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

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

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 [email protected]
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)

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].

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

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

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

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

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
+

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

-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

+
-
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
+

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

+
/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

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

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±

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

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

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
+

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

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