2
.
7
. Electrophoretic analysis Polyacrylamide gel electrophoresis PAGE was
carried out in 2.5 polyacrylamide gel containing 0.5 agarose under nondenaturing conditions.
SDS-PAGE was carried out by the method of Laemmli [31] in 12.5 slab gel. Protein was de-
tected by staining with Coomassie Brilliant Blue G-250. Kaleidoscope prestained standards Bio-
Rad Laboratories were used for SDS-PAGE.
2
.
8
. Immunological analysis Polyclonal antibody against the spinach 26S
proteasome has been described previously [32]. An immunoelectrophoretic blot analysis was carried
out according to the method of Towbin et al. [33]. Samples were separated by PAGE and transferred
electrophoretically to Immobilon polyvinylidene difluoride membranes Millipore, Bedford, USA.
Anti-rabbit immunoglobulin G conjugated with alkaline phosphatase Promega, Madison, USA
was used as a secondary antibody. Nitroblue tetra- zolium and 5-bromo-4-chloro-3-indolyl phosphate
were used as substrates of alkaline phosphatase.
3. Results
3
.
1
. Purification of the
26
S proteasome from rice cells
To purify the 26S proteasome on a large-scale, we used rice suspension-cultured cells collected in
the logarithmic phase of growth. All purification procedures were performed at 4°C, using standard
buffer consisting of 50 mM Tris – HCl pH 7.5, 10 mM 2-mercaptoethanol, 2 mM ATP, 5 mM
MgCl
2
, 20 glycerol, and 10 mM leupeptin, unless otherwise specified.
Fresh rice cells 40 g were homogenized with 100 ml of standard buffer in a Teflon homoge-
nizer. The homogenate was centrifuged for 1 h at 30,000 rpm in a Hitachi RP50A rotor average,
70,000 × g, and the resulting supernatant was fur- ther centrifuged for 5 h at 33,000 rpm in a Hitachi
RP50A rotor average, 85,000 × g. The crude ex- tract was prepared by dissolving the precipitate in
a suitable volume about 1 – 2 ml of standard buffer. This crude extract approximately 217 mg
of protein was applied to a Biogel A-1.5m column 2.5 × 100 cm equilibrated with standard buffer.
Three-milliliter fractions of the eluate were col- lected at a flow rate of 20 mlh. A single peak of
material showing Suc-LLVY-MCA-degrading ac- tivity was obtained. The active fraction from the
Biogel A-1.5m gel filtration column was dialyzed against standard buffer without glycerol and leu-
peptin, and then concentrated in a Centriprep-50 concentrator Amicon, Beverly, USA. The con-
centrated sample was loaded onto a linear gradient of 10 – 40 vv glycerol in standard buffer with-
out glycerol and leupeptin. After centrifugation at 25,000 rpm for 22 h in a Hitachi SRP28SA rotor
Hitachi, Tokyo, Japan, the gradient was sepa- rated into 30 fractions of 1 ml each, and peptidase
activity was assayed. As shown in Fig. 1, a single major peak of peptidase activity in the absence of
SDS was eluted in about fraction 11, but a small shoulder of Suc-LLVY-MCA degrading activity in
the presence of 0.02 SDS was observed in a lighter fraction. By SDS-PAGE analysis, the rice
26S proteasome gave multiple bands with molecu- lar masses of 25 – 120 kDa Fig. 1, inset. Those
with molecular masses of 25 – 35 kDa were as- sumed to be components of the 20S proteasomal
portion, and those of 42 – 120 kDa were assumed to be components of the regulatory part, called
Fig. 1. Fractionation of the 26S proteasome from cultured rice Oryza sati6a cells by glycerol density-gradient centrifu-
gation analysis. Samples 12 mg protein obtained by Biogel A-1.5m column chromatography were analyzed according to
the procedure described in the text. Suc-LLVY-MCA break- down was measured with or without 0.02 SDS.
The bar at the top indicates the fraction pooled. The inset shows the SDS-PAGE profile of the purified 26S proteasome
from rice cells. Protein was stained with Coomassie Brilliant Blue.
Fig. 2. Electrophoretic analysis of degradation of poly-Ub-
125
I-lysozyme conjugates by the purified 26S proteasome. A The purified 26S proteasome 7.5 mg was incubated with 0.2
mg of poly-Ub-
125
I-lysozyme conjugates for the indicated times in the presence of 10 mM Mg
2 +
and 2 mM ATP. The resulting products were then separated by SDS-PAGE and
detected by a Fuji Bio-imaging analyzer BAS 2000. B Anal- ysis was performed as for A, except that 20 mM EDTA was
added to the reaction mixture instead of Mg
2 +
and ATP. The arrow indicates the position of
125
I-lysozyme.
of the 26S proteasome. The purification method described usually led to
a 14.7-fold increase in specific Suc-LLVY-MCA- degrading activity in the absence of SDS i.e. that
of the 26S proteasome from the crude extract, and the overall yield was approximately 4.0.
3
.
2
. Degradation of ubiquitinated lysozyme by the
26
S proteasome To clarify whether the 26S proteasome purified
from rice is capable of degrading ubiqitinated proteins, we electrophoretically monitored changes
in poly-Ub-
125
I-lysozyme conjugates during incu- bation with the 26S proteasome. Incubation of
poly-Ub-
125
I-lysozyme conjugates with the 26S proteasome in the presence of Mg
2 +
and ATP resulted in a marked loss of material in main
bands approximately 200 kDa of conjugates Fig. 2A, while little significant degradation was
observed without Mg
2 +
Fig. 2B. Moreover, un- modified
125
I-lysozyme was not degraded apprecia- bly when incubated with the 26S proteasome in
the presence of Mg
2 +
and ATP data not shown. These results strongly suggest the idea that the
purified 26S proteasome complex hydrolyzes Ub- ligated proteins ATP dependently.
3
.
3
. Identification of two isoforms of the
26
S proteasome complex
As reported for the enzymes from rat liver [28], rabbit reticulocytes [34], spinach leaves [10], and
yeast [35], the purified rice 26S proteasome gave two protein bands on nondenaturing PAGE Fig.
3A. As shown in Fig. 3B, the anti-26S protea- some antibodies from spinach reacted with both
bands, indicating that the protein is present as two isoforms, because preimmune serum reacted with
neither band data not shown. We named these isoforms rice 26Sa and 26Sb proteasomes, accord-
ing to our nomenclature for the rat liver enzyme [28]. To determine whether these two isoforms
have proteolytic activity, after their separation by electrophoresis, the polyacrylamide gel was over-
loaded with a solution of a fluorogenic substrate Suc-LLVY-MCA and incubated for about 15
min at room temperature, and peptide degrading activity was detected under ultraviolet light. Suc-
LLVY-MCA degrading activity was detected in both bands Fig. 3C. Thus, rice cells contain two
Fig. 3. Electrophoretic and immunological analyses of the 26S proteasome. The purified 26S proteasome 2 mg was elec-
trophoresced in 2.5 polyacrylamide gel – 0.5 agarose gel. A The gel was stained for protein with Coomassie Brilliant
Blue; B immunoblot analysis was performed with anti-26S proteasomal polyclonal antibodies from spinach; C Suc-
LLVY-MCA degrading activity was detected in the gel as described in Section 2. Arrows show the position of the
isoforms of the 26S proteasome 26Sa and 26Sb.
PA700. Pooled active fractions approximately 600 mg protein were used for further characterization
Fig. 4. Effect of pH on three peptidase activities. The degra- dation activities by rice A and spinach B 26S proteasomes
for the indicated substrates were measured at various pH values in the absence of SDS. Values are shown as percent-
ages of the maximum activity.
was 9.0. Comparison of the effects of pH on the rice 26S proteasome with those on the spinach
enzyme Fig. 4B revealed that the effects of pH are similar in monocotyledonous and dicotyle-
donous plants, although the rice 26S proteasome was slightly more active at acidic pH values than
the spinach one.
3
.
5
. Inhibitor sensiti6ity of the purified
26
S proteasome
Chymotrypsin-like and trypsin-like activities of rice and spinach 26S proteasomes were strongly
inhibited by PSI, MG132, and MG115 Table 1. Peptidylglutamyl-peptide hydrolase activities of
rice and spinach 26S proteasomes were inhibited approximately 10 and 50 by these inhibitors,
respectively. Chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolase activities of
these 26S proteasomes were also partially inhibited by 10 mM lactacystin.
3
.
6
. ODC degradation by
26
S proteasomes from plants
Previously, ODC was shown to be degraded by the 26S proteasome purified from rat liver without
ubiquitination, but requiring antizyme and ODC inhibitory protein instead of ubiquitin [29]. We
examined whether plant 26S proteasomes degrade
35
S-labeled ODC in vitro. As shown in Fig. 5, ODC was degraded rapidly by purified rat liver
26S proteasome in an antizyme- and ATP-depen- dent fashion, as previously reported [29]. How-
ever, unexpectedly, no appreciable degradation of ODC by 26S proteasomes from rice and spinach
was observed, even in the presence of antizyme and ATP, indicating strongly that 26S protea-
somes from plants are functionally different from active isoforms of the 26S proteasome, 26Sa and
26Sb.
3
.
4
. Effect of pH on peptidase acti6ities Fig. 4 shows the influence of pH on the activi-
ties of the enzyme with three substrates. In rice 26S proteasome, as shown in Fig. 4A, the opti-
mum pH value for chymotrypsin-like and pep- tidylglutamyl-peptide hydrolase activities was 8.0.
The optimum pH value for trypsin-like activity
Table 1 Effect of inhibitors of rice and spinach 26S proteasomes
a
Compound Concentration
Inhibition Chymotrypsin-like activity
PGPH activity Trypsin-like activity
Rice Spinach
Rice Spinach
Rice Spinach
97.1 9 1.85 98.2 9 1.09
82.2 9 1.27 79.2 9 9.52
10.2 9 1.16 PSI
43.7 9 4.27 10
53.7 9 3.26 7.90 9 3.52
81.7 9 0.99 78.3 9 2.83
MG132 96.8 9 1.79
81.3 9 0.96 10
10 MG115
95.9 9 2.04 96.8 9 0.73
85.1 9 2.97 94.7 9 2.20
25.3 9 6.71 55.2 9 2.81
67.3 9 0.35 16.1 9 1.46
2.60 9 2.54 30.0 9 3.80
23.7 9 3.32 10
71.6 9 1.85 Lactacystin
a
The peptidase assay was performed in the absence of SDS. Results are means 9 S.D. of three independent experiments.
Fig. 5. Degradation of rat ODC by purified 26S proteasomes from rice, spinach and rat. The ATP- and antizyme-depen-
dent degradation of
35
S-labeled ODC with purified 26S proteasomes 1 mg was assayed by measuring the conversion
into TCA-soluble fraction as described in Section 2.
26S proteasome from rice cells, however, could only be isolated with buffers containing 10 mM
leupeptin, as well as 2 mM ATP, since dissociation of the complex into smaller particles occurred very
rapidly in the absence of leupeptin. This dissocia- tion was also suppressed by casein, but not by
pepstatin A or ATP alone data not shown. Therefore, we suggest that some proteases should
be taken into consideration during the purification of 26S proteasomes from plants.
In this work, we have shown that the purified rice 26S proteasome can be separated into two
forms by slab electrophoresis on a nondenaturing polyacrylamide gel Fig. 3. These isoforms proba-
bly include double 26Sa and single 26Sb regu- latory particles, as the spinach 26S proteasome
[10]. Using SDS-PAGE analysis, we found that the enzyme is a multisubunit complex consisting of
subunits with molecular masses of 25 – 120 kDa. The heterogeneity of subunit composition ana-
lyzed by SDS-PAGE is highly conserved among 26S proteasomal complexes isolated from other
sources [11 – 13,35].
In addition, substrate specificity and pH depen- dence of the 26S proteasome indicate that the
enzyme has at least three distinct activities, namely chymotrypsin-like, trypsin-like, and peptidylglu-
tamyl-peptide hydrolase activities. Each of the en- zyme activities exhibits a maximum at neutral and
weakly alkaline pH values, similar to the 20S proteasome from spinach leaves. However, it is
noticeable that three peptidase activities of spinach 20S proteasome exhibit a maximum at higher pH
values than those of rice and spinach 26S protea- somes [7]. Chymotrypsin-like, trypsin-like, and
peptidylglutamyl-peptide hydrolase activities of 26S proteasomes from rice and spinach were sensi-
tive to PSI, MG132, MG115 and lactacystin; how- ever, peptidylglutamyl-peptide hydrolase activity
exhibited only weak sensitivity to them. The sensi- tivities of plant 26S proteasomes are similar to
that of mammalian 26S proteasome.
The purified 26S proteasome complex rapidly degraded poly-Ub-
125
I-lysozyme conjugates in an ATP-dependent fashion Fig. 2, suggesting that
covalent modification
of substrate
proteins through ubiquitination is essential for their prote-
olysis by the 26S proteasome. Recently, we also found a multiubiquitin-binding subunit of the
proteasome, a homolog to S5a, from rice [19]. Thus, the rice 26S proteasome may have the abil-
those from mammals. Nonetheless, they can hy- drolyze Ub-ligated proteins and their subunit mul-
tiplicity is common in the size range of 22 – 120 kDa among all eukaryotic 26S proteasomes from
yeast, plants and mammals [10 – 12,35] this study.
4. Discussion
