP-Tris, 16 m M KCl, 25 m M sucrose, 4 m M MgCl 2 ,0.1m M EDTA in a total volume of 2 mL. The reaction was initiated by the addition of 2 lgenzyme. Fluorescence measurements Intrinsic ¯uorescence was monitored at 300±400 nm with an excitation at 280 nm. The sa mple solution contained 10 lg of Na + /K + -ATPase, 16 m M KCl and/or 16 m M NaCl, 25 m M Tris/HCl, pH 7.4, 0.43 m M MgCl 2 ,25m M sucrose, and 0.1 m M EDTA in a total volume of 2 mL. BIPM and FITC ¯uorescences were monitored a t 330±560 nm with an excitation at 305 nm and at 500±560 nm with an excitation at 470 nm, respectively. The sample solution contained 10 lg of BIPM- or FITC-labeled enzyme, 160 m M NaCl or 16 m M KCl, 25 m M Tris/HCl, pH 7.4, 0.43 m M MgCl 2 , 25 m M sucrose, a nd 0.1 m M EDTA in a total volume of 2 m L. Fluorescence energy transfer was determined by measuring the ratio of FITC ¯uorescence emitted as a result of excitation at 305 nm to the FITC ¯uorescence emitted by the excitation at 470 nm (BF/F). The sample solution contained 10 lg of BIPM/FITC doubly labeled enzyme and the other compounds as described above. All ¯uorescence emission spectra were recorded after the sample solutions had been maintained at various pressures and 37 °Cfor 15 min. Preparation of solubilized Na + /K + -ATPase Two milligrams per milliliter Na + /K + -ATPase was incu- bated i n the solution containing 6 mgámL )1 C 12 E 8 ,0.05 M KCl, 2 m M dithioerythritol, 10% (w/v) glycerol, 13 m M imidazole, and 8 m M Hepes, pH 7.0, in a total volume of 2mLat0°C for 5 min [28]. The solution was centrifuged at 170 000 g and 0 °C for 20 min. The supernatant was collected and i s hereafter re ferred t o as t he solubilized Na + /K + -ATPase,whichretained30%oftheoriginal activity. Tritium±hydrogen exchange Twenty microliters (100 lg) of Na + /K + -ATPase were mixedwith100lL of tritiated water (3.7 MBqámL )1 )and 120 lLof40m M Tris/HCl buffer, pH 7.5, and a 240-lL aliquot of the mixture was p laced in a small plastic tube (4 mm internal diameter ´ 19 mm) and the tube covered with a polyethylene-based stretch ®lm obtained f rom T oho Co. (Tokyo, Japan), after carefully removing the air bubble in the headspace. The sealed tube was placed in the h igh- pressure vessel and pressurized at 37 °Cfor15min.After the release of the pressure, a 200-lL aliquot of the mixture was ®ltered with a Millipore ®ltration system with a ®lter (diameter: 25 mm) and a ®lter membrane (pore size: 0.45 lm). The ®lter membrane was washed ®ve times with 500 lLof40m M Tris/HCl buffer, pH 7.5, which had previously been warmed to 37 °C and then transferred into Ó FEBS 2002 High pressure eects on biological membrane (Eur. J. Biochem. 269) 111 a disposable scintillation vial with 3 mL of scintillator (Clear-sol I, Nacalai Tesque, Japan). The radioactivity of the tritium was m easured using an LKB liquid scintillation counter (Bromma, Sweden). One hundred microliters of albumin (1 mgámL )1 )or 100 lLof1m M soy-PtdCho liposome (see b elow), 100 lL of tritiated water (3.7 MBqámL )1 ), and 40 lLof40m M Tris/HCl buffer, pH 7.5, were mixed and pressurized at 37 °C for 15 min. A 200-lL aliquot of the mixture was then ®ltered with a Millipore centrifugal ®lter unit. The ®lter membrane was w ashed a nd its tritium activity meas ured as described above. Tryptic digestion After the enzym e was pressurized in the buffer containing tritiated water at 200 MPa and 37 °Cfor15minas described above, a 200-lL aliquot of the solution containing 83 lg o f enzyme was incubated with 1 0 lgoftrypsinat 37 °C. At appropriate intervals, an aliquot of the mixture was removed, ®ltered and washed. The tritium activity on the ® lter membrane was measured as described above. To con®rm the tryptic digestion, 83 lg o f F ITC/BIPM doubly labeled ATPase were d igested with 10 lg of trypsin and the amounts of FITC o r B IPM released f rom the pr otein w ere determined ¯uorometrically. Preparation of liposome Soy-PtdCho (3.08 mg) was dissolved in a small amount of a 1 : 4 (v/v) mixture of methanol and chloroform in a test tube and the solvent was then evaporated with nitrogen gas ¯ushing. The ®lm of soy-PtdCho which formed on the inner wall of the test tube was mixed with 4 mL of 50 m M Tris/HCl buffer, pH 7.5, by a Vortex mixer and s ubjected to ultrasonication. RESULTS Effects of pressure on Na + /K + -ATPase, Na + -dependent ATPase and K + -activated phosphatase activities. It could be con®rmed that the Na + /K + -ATPase activity, as determined by the c oupled assay system, increased linearly with an increase in the incubation time and enzyme concentration under a pressure of 300 MPa or lower for 20 min. Thus, the present coupled assay system i s judged to correctly re¯ect Na + /K + -ATPase activity under the pres- sures used in this study. Relationships between pressure dependencies a nd the three activities a re shown in F ig. 1. The Na + /K + -ATPase activity decreased with increasing applied hydrostatic pres- sure, showing a three-step change at 0.1±100, 100±220, a nd 220±300 MPa ( Fig. 1, open circles). Clearly t his i s not a two-state transition, which has been observed in the case of general protein denaturation [29]. The large activity decrease in the ®rst step is consistent with ®nding reported by Chong [22] and De S medt [23], and are probably due to a decrease in the ¯uidity of the lipid bilayer membrane [22]. The reduced activity in the s econd step was nearly independent of pressure change, followed by a large activity decrease in the third step, with a complete loss of the activity at 300 M Pa. The enzymatic activity was completely recovered after a 1-h incubation at 0.1 MPa after pressurization up to 220 MPa for 15 min but activity was not recovered after the same incubation for the case of pressurization at 220± 300 M Pa (Fig. 1, closed circles). Na + -dependent ATPase activity showed almost no change at 0.1±100 MPa , but decreased with i ncreasing pressure of 100±300 MPa (Fig. 1, closed triangles). K + - activated phosphatase activity decreased gradually with increasing pressure up to 300 MPa ( Fig. 1, open triangles). As the different pressure-dependency of Na + -dependent ATPase and K + -activated phosphatase may be related to the l oose o r t ight association of t he diprotomer [28], pressure effects on these two r eactions hold promise for use in m ethodology in discriminating conformational changes. Effects of pressure on intrinsic ¯uorescence The emission maximum (k max ) and intensity of intrinsic ¯uorescence of Na + /K + -ATPase under increasing pressures are s hown in F ig. 2A. The value for k max was shifted from 341 to 345 nm with increasing pressure, up t o 220 M Pa, and then decreased f or pressures of 220 MPa or higher, being independent on ligands, Na + and/or K + . However, the ¯uorescence intensity showed a small decrease up to 220 M Pa, followed by a large decrease at 220 MPa or higher, and was independent on ligands. In general, it appears t hat the red-shift of k max ,whichis accompanied b y a decrease in ¯uorescence intensity re¯ects environmental changes of the a romatic residues in going from a hydrophobic to hydrophilic environment [30,31]. ThisisthecaseforthepresentNa + /K + -ATPase at pressures of 220 MPa or lower. However, the parallel decrease in the k max and the intensity at 220 MPa or higher re¯ects changes in the lipid bilayer, in addition to protein conformational changes (see Discussion). Fig. 1. Eects of pressure on Na + /K + -ATPase (s), K + -activated phosphatase (n), and Na + -dependent ATPase activities (m). Each activity was measured a t various pressures and 37 °C as described in Experimental procedures. Spe ci®c activities of Na + /K + -ATPase, K + -activated phosphatase , and Na + -dependent ATPase at 0.1 MPa were 1240, 110, and 29 lmoláh )1 ámg )1 , respectively. Na + /K + -ATPase activity was determined 1 h later after the pressurization at designated pressures for 15 min (d). Based on three independent experiments, the means and standard deviations are shown by error bars. Error bars are included in the symbols. 112 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Pressure effects on the ¯uorescence properties o f t he solubilized-enzyme are shown in Fig. 2 B. With an increase in pressure up to 400 MPa, k max shifted from 334 to 340 nm with a small decrease in ¯uorescence intensity. This indicates that aromatic amino-acid residues located inside the protein molecule are exposed to an aqueous environment by the pressure-induced unfolding, as has been previously report- ed. Parallel changes up to 220 MPa in Fig. 2 A,B show similar pressure effects on both the membrane-bound and the solubilized forms. Effects of pressure on ¯uorescence probes Pressure effects on the ¯uorescence of the BIPM- and FITC- labeled enzymes ar e shown in F ig. 3. T he BIPM ¯uores- cence intensity of K + -activated phosphatase (Fig. 3A) was higher by 5% t han that of Na + -dependent ATPase (Fig. 3B) at 0.1 M Pa as previously reported [32] and was little changed by up to 220 M Pa, followed by a signi®cant decrease with further increases in pressure. The BIPM ¯uorescence intensity of Na + -dependent ATPase increased somewhat with increasing pressure up to 100 MPa, and was nearly independently changed up to 220 MPa, followed by a signi®cant decrease with an increase in pressure (Fig. 3B). This change parallels the BIPM ¯uorescence of K + - activated phosphatase. It is of interest that the difference in BIPM ¯uorescence intensity of Na + -dependent ATPase and K + -activated phosphatase at 0.1 MPa disappeared at pressures in the range of 100±220 MPa. This may re¯ect conformational differences of enzyme in the presence of Na + or K + . The FITC ¯uorescence intensity of K + -activated phos- phatase decreased somewhat, up to 100 MPa and then increased with increasing p ressure from 100 to 220 MPa, Fig. 2. Eect of pressure on the intensity and k max of intrinsic ¯uores- cence of membrane-bound Na + /K + -ATPase (A) and solubilized Na + / K + -ATPase (B). Ten m icrograms of enzyme were suspended in 2 mL of a solution containing 16 m M KCl, and/or 16 m M NaCl, 25 m M Tris/ HCl, 0.43 m M MgCl 2 ,25m M sucrose, and 0.1 m M EDTA (pH 7.4) and ¯uorescenc e of N a + /K + -ATPase (circles), Na + -dependent ATPase (triangles) and K + -activated phosphatase (squares) were measured under pressure at 37 °C. Fluorescence intens it y was shown by taking the value at 0.1 MPa as 100%. Open and closed symbols show ¯uorescence intensity and k max , respectively. See Experim ental procedures for details. Fig. 3. Eect of pressure on BIPM and FITC ¯uorescences of BIPM- and FITC-labeled Na + /K + -ATPase. TenmicrogramsoftheBIPM-orFITC- labeled enzyme were suspended in 2 mL of a solution containing 160 m M NaCl or 16 m M KCl, 25 m M Tris/HCl, 0.43 m M MgCl 2 ,25m M sucrose, and 0.1 m M EDTA (pH 7.4). Fluorescences of BIPM (A and B) and FITC (C and D) were measured with K + -activated phosphatase (A and C) and Na + -dependent ATPase (B and D) under pressures at 37 °C(d). The BIPM ¯u orescence wa s detecte d at 361 nm with excitation at 3 05 n m and the FITC ¯uorescence we re detected at 520 nm with excitation at 470 nm. After r elease of the pressure , the pressurized s amples were maintained at 37 °C for 20 m in and then ¯uorescence due to BIPM (A and B) and FITC (C and D) was determined (s). Fluorescence intensity of K + -activated phosphatase at 0.1 MPa for BIPM and FITC was taken as 100%. Based on three i ndependent experiments, the means and s tandard deviations a re shown by error bars. Ó FEBS 2002 High pressure eects on biological membrane (Eur. J. Biochem. 269) 113 followed by a decrease with further increase in pressure (Fig. 3 C). The FITC ¯uorescence intensity of Na + -depen- dent ATPase (Fig. 3D), which was higher by 20% than that of K + -activated phosphatase (Fig. 3C) a t 0.1 M Pa [18,33] showed no change with increasing pressures up to 220 MPa, but signi®cantly decreased for pressures of 220 MPa or higher (Fig. 3D). The FITC ¯uorescence of K + -activated phosphatase and N a + -dependent ATPase at 100±220 MPa is different but decreased in parallel a t 220 MPa or higher. The difference may be explained by the loose or tight association of the diprotomer. The pressure-dependency for the ¯uorescence intensity of Na + /K + -ATPase paralleled that of K + -activated phos- phatase throughout all p ressures applied ( data not shown ). These results can be simply explained by assuming that the hydrophobic environment of BIPM becomes more hydrophobic at pressures of up to 100 MPa and then hydrophilic at 100 M Pa or higher and FITC becomes hydrophilic with increasing pressure. To examine reversibility o f pressure-induced ¯uorescence changes, BIPM and FITC ¯uorescence intensities were measured after p ressure treatments a t various pressures for 15 min (Fig. 3, open circles). As seen in Fig. 3A,B, BIPM ¯uorescence of both K + -activated phosphatase and Na + - dependent ATPase were nearly completely recovered after pressure treatment up to 400 MPa, while as shown in Fig. 3C,D, the FITC ¯uorescence of the two enzymes showed a complete rec overy for pressure treatments up to 220 M Pa but an incomplete recovery for the case of pressure treatments of 220 MPa o r higher. The results shown in F ig. 3 indicate that a pressure of 220 M Pa or below affects structures o f both t he transmem- brane segment and the soluble domain in a reversible manner and at pressures o f 220 MPa or higher a phase transition of lipid bilayer is i nduced and a conformational change in the transmembrane segment occurs in a reversible manner, while it induces a conformational change i n the soluble domain in an irreversible manner. It is possible, however, that the reversible change in BIPM ¯uorescence a t 220 M Pa or higher may re¯ect a complex mixture of disrupted membrane fragments and unfolded protein (see Discussion). Effect of pressure on ¯uorescence energy transfer In order to estimate the distance between Cys964 and Lys501, the energy transfer from BIPM to FITC (BF/F) was measured as the ratio of FITC ¯uorescence emitted at the excitation wavelength for BIPM (305 nm) to FITC ¯uorescence emitted a t the excitation wavelength for FITC (470 nm). Pressure-induced changes in BF/F are shown in Fig. 4. The pressure dependence of BF/F for the Na + /K + - ATPase in the p resence of Na + or K + were in parallel, resulting in a concave curve: a slight decrease up to 100 MPa, a small change i n the 100±220 MPa r ange, followed by an increase at 220 MPa or higher. It was con®rmed that the excitation spectra of BIPM and FITC do not change under these high pressures. Hydrogen±tritium exchange under high pressure Effects of high pressure on irreversible tritium incorporation into Na + /K + -ATPase are shown i n Fig. 5. Tritium incor- poration was small under pressures of 100 MPa or b elow, but increased with i ncreasing pressure in t he 100±220 MPa range (800 t ritium per m ole of A TPase at the maximum), followed by a decrease at 220 M Pa or higher. A ny tritium once trapped at 220 MPa was not removed by repeated washings and by tryptic digestion as shown in Fig. 6. During tryptic digestion, FITC was r eleased but BIPM was not (Fig. 6, open triangles). This indicates that the intracel- lular water-soluble domain was digested by trypsin but the transmembrane segments were not, consistent with previous results [17,34]. Fig. 5. Eect of pressure on tritium incorporation into membrane-bound Na + /K + -ATPase. Mem brane-bound Na + /K + -ATPase was mixed with tritiated water at various pressures and at 37 °Cfor15minand ®ltered by M illipore ®lter membranes. After washing the ® lter mem- brane by tritium-free buer, radioactivity o f the ®lter membrane was determined (d). T he membrane-bound ATPase trapping tritium by pressurization at 220 MP a was re-pressurized in tritium-free medium at 220 MPa and 37 °C for 15 min and the radioactivity of bound tritium was measured (s). See Experimental procedures for details. Fig. 4. Eect o f pressure on ¯uorescence energy transfer from BIPM to FITC of BIPM/FITC doubly labeled Na + /K + -ATPase. Ten micro- grams o f the BIPM/FITC doubly labeled enzyme were suspended in 2 mL of the solution containing 160 m M NaCl (d)or16 m M KCl (s), 25 m M Tris/HCl, 0.43 m M MgCl 2 ,25m M sucrose, and 0.1 m M EDTA (pH 7 .4). BF/F i ndicates the r atio of FITC ¯u orescenc e caused by energy transfer from BIPM to FITC (ex. 305 nm, em. 520 nm) to FITC ¯uorescence (ex. 470 nm, em. 520 nm). Based on three inde- pendent experiments, the means and standard deviations are sh own by error bars. 114 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Tritium once incorporated was released by the re-pressurization of the tritium-trapped Na + /K + -ATPase in a t ritium-free medium at 220 MPa (Fig. 5, open circles). These results indicate that no tritium was trapped i n either the intracellular soluble domain of the enzyme, w hich is digestible by trypsin, under the present pressure conditions. In addition, no tritium was trapped irreversibly i n either the lipid bilayer or in the cores of the protein molecule b y the short expose to tritium used herein [35]. This was con®rmed by experiments with tritium incorporation into phosphat- idylcholine liposome and egg albumin: neither liposome nor albumin irreve rsibly incorporated tritium a s the result of pressure-treatment at 0.1, 100, 200, 300, and 40 0 MPa for 15 min ( data not shown). DISCUSSION PigkidneyNa + /K + -ATPase activity showed at least a three-step change, depending on the pressure applied at 0.1± 100 MPa, 100±220 MPa, and 220 MPa or higher (Fig. 1). The t hree pressure-sensitive states include the lipid phase transition, which is the most sensitive to pressure, s ubunit dissociation, conformational c hange of p rotein, and/or the destruction of integral structure (lipid bilayer and protein). The issue of clarifying the precise nature of the interrela- tionship between these pressure-induced changes and enzyme activity is of concern. General features of pressure effects on protein and the lipid bilayer High hydrostatic pressure causes the dissociation of the protein subunit in aqueous solution at pressures of 200 MPa or less [36]. F or example, lactate dehydrogenase (four identical subunits) [37], yeast hexokinase (two identical subunits) [38], glyceraldehydephosphate dehydrogenase (four identical subunits) [39] dissociates into subunits under pressures around 150 MPa. Dissociation of the ATPase subunits used here is also possible under pressures in the range of 100±220 M Pa as the functional unit of this enzyme is composed of four ab protomers [ 40±42]. Although t he issue of how the lipid bilayer affects the pressure-induced dissociation of protein subunits is not presently known, it should be remembered the ab protomer is largely exposed to an aqueous environment [11] and, as a result, such water- soluble domains are directly affected by pressure effects. The phas e transition of a lipid bilayer f rom the liquid crystalline phase to a gel phase is accompanied by an increase in the thickness of the lipid bilayer and a decrease in the cross-sectional area, due to the ordering of hydrocarbon chains [43±50]. For example, the thickness of a bilayer which is composed of 1-palmitoyl-2-oleoyl-sn -glycero-3-phospho- choline increases by 15% from 63.2 to 72.3 A Ê [43] and the cross-sectional area occupied by hydrocarbon chains of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine bilayer decreases by 20% from 47 .0 to 37.9 A Ê 2 ámol )1 [44] during their phase transition. Pressure and temperature are factors for inducing such phase transitions of the lipid bilayer, which is accom- panied by changes in thickness and the cross-sectional a rea of the hydrocarbon region [43,45,46]. These changes would be expected to alter the environment of transmembrane proteins in the contact surfaces of proteins and water, and/or protein and lipid bilayer: an increase in the thickness of the lipid bilayer p artially covers the transmembrane proteins and, th us, decreases the water-soluble region, or its l ateral shrinkage separates protein from lipid. Pressure-induced changes of membrane-bound Na + /K + -ATPase Although a consistent explanation of all the present experimental results is dif®cult, pressure-induced changes in membrane-bound Na + /K + -ATPase c an be summarized as follows. Pressure eects at 100 MPa or lower. The decreased reaction rate of the Na + /K + -ATPasewithanincreasein pressure can be rationalized based on a decrease in ¯uidity of the lipid bilayer with a concomitant decrease in protein conformational ¯exibility as described previously [22]. However, with an increase in pressu re, t he k max of intrinsic ¯uorescence was shifted to a longer wavelength with a small change in intrinsic ¯uorescence intensity (Fig. 2), probably the r esult of an increase i n the hydrophilic region of the enzyme. A slight decrease in en ergy transfer which is accompanied by a pressure increase (Fig. 4) i ndicates a protein conformational change, which d etaches Lys501 from Cys964. These results show that a pressure o f 100 MPa or lower causes, not only a d ecrease in c onformational ¯exibility as previous report [22], but also conformational changes in the transmembrane protein especially in the water-soluble domain. Fluorescence with a nd without lipid bilayer is paralleled with an increase in pressure (Fig. 2), showing that a romatic residues, which are present in the water-soluble domain of Na + /K + -ATPase, are exposed to an aqueous environment by receiving a direct pressure-effect. However, the ¯uores- cence of FITC and BIPM, when bound to the water-soluble domain and the transmembrane segment, respectively, shows only a small pressure-induced change (Fig. 3 ). Therefore, it can be concluded that the conformational change of Na + /K + -ATPase, if any, would be small at 100 MPa or lower and would not disturb t he gross structure Fig. 6. Changes in bound tritium during tryptic digestion of tritium- bound membrane-Na + /K + -ATPase. Tritium-bound Na + /K + -ATPase was prepared by pressurization at 200 MPa for 15 min as described in Fig. 5 and digested with trypsin. Bound tritium was measured during tryptic digestion as described in Experimental procedures (d). The process of tryptic digestion was monitored by FITC (s)andBIPM(n) releasing from FITC/BIPM doubly labeled ATPase. See Experimental procedures for details. Ó FEBS 2002 High pressure eects on biological membrane (Eur. J. Biochem. 269) 115 of the protein/membrane s yste m. This conclusion is sup- ported by the reversibility of these changes (Figs 1±3) and that no trapping of tritium was detected (Fig. 5) in this pressure-range. Pressure eects of 100±220 MPa. Na + /K + -ATPase ac- tivity showed little or no change i n this pressure r ange, a nd the lower level of activity was maintained, while activities of Na + -dependent ATPase and K + -activated phosphatase showed a pressure-dependent decrease (Fig. 1). The k max for intrinsic ¯uorescence shifted to a longer wavelength with a small decrease in ¯uorescence intensity, indicating that aromatic residues in the protein moved from hydrophobic to hydrophilic environments. As the pressure was increased from 100 to 220 MPa, the BIPM and FITC ¯uorescence showed changes, which were similar to those observed at 100 MPa or lower (Fig. 3, closed circles). The increase in energy transfer induced by a pressure of 100 M Pa or higher (Fig. 4) may be explained simply by a decrease in the distance between Cys964 and Lys501 residues, due to a partial dissociation or re-arrangement of subunits, a n i ncrease in rotational f reedom, f ragmentation of the lipid bilayer, and/or unfolding of the protein molecule, although t he precise identi®cation of this r emains for further study. The most noticeable change in this pressure range is tritium i ncorporation (Fig. 5). Tritium inc orporation i s a n irreversible process, while pressure-induced changes in activity and ¯uorescence are reversible. The i rreversible b inding of tritium in the pressure range studied can be explained as a change in the N a + /K + - ATPase-lipid bilayer system. That is, a high pressure of 100± 220 M Pa causes dissociatio n of a and b subunits, dis- assembly of transmembrane a helices, a nd a separation in the c ontact surface o f membrane and protein due to the thickening and shrinkage of lipid bilayer. For the last case, a quantitative estimation of the thickness and cross-sectional area of the lipid bilayer of the present p ig kidney Na + /K + - ATPase is dif®cult because the precise lipid compositio n is unknown. Generally, phospholipids of pig kidney contain 23.5% of palmitic acid and 26.4% of oleic acid. H owever, the s hrinkage of the c ross-sectional a rea i s p artly c aused b y the phase transition of lipids a nd is accumulative in the lateral direction, and lastly, the shrinkage is suf®ciently large to separate the contact s urface between the transmembrane segment and the lipid bilayer to permit to take up o f w ater from the medium. H±T exchange could occur on these contact surfaces. After release of the pressure, the exchanged tritium cannot escape from the reversibly closed contact surface, even after repeating washing (Scheme 1). As no tritium trapped was removed by tryptic digestion (Fig. 6 ) and was trapped in egg albumin, the possibility that the core part of protein molecule, including the transmem- brane helice s, irreversib ly incorporates tritium atoms at Scheme 1. Pressure-induced separation of the contact surface between protein and lipid bilayer and H±T exchange in the membrane-bound protein system. With an increase in pressure from 0.1 t o 220 MPa, the thickness of the lipid bilayer increases from L t o Lp and its cross-sectional area decreases from W to Wp du e to the pre ssure-induc ed phase transition o f a portion of the lipid bilayer (A and B: see the text for de tails). As a result, the contact surface between protein and lipid bilayer is separated, producing tunnels (B). In the presence of tritiated water (T 2 O), T 2 Oistakenupby these tunnels and H±T e xchange occurs on the protein surface. As such changes are completely reversible in t his pressure range, the trapped tritium cannot be removed from the reversibly closed contact surface after releasing the pressure and washing with H 2 O (C), and even after tryptic digestion (D). At 400 MP a or higher, the lipid bilayer undergoes fragmen tation (E), accompan ied by protein denaturation and further phase transition and interdigitation of t he lipid bilayer. 116 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002 100±200 MPa may be excluded. The lack of tritium release by tryptic digestion also suggests that there are few tritium atoms in the contact surface of a and b subunits. A ll things considered, t ritium incorporation mainly o ccurs in t he interface of protein and lipid bilayer. Pressure eect at 220 MPa or higher. Enzyme activities decreased in this pressure range, accompanied by a decrease in the intensity of intrinsic ¯uorescence a nd a blue-shift of k max (Fig. 2); these changes were irreversible. Tritium incorporation into enzyme decreased substantially (Fig. 5 ). These results show that the lipid bilayer, which c ontains the transmembrane protein, is disrupted and fragmented re- sulting in the destruction of the organized structure of protein and lipid bilayer. The p ressure-induced destruction of a biomembrane composed of a lipid bilayer and a protein will be brought about by separation of the interface between the trans- membrane protein and the lipid bilayer, which is ampli®ed by an increase in pressure. Some tritium irreversibly binds with aggregates of unfolded protein and the fragmented lipid bilayer. CONCLUSION High pressure induces a three-step change in membrane- bound Na + /K + -ATPase. Pressure o f 100 MPa or lower induces a d ecrease in the ¯uidity of the lipid bilayer and reversible conformational changes of the transmembrane protein, resultin g i n t he functional disorder o f the enzymes. Pressure of 100±220 M Pa causes a reversible phase transition of the lipid bilayer a nd the dissociation of protein s ubunits. T hese changes bring about the separation of protein and the lipid bilayer, producing transmembrane tunnels. Pressure of 220 MPa or higher is accompanied by irreversible protein unfolding and fragmentation of the lipid bilayer, thus destroying the gross membrane struc- ture. The present perspective o f the p ressure-induced changes in a membrane-protein system is novel and unique and gives a clue to our understanding of biochemical phenomena in cells and tissues, which are induced by high pressure-treatment. Fruitful information will be also obtained relative to m embrane dynamics f rom studies of the effects of pressure on membrane-associated proteins. 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