proteasome subunits, including all of the 20S proteasome, have been reported [14 – 20].
The properties of some enzymes are distinct between monocotyledonous and dicotyledonous
plants; for example, acetyl-CoA carboxylase is particularly distinct in gramineae such as rice [21].
Although there have been a considerable number of genetic studies on rice [14,17,19,22], little is
known of the purification and properties of the rice 26S proteasome. The relationship between the
properties of rice and spinach 26S proteasomes has never been clarified. In this paper, we report
the purification and some properties of the 26S proteasome from cultured rice cells, compare the
properties of rice and spinach 26S proteasomes, and discuss the functional differences between
higher plant and mammalian 26S proteasomes.
2. Materials and methods
2
.
1
. Materials The materials used were as follows: succinyl-
Leu-Leu-Val-Tyr-4-methyl-coumaryl-7 amide
Suc-LLVY-MCA Peptide
Institute, Minoh,
Japan; t-butyloxycarbonyl-Phe-Ser-Arg-4-methyl- coumaryl-7 amide Boc-FSR-MCA Peptide Insti-
tute; carbobenzoxy-Leu-Leu-Glu-4-methyl-cou-
maryl-7 amide Z-LLE-MCA Peptide Institute; Biogel A-1.5m Bio-Rad Laboratories, Richmond,
CA, USA; carbobenzoxy-Ile-GluOBut-Ala-Leu- H aldehyde PSI Peptide Institute, carboben-
zoxy-Leu-Leu-Leu-H
aldehyde MG132
Peptide Institute; carbobenzoxy-Leu-Leu-Nva-H MG115 Peptide Institute; lactacystin Kyowa
medix, Tokyo, Japan; leupeptin Peptide Insti- tute; ATP Oriental Yeast, Osaka, Japan; ubiqui-
tin Ub Sigma; and Na
125
I New England Nuclear.
2
.
2
. Plant materials Suspension cultures of rice cells Oryza sati6a L.
cv. Nipponbare were carried out according to Muller and Grafe [23]. The growth of rice cells
was initiated by cultivation of seed embryos [24]. After transfer to fresh media, the cells were sub-
cultured with shaking at 100 rpm. Protein concen- tration was determined by the method of Bradford
[25] with bovine serum albumin as standard.
2
.
3
. Assay of peptidase acti6ity Fluorogenic substrates, such as Suc-LLVY-
MCA, Z-LLE-MCA, or Boc-FSR-MCA were in- cubated with a test preparation for 10 min at 37°C
in the presence or absence of 0.02 sodium dode- cyl sulfate SDS in 100 mM Tris – HCl pH 8.0,
as described previously [26]. The reaction was stopped by the addition of 100 ml 10 SDS and 2
ml 100 mM Tris – HCl pH 9.0, and the fluores- cence of the reaction products was measured.
Protease inhibitors, such as PSI, MG132, MG115, or lactacystin were dissolved in dimethyl sulfoxide
and added at a final concentration of 10 mM.
2
.
4
. Assay of protease acti6ity About 18,000 cpm of poly-Ub-
125
I-lysozyme conjugates were incubated at 37°C for 0 – 60 min
in a total volume of 100 ml reaction mixture con- sisting of 50 mM Tris – HCl pH 7.8, 10 mM
MgCl
2
, 2 mM ATP, 1 mM dithiothreitol, and a suitable amount of test preparation. The reaction
was then terminated by adding the SDS sample buffer. For exact measurement of the activity
without Mg
2 +
, 20 mM EDTA was added to the assay mixture. The method for preparation of
radiolabeled Ub-lysozyme conjugates was as previ- ously described [27].
2
.
5
. Purification of
26
S proteasomes from spinach and rat
The 26S proteasomes from spinach leaves [10] and rat liver [28] were purified as described
previously.
2
.
6
. Assay of ornithine decarboxylase degrading acti6ity
Degradation of ornithine decarboxylase ODC in vitro was determined in the presence of recom-
binant antizyme and ATP, as described previously [29].
35
S-ODC was prepared by in vitro translation using rabbit reticulocyte lysate Wako, Osaka,
Japan, rat ODC mRNA and
35
S-labeled methion- ine
DuPont NEN,
and purified
by im-
munoaffinity chromatography
as described
previously [30].
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
