Effect of proteasome inhibitor on sarcoplasmic protein of
bovine skeletal muscle during storage
M. Sekikawa, M. Yamamoto, M. Fukushima, K. Shimada, *T.
Ishikawa, and
M. Mikami
Laboratory of Meat Science, Department of Bioresource Science,
Obihiro University of Agriculture and Veterinary Medicine
Inada, Obihiro 080-8555, Japan
*Laboratory of Biology, Nihon University, School of Dentistry
at
Matsudo, Sakae, Matsudo 271-0061, Japan
Abstract
In previous report we showed that the ubiquitin existed both
in bovine skeletal and cardiac muscle after slaughter and this protein degraded
during storage. We prepared the homogenized bovine skeletal muscle immediately
after slaughter (1.5 h; 0 d), samples of quadriceps femoris muscle were
homogenized in 5-fold (w/v) of 0.2 M Tris-HCl buffer containing 0.05 % NaN3 , pH
7.4, and then proteasome inhibitor (10ΚM MG132 or 10ΚM Lactacystin) was
individually added , and stored samples at 2 d post-mortem (4 } 1 C) for SDS-PAGE
and Western blotting analysis.
Among the sarcoplasmic proteins prepared from stored homogenized muscle sample
at 0 and 2 d, the ubiquitin antiserum (Sigma, St Louis) reacted with bands
corresponding to purified ubiquitin and small amounts of some other,
higher-molecular-mass proteins (about 30 and 40 kDa) which were considered to be
ubiquitin-protein conjugates. These bands were faint in the control 2 d sample,
suggesting that they had degraded. However these decrease tendencies of the
ubiquitin positive bands were not clearly observed in the sample added
proteasome inhibitors (MG132 and Lactacystin). These results suggest that both
ubiquitin and the ubiquitin-protein conjugates were presented in the skeletal
muscle immediately after slaughter and they were then degraded during storage,
and this degradation was partially due to the action of proteasome.
Introduction
Although various meats from skeletal muscles are obtained from
animals after slaughter, the intracellular proteolytic system of muscle cells is
considered to play an important role in improving the texture and taste of meat.
It is well known that low-molecular-mass peptides and free amino acids
accumulate in the sarcoplasmic fraction of various meats during conditioning
(Field et al. 1971, Nishimura et al. 1988, Sekikawa et al. 1999). The structures
and sources of these peptides and amino acids have not been definitively
identified, although it is generally considered that these compounds accumulate
because of the action of proteases (Dransfield 1994, Etherington et al. 1990,
Koohmarie 1994, Mikami et al. 1994). This intracellular protein degradation by
different proteolytic mechanisms occurs through either lysosomal or cytoplasmic
pathways in living cells (Ciechanover et al. 1990). In the cytoplasmic pathway,
there are ATP-dependent and independent mechanisms. Recent studies have
suggested that the ubiquitin system, consisting of ATP, proteasomes and
ubiquitin, plays an important role in the degradation of muscle proteins under
various catabolic conditions. This system is involved in various cellular
functions: the regulation of intracellular protein degradation, cell cycle
regulation, and the stress response (Fang. et al. 1995, Sharma et al. 1996). The
ubiquitin, a highly conserved 76-residue protein found in all eukaryotic cells,
is covalently ligated to the target protein. Protein ligated to multiple units
of ubiquitin is degraded by the 26S proteasome. The 26S proteasome is involved
in ATP-ubiquitin-dependent proteolysis, and the 20S proteasome is the catalytic
core of the 26S proteasome. Recently, Taylor et al. (1995), Matsuishi and
Okitani (1997) and Robert et al. (1998) reported that their purified 20S
proteasome degraded myofibrils and/or myofibrillar proteins. The function of the
20S proteasome in the living body is not yet clear, but ATP depletion, which is
a condition of postmortem muscle cells, results in reversible dissociation of
the 26S into the 20S proteasome (Robert et al. 1998). However, it is uncertain
whether the ubiquitin-proteasome system functions in the muscle cell after
slaughter. There is a small amount of ATP in the muscle cell until rigor mortis
occurs, e.g. 24 h after slaughter in beef. During this period, immediately after
slaughter until rigor mortis, there is a possibility that the ubiquitin system
could be acting in the bovine muscle cell. Moreover, our previous work on bovine
skeletal and cardiac muscles indicated that ubiquitin and ubiquitin linked
proteins were present in muscle immediately after slaughter, but almost
disappeared during storage, suggesting that the ubiquitin and linked proteins
had been degraded by proteinases (Sekikawa et al. 1998, Sekikawa et al. 2000).
The purpose of this study was to demonstrate electrophoretically the effects of
proteasome inhibitor on sarcoplasmic protein of bovine skeletal muscle during
storage.
Materials and Methods
Samples of quadriceps femoris muscle were obtained from three
Holstein cows (average age 38 mo) within 90 min after slaughter, and the excess
fat, the fascia and the large blood vessels were removed. The samples were
homogenized in 5-fold (w/v) of 0.2 M Tris-HCl buffer containing 0.05 % NaN3 , pH
7.4, with or without proteasome inhibitor and then stored at 4 } 1 C for 48 h
(2 d). The inhibitor used were 10ΚM MG132 (Calbiochem, USA), 10ΚM lactacystin
(Kyowa Medics, Japan) and completeTM (Boehringer Mannheim, Germany, proteases
inhibitor cocktail, one tablet was dissoluble in 50 ml Tris-HCl buffer). Each
inhibitor was individually added.
Sarcoplasmic proteins were prepared from aliquot volumes of the homogenized
samples at 0 h and 48 h after storage. These samples were boiled for 5 min, and
then allowed to cool to room temperature. Precipitates were removed by
centrifugation (0 , 8,000 x g, 30 min) and the supernatant was dialyzed
against distilled water at 4 } 1 for 24 h. The dialysate obtained was
freeze-dried, and the lyophilized sample was considered to be the sarcoplasmic
fraction for the present study.
Muscle pH was measured 90 min after slaughter by a metal electrode (HM-17MS,
Toa, Tokyo) directly inserted into the sample, and the pH of the homogenized
samples was measured two days after slaughter using a pH meter with a glass
electrode (Toa, HM-5s).
The prepared sarcoplasmic fractions were analyzed in a 15% Tris-HCl slab gel
with a 6% stacking gel (Laemmli 1970). The gel (85~90 ~1.5 mm) was run at 20
mA for ca. 2 h and then stained with 0.1% Coomassie Brilliant Blue R-250 in 30%
methanol and 10% acetic acid.
Proteins were transferred from the slab gel to a nitrocellulose membrane (ADVANTEC,
Japan) by a buffer-transfer method (Towbin et al. 1979, Negishi et al. 1996).
After transfer, the membrane was incubated in phosphate buffered saline (PBS, pH
7.4) containing 5.0% skim milk overnight at 4C, and then washed three times in
PBS for 5 min at room temperature. The transferred membrane was incubated with
rabbit ubiquitin antiserum (Sigma, USA) for 1 h at 37 C, washed three times in
PBS, and then incubated with peroxidase-labeled goat anti-rabbit secondary
antibody (BIO-RAD, USA) for 1 h at 37 C, followed by two washes in PBS. The
peroxidase was detected with 0.6% (w/v) 4-chloro-1-naphthol, 20% ethanol and
0.02% H2O2. Each analysis was done at least in duplicate.
Results and Discussion
In the current study, the average pH (SE; standard error) of a
sample of quadriceps femoris muscle 90 min after slaughter was 7.07 (+0.03) and
that of the homogenized muscle samples after storage for 48 h was 6.79 (+0.04)
in the control sample, and the range was from 6.60 to 6.76 (+0.04) in the
samples with added inhibitor. There were no significant mean differences among
groups at the 5% level using Students t test.
The color of the homogenized muscle in the control became darker due to
met-myoglobin formation, whereas the MG132-treated sample showed a bright red
color after storage for 48 h. Although the reason for this is unclear, MG132 is
the peptide aldehyde inhibitor N-carbobenzoxyl-Leu-Leu-leucinal, in general,
aldehyde is considered a reducing agent.
It is well known that low-molecular-mass peptides and free amino acids
accumulate in the sarcoplasmic fraction of various skeletal muscles during
conditioning (Field et al. 1971, Nishimura et al. 1988, Sekikawa et al. 1999).
This tendency was observed in the SDS-PAGE profiles of the sarcoplasmic
proteins, especially the under 15-kDa band (Fig. 1: lanes 1 and 2) in the
control. However, in comparison with our previous results for bovine skeletal
muscle (Sekikawa et al. 1998), there was a smaller increase in the intensity of
CBB staining, and few new bands appeared in the present study, indicating that
the production of low-molecular-mass proteins was lower than in the previous
study. This difference of SDS-PAGE profiles might be related to the age of the
animals and/or storage conditions. In the present study muscle samples were
obtained from old milking cows and were homogenized with buffer and then stored.
In our previous study muscle samples were taken from young steers and these were
stored intact. Distilled water extraction was also used.
There were no apparent differences of the SDS-PAGE profiles between the control
and MG132 or lactacystin treatments (Fig.1: lanes 4-5), indicating that
proteasome inhibitors, MG132 and lactacystin, had no effect on the SDS-PAGE
profiles stained with CBB.
The peptide aldehyde inhibitor MG132 can also inhibit calpains and certain
lysosomal cysteine proteases (Tawa et al. 1997, Lee and Goldberg, 1998),
although there was no apparent difference of the SDS-PAGE profiles between the
control and this inhibitor treatment (Fig 1: lanes 2 and 4). However,
lactacystin, originally isolated from actinomycetes, is considered to be a
specific inhibitor of proteasomes. This reagent is a natural product, and not
considered to affect other proteases (Tawa et al. 1997, Lee and Goldberg, 1998).
The SDS-PAGE profile after lactacystin treatment was similar to that of the
control (Fig 1: lanes 2 and 5). This present results suggest that both MG132 and
lactacystin do not inhibit the low-molecular-mass peptide production of the
sarcoplasmic fraction caused by proteases in bovine skeletal muscles after
slaughter. However, the SDS-PAGE profile after treatment with CompleteTM for 48
h was similar to that of the control at 0 h (Fig 1: lanes 1 and 3). The
CompleteTM inhibited the production of low-molecular-mass peptide, indicating
that the latter was produced by proteases, which were inhibited by this
inhibitor cocktail.
In our previous study (Sekikawa et al., 1998), the characterization of ubiquitin
antiserum (Sigma, USA) showed that it clearly and strongly recognized the
ubiquitin band (8.7 kDa) in the purified ubiquitin sample (Sigma, USA). We have
also reported that when the 8-kDa band purified by preparative SDS-PAGE (Prep
cell, BioRAD) was subjected to amino acid sequence analysis by Edman degradation
(model 470, ABI), the sequence of the five N-terminal residues (MQIFV) was the
same as that of ubiquitin (Schlesinger et al. 1975). These results suggest that
the 8-kDa band among the sarcoplasmic proteins includes ubiquitin as a major
component.
The ubiquitin antiserum reacted with bands corresponding to ubiquitin (about 8
kDa) and small amounts of other higher-molecular-mass proteins (about 30 and 40
kDa), which were considered to be ubiquitin-protein conjugates (Fig. 2: lane 1).
In a preliminary experiment, the 30-kDa band was purified by two-dimensional
electrophoresis and its amino acid sequence was analyzed. However, at this stage
the N-terminal amino acid was not detected. After pyroglutamyl peptidase
digestion, the amino acid sequence of six residues was determined as RQTAAG.
When this sequence was compared with the ubiquitin reported previously (Schlesinger
et al. 1975), there was no correlation. This suggests that the 30-kDa protein is
a ubiquitin-protein conjugate, and not poly-ubiquitin.
The results of western blotting suggested that both the 8-kDa ubiquitin and the
ubiquitin-protein conjugates existed and/or were released in the sarcoplasmic
fraction of skeletal muscle cells immediately after circulatory arrest, and that
they then degraded during storage (Fig. 2: lane 2). This was similar to the
trend observed in our previous study (Sekikawa et al., 1998). However, when
using inhibitor treatments (CompleteTM, MG132 and lactacystin, Fig. 2: lanes 3-5
respectively) the positive bands stained with ubiquitin untiserum corresponding
to ubiquitin and ubiquitin-conjugated proteins were not affected by storage for
48 h, especially after CompleteTM and MG132 treatments. The patterns of western
blotting after lactacystin treatment were similar to, but weaker in intensity
than those after CompleteTM and MG132 (Fig.2: lanes 3-5).
In summary, CompleteTM almost completely inhibited the production of
low-molecular-mass protein observed in SDS-PAGE profiles, and in addition, no
change was observed in the ubiquitin-positive band upon western blotting.
However, neither MG132 nor lactacystin had a large influence on
low-molecular-mass protein production, or even changed the ubiquitin-positive
band upon western blotting. These results suggest that the ubiquitin and
ubiquitin-conjugated proteins were degraded by proteases, including proteasomes
as well as calpain and cathepsins. The results also suggest the possibility that
the ubiquitin-proteasome system functioned from immediately after slaughter
until ATP depletion. In beef, this was generally when maximum rigor mortis had
occurred.
The 26S proteasome is involved in ATP-ubiquitin-dependent proteolysis, and the
20S proteasome is the catalytic core of the 26S proteasome. The function of the
20S proteasome in the living body is not yet clear, but ATP depletion results in
reversible dissociation of 26S into the 20S proteasome (Peters et al. 1994).
Recently, Robert et al. (1999) reported that the 20S proteasome degraded
myofibrillar proteins, and that after the ATP level had decreased, the 26S
proteasome appeared to dissociate into the 20S proteasome and PA700, thus
increasing the 20S concentration in muscle cells. As this 20S proteasome does
not require ATP and ubiquitin, its proteolytic actions contribute to meat
conditioning after maximum rigor mortis has occurred.
Although the ubiquitin system of cellular protein degradation has been
investigated in various fields, such as clinical medicine and cellular biology
(Fang et al. 1995), the state of this peptide in muscle cells postmortem seems
to have received little attention because of ATP depletion. Riley et al. (1988)
and Hilenski et al. (1992) reported that ubiquitin was conjugated to the Z-bands
of normal skeletal muscle, enhancing the ubiquitin-mediated pathway of protein
turnover and causing degradation of striated muscle.
It is considered that protein degradation with the ubiquitin system comprising
ubiquitin, ATP and proteasomes, which act in living muscle cells, is one of the
primary factors affecting the ischemia conditions of muscle cells (Sharma et al.
1996). It is also apparent that the mechanism of action of ubiquitin is
important not only in meat science, but also in general biochemical studies.
Therefore, further experiments are needed to demonstrate the contribution of the
ubiquitin system to meat conditioning.
Acknowledgments
This research was supported in part by a Grant-in-Aid of Scientific Research (A) from the Ministry of Education, Science, Sports and Culture of Japan (MS: #10660253).
Legends for figures
Figure 1. SDS-PAGE profiles of bovine skeletal sarcoplasmic proteins incubated with or without protease inhibitors. Each lane was loaded with 10 mg of sample proteins and stained with CBB. Lane 1and 2: incubated for 0 h and 48 h without (control) inhibitor respectively, lane 3: incubated for 48 h with CompleteTM, lane 4: incubated for 48 h with MG132, and lane 5: incubated for 48 h with lactacystin.
Figure 2. Western blot analysis of sarcoplasmic proteins
obtained from bovine skeletal
muscle. Each lane was loaded with 15 mg of sample proteins and stained with
rabbit anti-ubiquitin antiserum. Lane 1 and 2: incubated for 0 h and 48 h
without (control) inhibitor respectively, lane 3: incubated for 48 h with
CompleteTM, lane 4: incubated for 48 h with MG132, and lane 5: incubated for 48
h with lactacystin.