Effects of a high-pressure treatment on the activity and structure of rabbit muscle proteasome

Article abstract of DOI:10.1271/bbb.69.1239

The effects were assessed of high hydrostatic pressure on the activity and structure of rabbit skeletal muscle proteasome. The pressure effects on the activity were measured by the amount of fluorometric products released from synthetic substrates under pressure and from fluorescein isothiocyanate (FITC)-labeled casein after releasing the pressure. The effects on the structure were measured by fluorescene spectroscopy under pressure, and by circular dichroism (CD) spectroscopy and surface hydrophobicity after releasing the pressure. The optimal pressure for the hydrolyzing activity of synthetic peptides was 50 MPa. The degradation of FITC-labeled casein increased linearly with increasing pressure applied up to 200 MPa, and then markedly decreased up to at 400 MPa. The changes in the tertiary structure detected by fluorometric measurement were irreversible, whereas the changes in the secondary structure were small compared with those by heat treatment. The pressure-induced activation of proteasome therefore seems to have been due to a little unfolding of the active sites of proteasome.

Full text of DOI:10.1271/bbb.69.1239

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Effects of a High-Pressure Treatment on
the Activity and Structure of Rabbit Muscle
Proteasome
Shuhei YAMAMOTO, Yuichi OTSUKA, Gerelt BORJIGIN, Kanna MASUDA,
Yoshihide IKEUCHI, Tadayuki NISHIUMI & Atsushi SUZUKI
To cite this article: Shuhei YAMAMOTO, Yuichi OTSUKA, Gerelt BORJIGIN, Kanna MASUDA,
Yoshihide IKEUCHI, Tadayuki NISHIUMI & Atsushi SUZUKI (2005) Effects of a High-Pressure
Treatment on the Activity and Structure of Rabbit Muscle Proteasome, Bioscience,
Biotechnology, and Biochemistry, 69:7, 1239-1247, DOI: 10.1271/bbb.69.1239
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Biosci. Biotechnol. Biochem., 69 (7), 1239–1247, 2005
Effects of a High-Pressure Treatment on the Activity and Structure
of Rabbit Muscle Proteasome
Shuhei YAMAMOTO,1 Yuichi OTSUKA,1 Gerelt BORJIGIN,2 Kanna MASUDA,2
y
2;
Yoshihide IKEUCHI,3 Tadayuki NISHIUMI,2 and Atsushi SUZUKI
1Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan
2Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University,
Ikarashi, Niigata 950-2181, Japan
3Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University,
Hakozaki, Fukuoka 821-8581, Japan
Received November 11, 2004; Accepted March 26, 2005
The effects were assessed of high hydrostatic pressure
on the activity and structure of rabbit skeletal muscle
proteasome. The pressure effects on the activity were
measured by the amount of fluorometric products
released from synthetic substrates under pressure and
from fluorescein isothiocyanate (FITC)-labeled casein
after releasing the pressure. The effects on the structure
were measured by fluorescene spectroscopy under
pressure, and by circular dichroism (CD) spectroscopy
and surface hydrophobicity after releasing the pressure.
The optimal pressure for the hydrolyzing activity of
synthetic peptides was 50 MPa. The degradation of
FITC-labeled casein increased linearly with increasing
pressure applied up to 200 MPa, and then markedly
decreased up to at 400 MPa.
considered to be the core unit for the proteinase activity
of 26S proteasome, and other components are assumed
to be subunits that make the proteinase activity of 26S
proteasome ATP-dependent. It has also been shown that
ATP was essential for maintaining the 26S complex, and
that a depletion of ATP caused rapid dissociation of the
26S complex into 20S proteasome and other compo-
nents.6) However, under the post-mortem condition
without ATP, it is necessary to investigate the properties
of 20S proteasome.
20S proteasome has a cylinder-shaped structure
arranged as four axially stacked heptameric rings
composed exclusively of either ꢀ-subunits (two outer
rings, ꢀ1–ꢀ7) or ꢁ-subunits (two inner rings, ꢁ1–ꢁ7),
respectively (ꢀ7ꢁ7ꢁ7ꢀ7). The multiple catalytic sites of
this proteolytic complex are exclusively associated with
the ꢁ-subunits because of the particular structural
topology of the particle, and they are assumed to be
all sequestered within the hollow cavity of the cylin-
der.7) This proteasome has two unique enzymological
properties as a protease: multiple peptidase activities,
and a latent form. According to Mykles and Harie,8) the
proteasome has at least five activities — peptidylglu-
tamyl peptide hydrolase (PGPH), trypsin-like, chymo-
trypsin-like, branched-chain amino acid-preferring, and
small neutral amino acid-preferring — within the single
proteasome complex. Furthermore, the three major
activities (PGPH, trypsin-like and chymotrypsin-like)
have been respectively assigned to the three active
subunits, ꢁ1, ꢁ2 and ꢁ5, based on mutational and crystal
structural analysis studies.9,10) The proteasome isolated
from tissues in a latent form can be activated by various
chemicals and such treatments as polylysine,11,12)
SDS11,13–15) and fatty acids,13) heat15–18) and high hydro-
static pressure.15,19) These results indicate that protea-
some activity is stimulated by a mild denaturing
treatment. However, it has not yet been made clear
The changes in the tertiary structure detected by
fluorometric measurement were irreversible, whereas
the changes in the secondary structure were small
compared with those by heat treatment. The pressure-
induced activation of proteasome therefore seems to
have been due to a little unfolding of the active sites of
proteasome.
Key words: proteasome; high hydrostatic pressure; fluo-
rescein isothiocyanate (FITC)-labeled ca-
sein; circular dichroism (CD) spectra; sur-
face hydrophobicity
Proteasome or multicatalytic proteinase complex
(MCP) was first isolated from bovine pituitaries.1) This
enzyme is a high-molecular-mass intracellular protei-
nase (20S proteasome; 700 kDa) which has a complex
subunit composition and multicatalytic proteolytic ac-
tivities with different specificity.2–5)
Proteasomes have been classified into two isoforms
with apparent sedimentation coefficients of 20S and 26S
proteasome (1,600 kDa), respectively. 20S proteasome is
y
To whom correspondence should be addressed. Tel: +81-25-262-6693; Fax: +81-25-262-6854; E-mail: atsuzuki@agr.niigata-u.ac.jp

1240
S. YAMAMOTO et al.
why the proteasome is activated, especially by a high
hydrostatic pressure treatment.
proteasome solution was vacuum-sealed in a polyeth-
ylene bag, and this bag was transferred to a larger
polyethylene bag. The space between the two bags was
filled with water. Each double bag was then placed in a
pressure vessel with water and subjected to 0.1–400 MPa
at about 10 ꢀC for 10 min with a cold isostatic press
(CIP) from Nikkiso Co., Ltd., Tokyo, Japan.
High hydrostatic pressurization is one of the new
technologies for tendering or accelerating of meat aging
which is a very important process for the meat industry.
The availability of intramuscular proteinases during
meat aging has been well investigated. In particular,
cathepsin and calpain have been assumed to participate
in proteolysis during meat aging.20–22)
Heat treatment. The purified proteasome was heated
at 20–80 ꢀC for 30 min. After chilling on ice for 10 min,
the sample was subjected to the subsequent analyses.
Several studies have been made on the effects of high
hydrostatic pressurization to treat intramuscular protei-
nase. Homma et al. have reported that the pressure-
induced increase in proteolytic level in the muscle was
due to a release of cathepsins from lysosomes.23) In
respect of calpain systems, Suzuki et al. have provided
direct evidence for the pressure-induced Ca2þ release
from the sarcoplasmic reticulum by using electron
micrographs of the pyroantimonate-fixed fiber bundles
prepared from pressurized muscle tissue.24) Ca2þ dis-
persion into myofibrils may cause an increase in
activated calpain. Homma et al. have reported that the
calpain level was retained in muscle pressurized up to
200 MPa, whereas the calpastatin level was lowered by
pressurization. The total calpain activity in pressurized
muscle therefore appears to be increased by a pressure
treatment, and this may result in tenderization of the
meat.25,26)
In situ measurement of proteasome activities under
high pressure. The proteasome activities were assayed
by fluorometric measurement of the amount of 7-amino-
4-methylcoumarin (-NH-Mec; AMC) released from
synthetic substrates. The substrates used were Suc-
Leu-Leu-Val-Tyr-NH-Mec (LLVY; chymotrypsin-like
activity) and Ala-Ala-Phe-NH-Mec (AAF; chymotryp-
sin-like activity) purchased from Sigma (St. Louis, MO,
U.S.A.), and Boc-Leu-Sec-Thy-NH-Mec (LSTR; tryp-
sin-like activity) purchased from the Peptide Institute
(Osaka, Japan), these being selected to assay distinct
proteolytic activities of the proteasome. All methylcou-
marylamide substrates were stored as 1 mM solutions in
N,N-dimethylformamide at ꢁ20 ꢀC, and then diluted
with water to a 20-ml solution prior to use.
The activation of proteasome induced by high
pressure may be involved in the pressure-induced
modification or breakdown of muscle proteins.15,19)
However, it is not yet clear why proteasome is activated
by high hydrostatic pressure. This paper therefore
describes the effects of a high hydrostatic pressure
treatment on the structure and activities of rabbit skeletal
muscle proteasome in comparison with those of a heat
treatment.
An enzyme solution (300 ml) was mixed with 900 ml
of 100 mM Tris–HCl (pH 8.0) and 300 ml of 6 mM DTT.
After preincubating at 37 ꢀC for 5 min, 300 ml of a
substrate was added to the reaction mixture.
The activities under high pressure (0.1–300 MPa,
0–30 min) were measured in a high-pressure vessel with
a Hitachi F-2000 spectrofluorimeter at 480 nm, with
excitation at 370 nm. This device consisted of a temper-
ature-controlled high pressure vessel equipped with
sapphire windows and a TP-500 high-pressure hydro-
static pump capable of elevating the pressure to
400 MPa (Teramecs Co., Kyoto, Japan). The vessel
was positioned in the light beam of the spectrofluoro-
meter, and a quartz cuvette containing the sample
solution was placed inside the vessel.
Materials and Methods
Purification of proteasome. Proteasome was purified
from rabbit skeletal muscle by the method of Otsuka et
al.15) Briefly, a rabbit was anaesthetized with sodium
pentobarbital (2.5 ml) and d-tubocurarine chloride pen-
tahydrate (0.5 ml) just before exsanguination. The back
and leg muscles were immediately excised and sub-
jected to extraction. The crude proteasome that had been
extracted was purified by chromatography in a DEAE-
Sephacel column. The fractions with activity were
subsequently concentrated and further purified in a
phenyl Sepharose HP column. Those fractions with
activity were finally concentrated and rechromatograph-
ed once again in the phenyl Sepharose HP column. The
active fraction from the second chromatography in the
phenyl Sepharose HP colomn is designated as purified
proteasome.
Determination of the FITC-labeled casein hydrolyz-
ing activities under high pressure. The mixture of the
proteasome and FITC-labeled casein in 100 mM Tris–
HCl and 1 mM DTT (pH 8.0, 2.0 ml) was pressurized to
0.1–400 MPa at 37 ꢀC for 90 min. After releasing the
pressure, the products released from the FITC-labeled
casein were measured by modified method of that
presented by Lonergam et al.28) A 600-ml amount of the
reaction mixture was added to 600 ml of 5% trichloro-
acetic acid (TCA), and subsequently to 300 ml of 50 mg/
ml of a casein buffer (50 mM Na2CO3 and 150 mM NaCl
at pH 9.5) to be coprecipitated with the remaining FITC-
labeled casein. The TCA-insoluble protein was removed
by centrifugation at 10;000 ꢂ g for 20 min. The resulting
supernatant (1.0 ml) was again centrifuged under the
Pressurization. Pressurization was carried out with
the method described by Homma et al.27) Each purified

Effects of Pressure on Proteasome
1241
same conditions to clarify the supernatant. The super-
natant (500 ml) was diluted to 2.5 ml with 500 mM
Na2HPO4 (pH 8.5), before the fluorescence was meas-
ured at 515 nm, with excitation at 490 nm.
unpressurized sample (0.1 MPa). A pressure of 200 MPa
or more resulted in no progress in the degradation of
LLVY or AAF after the first reaction of 2 min (Fig. 1A
and C). However, LSTR degradation increased linearly
at 200 MPa, and no progress in degradation was
apparent after a first reaction for 3 min at 300 MPa
(Fig. 1B). As shown in Fig. 1A–C, the optimal pressure
for the hydrolyzing activity of the synthetic peptide used
in this experiment was 50 MPa. Otsuka et al.15) have
found that the optimal pressure for inducing the highest
synthetic peptide hydrolyzing activity in the pressurized
proteasome was about 100 MPa. This difference may
have been due to different analytical methods: the
former was measured in situ under high pressure, and
the latter was measured only after the pressure had
been released. The increase in proteasome activity was
perhaps due to an increase in the interaction between the
substrate and active site of the proteasome that had been
unfolded by the high-pressure treatment.
Measurement of the fluorescence spectra and center
of spectral mass of proteasome under high pressure. A
proteasome solution (0.03 mg/ml) in 40 mM Tris–HCl,
10 mM EDTA, 10 mM 2-mercaptoethanol and 100 mM
NaCl (pH 7.5) was subjected to a high pressure of 0.1–
400 MPa for 10 min. Changes in the fluorescence
spectrum of the proteasome were measured with a
Hitachi F-2000 spectrofluorometer fitted with a high-
pressure vessel. The fluorescence spectra of the protea-
some were recorded under pressure between 300–
420 nm, with excitation at 280 nm.
Changes in the center of spectral mass (hꢂi) were
calculated according to the method of Ruan et al.29)
hꢂi ¼ ꢀꢂi ꢃ Fi=ꢀFi
The Km and Vmax values of proteasome for the
synthetic peptides under high pressure are shown in
Table 1. The calculated Km and Vmax values for LLVY
and AAF were maximal at 50 MPa, the Km and Vmax
values then continuing to decrease up to 200 MPa or
higher. However, the Km and Vmax values for LSTR
were not influenced by the pressure treatment. In the
present experiment, it seems that the difference in the
change of Km and Vmax values for LLVY/AAF vs. those
for LSTR was caused by the difference in active
subunits of proteasome. It is known that the chymo-
trypsin-like (LLVY, AAF) and trypsin-like (LSTR)
activities of proteasome are respectively derived from
active subunits ꢁ5 and ꢁ2. Our results suggest that the
ꢁ5 subunits were more susceptible to pressure than the
ꢁ2 subunits, resulting in increased LLVY and AAF
hydrolyzing activity under a relatively low pressure.
The effects of pressurization on the casein-degrading
activity are shown in Fig. 2. During the 90-minute
reaction, the degradation of FITC-labeled casein in-
creased linearly with increasing pressure applied up to
200 MPa. When 400 MPa was applied, the degradation
of casein during the first 30 min of the reaction was
higher than at 0.1 MPa (control)–150 MPa. However,
after 30 min of the reaction, the progress of degradation
was slower than that of the proteasome subjected to
lower pressures, and finally after 90 min of the reaction,
the degradation was less than that of proteasome
pressurized at 50–200 MPa. This activation of casein-
olytic activity was probably due to an increase in the
interaction between the substrate and active site of the
proteasome unfolded by the high-pressure treatment.
The difference in optimal pressure for the hydrolyzing
activities of the synthetic peptides and FITC-labeled
casein may have been derived from two factors: the
difference in the active site of proteasome for the
substrate, and the higher susceptibility of denatured
protein to the proteasome than that of the nondenatured
proteins. In the case of casein, it seems that the
where ꢂi is the wavenumber and Fi the fluorescence
intensity at ꢂi.
Measurement of the secondary structure of protea-
some. The circular dichroism (CD) spectra of pressur-
ized or heated proteasome solutions (0.1 mg/ml) in
40 mM Tris–HCl, 10 mM EDTA, 10 mM 2-mercapto-
ethanol and 100 mM NaCl (pH 7.5) were recorded by
a Jasco J-725 spectropolarimeter at 20 ꢀC. The mean
residual ellipticity [ꢃ] is expressed in degreesꢄcm2ꢄ
d molꢁ1. [ꢃ] was calculated by using a molecular weight
of 700,000 and 7,000 residues. The secondary structure
of proteasome was analyzed with the program of Yang
et al.30)
Measurement of the surface hydrophobicity of pro-
teasome. The surface hydrophobicity of pressurized
or heated proteasome solutions (0.05 mg/ml, in 40 mM
Tris–HCl, 10 mM EDTA, 10 mM 2-mercaptoethanol and
100 mM NaCl (pH 7.5)) was measured according to the
method of Boyer et al.31)
Either 12.5 ml of 1 mM cis-parinaric acid (cPA) or
8 ml of 5 mM 8-anilino-1-naphthalene-sulphonic acid
(ANSA) was added to 2.5 ml of the proteasome solution.
The fluorescence intensity of the cPA-protein conjugates
was measured at 410 nm with excitation at 325 nm. The
excitation and emission wavelengths were 380 and
475 nm, respectively, for the ANSA-protein conjugates.
Results and Discussion
Effects of high pressure on the synthetic peptides and
FITC-labelled casein hydrolyzing activities
Changes in the LLVY, LSTR and AAF hydrolyzing
activities under high pressure are shown in Fig. 1A–C.
During the 30-minute reaction, the degradation of
synthetic peptides increased linearly with the applied
pressure up to 150 MPa, and was greater than that of the

1242
S. YAMAMOTO et al.
Fig. 1. Effect on the Hydrolyzing Activities of Synthetic Peptides.
A, LLVY hydrolysis; B, LSTR hydrolysis; C, AAF hydrolysis. , 0.1 MPa (Control); , 50 MPa; , 100 MPa; , 150 MPa; , 200 MPa;
, 300 MPa. Excitation wavelength = 370 nm. Emission wavelength = 480 nm.

Effects of Pressure on Proteasome
1243
Table 1. Km and Vmax Values for Proteasome under High Pressure
LLVY
LSTR
AAF
Pressure
(MPa)
Km
Vmax
Km
Vmax
Km
Vmax
(mM)
(pmol/min/ml)
(mM)
(pmol/min/ml)
(mM)
(pmol/min/ml)
0.1
50
30.63
41.95
34.09
31.14
8.02
397.08
685.32
509.25
453.80
67.95
16.97
17.18
17.14
17.13
15.01
10.20
139.63
195.49
167.19
160.84
117.65
83.60
12.36
16.06
15.33
11.41
5.01
173.13
270.36
209.10
161.19
54.05
100
150
200
300
7.89
61.98
3.23
27.18
Fig. 2. Effect on FITC-Labeled Casein Hydrolyzing Activities.
, 0.1 MPa (Control); , 50 MPa; , 100 MPa; , 150 MPa; , 200 MPa; , 400 MPa. Excitation wavelength = 490 nm. Emission
wavelength = 515 nm.
high-pressure treatment activated the proteasome
activities in parallel with casein denaturation. Therefore,
the caseinolytic activity of the proteasome was increas-
ed. However, when excessive pressure was applied,
the proteasome was denatured and inactivated. Gardrat
et al.19) have reported that the caseinolytic activity
progressively increased up to 150 MPa, and was inacti-
vated at 450 MPa. These differences in caseinolytic
activity may have been caused by differences in the
proteasome preparation, purification method and reac-
tion method.
suggest that high-pressure-induced structural change in
proteasome was irreversible (a minute structural change
led to activation of the proteasome).
Changes in the secondary structure of proteasome
Changes in the secondary structure of proteasome
induced by high pressure and heat treatment are shown
in Tables 2 and 3, respectively. The ꢀ-helix content of
the proteasome was lower at 400 MPa, although the
change was not significant. During heat treatment of the
proteasome, the ꢀ-helix content gradually decreased
with increasing temperature. The decrease in ꢀ-helix
content was especially significant at 70 ꢀC and 80 ꢀC.
The change obtained in this experiment in the secondary
structure of the proteasome does not correlate with the
results of Otsuka et al.15) Otsuka et al. reported that the
synthetic peptide hydrolyzing activity increased with
increasing applied pressure up to 150 MPa, and then
decreased at 200 MPa or higher. They suggested that the
activation of proteasome induced by high pressure might
be one cause of the pressure-induced modification of the
proteasome structure. However, from our results, such
pressure-induced modification of the proteasome struc-
ture was not apparent. On the other hand, Otsuka et al.
have reported that the inactivation of proteasome was
caused by excessive pressurization or heat treatment.
The results of our experiment suggest that the reduction
Changes in the fluorescence spectra and center of
spectral mass of proteasome under high pressure
Changes in the fluorescence spectra are shown in
Fig. 3. The red shift and decrease in fluorescence of the
proteasome gradually progressed with increasing high
pressure applied. This indicates that the environmental
polarity of the tryptophan residues became stronger due
to a pressure-induced change in the tertiary structure.
The center of mass (hꢂi) calculated from Fig. 3. is
shown in Fig. 4. The hꢂi value decreased gradually with
increasing pressure applied. Furthermore, the hꢂi values
during decompression were lower than those at the same
pressures during compression. In other words, hysteresis
in the recovery of the fluorescence parameters was not
apparent during decompression. The hꢂi values therefore

1244
S. YAMAMOTO et al.
Fig. 3. Fluorescence Spectra and Center of Spectral Mass of Proteasome under High Pressure.
A, 50 MPa; B, 100 MPa; C, 200 MPa; D, 400 MPa. —, before pressurization; , under pressure; ꢄ ꢄ ꢄ, after pressurization. Excitation
wavelength = 280 nm. Emission wavelength range = 280–420 nm.
Fig. 4. Pressure Dependence of the Center of Spectral Mass of Proteasome.
, compression; , decompression.
Table 2. Secondary Structure of Pressure-Treated Proteasome
Pressure
(MPa)
Helix
Beta
Turn
Random
Total
0.1
50
48:07 ꢅ 1:65
49:07 ꢅ 0:78
46:65 ꢅ 1:63
47:13 ꢅ 5:40
46:03 ꢅ 5:09
42:53 ꢅ 7:42
19:43 ꢅ 5:25
17:13 ꢅ 8:29
22:05 ꢅ 1:06
20:23 ꢅ 2:91
21:33 ꢅ 2:97
22:93 ꢅ 1:48
8:57 ꢅ 6:19
10:83 ꢅ 8:18
6:25 ꢅ 4:17
7:30 ꢅ 3:40
8:30 ꢅ 4:76
7:20 ꢅ 4:44
23:97 ꢅ 0:58
22:93 ꢅ 2:29
25:15 ꢅ 1:48
25:33 ꢅ 1:15
24:33 ꢅ 1:36
27:33 ꢅ 5:05
100.0
100.0
100.0
100.0
100.0
100.0
100
200
300
400
Each value is expressed as the mean ꢅ standard deviation (n ¼ 4); ꢃp < 0:05.

Effects of Pressure on Proteasome
1245
Table 3. Secondary Structure of Heat-Treated Proteasome
Temperature
(ꢀC)
Helix
Beta
Turn
Random
Total
20
40
50
60
70
80
47:27 ꢅ 2:99
46:83 ꢅ 4:69
45:98 ꢅ 5:82
46:98 ꢅ 5:95
38:38 ꢅ 5:91ꢃ
31:75 ꢅ 14:87ꢃ
19:90 ꢅ 6:29
19:38 ꢅ 2:15
20:03 ꢅ 4:29
18:30 ꢅ 8:18
26:28 ꢅ 10:02
8:90 ꢅ 8:00
10:40 ꢅ 10:82
8:45 ꢅ 3:76
9:85 ꢅ 4:89
9:00 ꢅ 5:76
7:45 ꢅ 7:44
19:15 ꢅ 8:89
22:43 ꢅ 4:65
24:58 ꢅ 2:83
24:20 ꢅ 3:69
25:73 ꢅ 3:11
27:85 ꢅ 3:72
40:18 ꢅ 14:13ꢃ
100.0
100.0
100.0
100.0
100.0
100.0
Each value is expressed as the mean ꢅ standard deviation (n ¼ 4); ꢃp < 0:05.
Fig. 5. Effect of Heat and Pressure Treatments on the Aliphatic Hydrophobicity of Proteasome.
, pressure treatment; , heat treatment. Excitation wavelength = 325 nm. Emission wavelength = 410 nm. Each value is expressed as the
mean ꢅ standard deviation (n ¼ 6); ꢃp < 0:01.
in the amount of ꢀ-helix in proteasome at 400 MPa or at
70 ꢀC and 80 ꢀC led to inactivation of the proteasome.
higher, the activity gradually decreased.15) Our results
indicate that when the proteasome was heated at 50–
60 ꢀC, increases in the surface aliphatic and aromatic
hydrophobicity led to an increase in the proteasome
activity. Above 70 ꢀC, however, this increase in the
aliphatic and aromatic hydrophobicity caused denatura-
tion of the proteasome.
The results of this study demonstrate that high
hydrostatic pressurization led to proteasome activation
and irreversible change (excessive structural change
leading inactivation of the proteasome) in the tertiary
structure as detected from fluorometric measurements,
whereas the change in secondary structure was slight
compared with that by heat treatment. Therefore,
pressure-induced activation of the proteasome seems to
have been due to a small amount of unfolding of the
active sites of proteasome. Further experiments will be
required to clarify this result.
Measurement of the surface hydrophobicity of pro-
teasome
The changes in surface hydrophobicity measured by
cPA and ANSA are shown in Figs. 5 and 6, respectively.
No significant change in aliphatic hydrophobicity was
apparent in the pressurized proteasome. However, an
increase in aliphatic hydrophobicity was observed in the
proteasome heated at temperatures over 60 ꢀC, especial-
ly at 80 ꢀC. Significant changes in the aromatic hydro-
phobicity were observed in both the pressurized and
heated proteasome. The aromatic hydrophobicity mark-
edly increased at above 70 ꢀC and above 300 MPa. It
seems that the increase in aliphatic and aromatic
hydrophobicity of the pressurized proteasome had al-
most no effect on the proteasome activity. Otsuka et al.
have reported that when the proteasome was heated at
60 ꢀC for 20 min and pressurized to 100–150 MPa, its
activity increased. However, when the proteasome was
heated at 70 ꢀC or higher and pressurized to 200 MPa or
We can presently conclude that proteasome may play
a role in high-pressure-induced meat tenderization or the
acceleration of meat aging, in addition to better-known
cathepsin and calpain.

1246
S. YAMAMOTO et al.
Fig. 6. Effect of Heat and Pressure Treatments on the Aromatic Hydrophobicity of Proteasome.
, pressure treatment; , heat treatment. Excitation wavelength = 380 nm. Emission wavelength = 475 nm. Each value is expressed as the
mean ꢅ standard deviation (n ¼ 6); ꢃp < 0:01.
Bartunik, H. D., and Huber, R., Structure of 20S
ꢁ
proteasome from yeast at 2.4 A resolution. Nature, 386,
463–471 (1997).
Acknowledgment
This work was supported in part by grant-aid to A.S.
for scientific research (A: 15208025) from the Japan
Society for the Promotion of Science (JSPS).
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Huber, R., The proteasome. Annu. Rev. Biophys. Biomol.
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