Plant Physiol. (1983) 71, 742-746
0032-0889/83/7 1/0742/05/$00.50/0
Alcohol Dehydrogenase Inactivator from Rice Seedlings'
PROPERTIES AND INTRACELLULAR LOCATION
Received for publication August 13, 1982 and in revised form November 29, 1982
SHOJI SHIMOMURA AND HARRY BEEVERS
Biology Department, University of California, Santa Cruz, California 95064
ABSTRACT
The alcohol dehydrogenase (ADH) inactivator from aerobically grown
rice (Oryza sativa) coleoptiles was shown to be associated with membranes
which were recovered in sucrose gradients at peak density 1.13 grams per
cubic centimeter.When Mge was included in the gradient, the inactivator
was recovered at peak density 1.16 grams per cubic centimeter coinciding
with the marker enzyme for endoplasmic reticulum, antimycin A-insensitive
NADH cytochrome c reductase. ADH was recovered exclusively in cytosol
fractions. The inactivator attacks ADH from several plant sources and
from yeast. There was no evidence for proteolysis when pure yeast ADH
was inactivated by the inactivator, but there was a loss of SH groups from
ADH during inactivation which was restored after incubation with dithiothreitol under denaturing conditions. The inactivator did not attack other
SH enzymes tested but did result in loss of SH groups from glutathione
and dithlothreitol which prevent ADH inactivation. When 02 was removed
from the inactivator assay medium, the inactivation as well as the loss of
SH groups from yeast ADH was significantly depressed.
In a previous paper, the inactivation of ADH3 by a component
from rice seedlings was described (15). This inactivator was produced only under aerobic conditions in roots and shoots. A
regulatory role on the intracellular level of ADH was suggested
for the inactivator in experiments in which seedlings were temporarily exposed to N2 and returned to air. However, in the crude
extract from aerobic seedlings, the amount of inactivator is sufficient to inactivate the ADH completely within 1 h, while in vivo
both components coexist for long periods without net loss of ADH
activity. Therefore, it was argued that they might normally be
separately compartmented.
In this paper, we show the intracellular localization of the ADH
inactivator and ADH and investigate the mechanism of inactivation.
MATERIALS AND METHODS
Seed Germination. Hulled seeds of rice (Oryza sativa cv S-6)
were sterilized as described previously (15) and germinated in
sterilized 250-ml Erlenmeyer flasks (50 seeds/flask) containing
two sheets of filter paper and 10 ml of deionized H20 in the dark
and at room temperature. For anaerobic experiments, high purity
N2 gas was passed continuously through the flasks.
Sucrose Density Gradient Centrifugation. One hundred coleoptiles were chopped for 25 min with razor blades in 4.5 ml of
chilled grinding medium composed of 13% (w/w) sucrose, 0.15 M
Tricine KOH (pH 7.5), 10 mM KCI, and the indicated concentra' Supported by Department of Energy contract EY 76-S-03-0034.
2
Recipient of a travel grant from the Institute of Scientific and Industrial Research, Osaka University.
3Abbreviation: ADH, alcohol dehydrogenase.
tions of EDTA, MgCl2, and DTT. The homogenate was filtered
through one layer of nylon cloth and centrifuged at 270g for 10
min to remove cell debris. The homogenate (4.0 ml) was layered
on a gradient composed of 2 ml of 15% (w/w) sucrose and 12 ml
of sucrose solution increasing linearly in concentration from 15 to
60%o (w/w) in an 18-ml cellulose nitrate tube. All sucrose solutions
contain 1 mm EDTA (pH 7.5) unless otherwise stated. The gradients were centrifuged at 21,000 rpm for 4 h in a Beckman L265B centrifuge (Spinco SW 27-1 rotor) at 4°C, and 0.4-ml fractions
were collected using an ISCO density gradient fractionator, model
640. Sucrose content of the fractions was determined refractometrically.
Enzyme Assays. ADH activity was measured spectrophotometrically as described previously (15). The assay solution (1 ml)
contained 0.1 M sodium glycine (pH 9.0), 0.2 mi NAD, and 0.1
M ethanol for the plant ADH, and, in addition, 1 mg/ml BSA and
0.5 mM EDTA for yeast ADH.
ADH inactivator activity was measured as described previously
(15). The assay medium contained 10 mm Tris-HCl buffer (pH
7.5) for plant ADH and 10 mm Tris-HCl plus 1 nm EDTA (pH
7.5) for yeast ADH.
Other enzyme assays were those described in the literature as
follows: aldolase (3), glutamate dehydrogenase (20), lactate dehydrogenase (16), catalase (10), NADH Cyt c reductase (9), and
fumarase (12).
Preparation of ADH Inactivator. One hundred coleoptiles from
aerobically grown 7-d-old rice seedlings were homogenized in a
chilled mortar using 10 ml 10 mm Tris-HCl, 1 mm EDTA (pH
7.5). The homogenate was centrifuged at 27,000g for 30 min at
4°C. In the previous paper (15), the supernatant solution was used
as the source of inactivator, but, inasmuch as the precipitate was
found to contain >70%o of the inactivator, this was used as a
convenient and concentrated source for the experiments in the
present paper. The precipitate was again homogenized in 10 ml of
the above buffer and then centrifuged. The precipitate was mixed
with 5 ml 10 mm Tris-HCl, 1 mm EDTA, 0.2% Triton X-100 (pH
7.5). After 30 min at 0°C, the slurry was centrifuged. The supernatant was dialyzed against 500 ml 10 mm Tris-HCl, 1 mim EDTA
at 4°C overnight, and used as ADH inactivator.
Other Methods. BSA (20 mg/ml) in 10 mm Tris-HCl buffer
(pH 7.5) was modified with a 10-fold molar excess of iodoacetamide for 1.5 h at 30°C in the dark and then dialyzed against the
same buffer. The dialysis buffer was exchanged four times to
remove unreacted iodoacetamide. The content of thiol groups was
determined with 5,5'-dithiobis(2-nitrobenzoic acid) (1) in the presence of 7.2 M urea. SDS-polyacrylamide gel electrophoresis was
carried out by the method of Weber et aL (19).
Chemicals. Yeast ADH (crystallized and lyophilized powder)
was purchased from Sigma, and dissolved in 10 mM Tris-HCl, 1
mM EDTA (pH 7.5). The concentration of yeast ADH was determined spectrophotometrically using an absorption index A % nm
= 12.6 (4), or using the Bio-Rad Protein Assay kit. BSA (>99%
pure) was obtained from Calbiochem. Aldolase from rabbit mus-
742
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743
ADH INACTIVATOR FROM RICE
similar to reported values for ER membranes (1 1), but the distribution of the inactivator does not coincide precisely with that of
the ER marker NADH Cyt c reductase (antimycin insensitive).
Nevertheless, when Mg2' was included in the gradient (9, 13),
both components were recovered together at peak density 1.16 g/
RESULTS
cm3
(Fig. 2). No loss of inactivator occurred during centrifugation
Intracellular Localization. The subcellular distribution of ADH
gradients.
and its inactivator was investigated by sucrose density gradient onThe
activity in peak fractions from the sucrose
centrifugation of extracts made with osmotic protection. In co- gradientinactivator
after disruption of the membranes
not
enhanced
was
leoptiles from aerobically germinated 7-d-old rice seedlings, al- either by osmotic shock or by
of detergent (Table I).
most all of the ADH activity was detected in the supernatant When the rice coleoptiles were addition
homogenized in grinding buffer
fractions (Fig. IA). When aerobic 5-d-old rice seedlings were without sucrose and the homogenate
was centrifuged at 27,000g,
subjected to anaerobic treatment for 2 d, the ADH activity of the about 20%o of inhibitor activity was recovered
in the supernatant
coleoptile extract was considerably larger than that of the aerobic fraction.
centrifuon
declines
This
prolonged
however,
activity,
coleoptile extract, but again no significant activity was present in gation and, furthermore, when (NH4)2SO4 was added to bring
the
the particulate fractions of the gradient (Fig. IA).
was
salted
of
the
inactivator
all
to
35%
of
saturation,
concentration
The marker enzymes of mitochondria, fumarase (Fig. 1 B), and
antimycin A-sensitive NADH Cyt c reductase (Fig. IE) were out. The inactivator is solubilized by addition of 0.1% Triton Xpresent in a fraction at peak density 1.16 g/cm3, and the peak 100 with 80% recovery, but high concentrations of Triton X-100
shifted slightly to 1.17 g/cm3 due to the anaerobic treatment. This lead to inactivation. Deoxycholate (1%) solubilizes the inactivator
shift may coincide with the fact that the mitochondrial ultrastruc- but cholate inactivates.
Properties of the ADH Inactivator. The inactivator shows opture, its shape and size, change on transfer of aerobically grown
timum activity at acidic pH (Fig. 3). It is unstable at 30°C and the
rice seedlings to conditions of anoxia (18).
Catalase, the marker enzyme of microbodies, is found in a addition of 10 mM MgCl2, 30%o glycerol, 10%Yo acetone or 0.2 M KCI
fraction at peak density 1.23 g/cm3 as well as in the supernatant further decrease the stability. However, addition of EDTA or
dialysis significantly increase the stability. Ninety-eight % of the
fraction and near the density 1.17 g/cm3 (Fig. 1, C and F).
The ADH inactivator was detected mainly in a fraction at peak inactivator was lost after boiling for 3 min. The inactivator atdensity 1.13 g/cm3 and to a lesser degree near the top of the tacked ADH from oats, wheat, corn, soybean, pea, and watergradient (Fig. ID). The inactivator activity of the coleoptile from melon seeds (Table II) as well as yeast ADH (Table III). The
seedlings exposed to anoxia is smaller than that of the aerobic inactivator activity was depressed by DTT, mercaptoethanol, and
coleoptile (15) but it behaved similarly during centrifugation (Fig. reduced GSH (Fig. 4). BSA also depressed activity (Fig. 4), but
ID). The peak density (1.13 g/cm3) of the ADH inactivator is this was not due to its constituent SH groups since modified BSA,
cle, glutamate dehydrogenase from bovine liver, lactate dehydrogenase from bovine heart, and 5,5'-dithiobis(2-nitrobenzoic acid)
were from Sigma.
0
0
0-
0
0
0
U
Uf)
0
20
30
10
Fraction Number
40
0
30 40
10
20
Fraction Number
FIG. 1. Distribution of (A) ADH, (B) fumarase, (C) catalase, (D) ADH inactivator, (E) NADH Cyt c reductase ([O, *1, antimycin A-insensitive;
[A, Al, antimycin A-sensitive), (F) catalase in sucrose gradients of rice coleoptile extracts. (0, A), data from 7-d-old aerobic seedlings; (@, A), data from
5-d-old aerobic seedlings exposed to N2 for an additional 2 d. A-C, 5 mm DTT and 10 mM EDTA were present in the grinding medium and I mM DTT
and I mM EDTA in the gradient to suppress ADH inactivation during centrifugation. D-F, DTT was omitted from grinding medium and gradient.
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744
SHIMOMURA AND BEEVERS
E
6
0
CY
_
4
a
.a
2
N
0
I
Plant Physiol. Vol. 71, 1983
I
I
I
801
._11
4-
I00
4
0
4
c
601
\\ lnactivator
4-
0
5
- 100
0
401
(Activity)
0
0
a
201
-
\
I
50
I
0
(I
4
I
-4 \
M
5
6
0
7
8
9
0o
pH
S
0
FIG. 3. Effect of pH on ADH inactivator activity from rice coleoptiles
(A) and on ADH activity in the crude extract from rice seeds (0).
00
C0
Table II. Effect of the Inactivatorfrom Rice Seedlings on ADHfrom
Different Seeds
For rice ADH, the crude extract from dry seeds was used; the other
seeds were soaked in water for I d before preparation of extracts. Solutions
containing 0.2 unit ADH/ml were mixed with 17 units/ml inactivator and
incubated at 30°C for 40 min.
Substrate ADH
Residual ADH Activity
50
2:
S
0
4
z
I
5ADH (Stability)
14
1001
I
I
1
10
20
30
40
50
60
Concentration of Sucrose (%,w/w)
FIG. 2. Effect of MgCi2 on distribution of components in the sucrose
gradient. A, A at 280 nm; B, ADH inactivator; C, antimycin A-insensitive
NADH Cyt c reductase. Coleoptiles from 6-d-old aerobic seedlings were
homogenized in grinding medium containing 10 mm EDTA and centrifuged on a sucrose gradient containing 0.15 M Tricine, 10 mm KC1, and I
mM EDTA, pH 7.5 (0). Coleoptiles homogenized in grinding medium
containing I mM EDTA, I mM MgCI2, and centrifuged on a sucrose
containing 0.15 mM Tricine, 10 mm KCI, 1 mm EDTA, and 3 mm MgC12
(*)Table I. Effect of Treatments on ADH Inactivator in Membrane Fractions
ADH inactivator (4.2 units/ml) in peak fractions (30%o sucrose) after
sucrose density centrifugation (Fig. 2) was mixed with 0.27 unit/ml rice
ADH under the indicated assay conditions. All incubations contained 10
iM Tris-HCI, pH 7.5.
Relative Inactivator Activity
Assay Conditions
Sucrose, 0.75%
Sucrose, 30%
Sucrose, 0.75%; Triton X-100, 0.05%
Sucrose, 30%; Triton X-100, 0.05%
92
100
80
92
in which 94% of the SH groups had been reacted with iodoacetamide, produced the same effect. Aldolase, glutamate dehydrogenase, and lactate dehydrogenase depend on SH groups for their
activity (6, 14, 20), but these enzymes were completely unaffected
by the ADH inactivator (Table III).
Mechanism of Inactivation. When yeast ADH was incubated
Rice
Oats
Wheat
Corn
Soybean
Pea
Watermelon
1.0
0.0
3.5
6.1
13.5
41.1
66.8
Table III. Substrate Specfifcity of Rice Coleoptile ADH Inactivator
Each substrate solution (0.5-0.7 mg/ml) was mixed with 74 units/ml of
rice inactivator and incubated at 30°C for 4 h.
Protein as Substrate
ADH from yeast
Aldolase from rabbit muscle
Glutamate dehydrogenase from bovine liver
Lactate dehydrogenase from bovine heart
Residual Enzyme
Activity
32
100
96
92
with the inactivator, there was a progressive loss of SH groups in
parallel with the loss of enzyme activity (Fig. 5) and, as shown in
Table IV, SH groups of DTT, reduced GSH and BSA are also
modified by the inactivator. The removal of 02 from the assay
medium strongly depressed inactivation and at the same time
prevented most of the loss of SH groups in the ADH (Table V).
When inactivated yeast ADH was incubated with excess DTT in
denaturing conditions, the SH groups were reformed (Table VI).
To investigate the possible proteolytic action of the inactivator,
yeast ADH treated with the inactivator was analyzed by SDS gel
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745
ADH INACTIVATOR FROM RICE
120
w
.-1-%
U
0
4-A
C
d
t
80~~~~~~~
BSA
0~~~~~~~~
~~~~GSH
00
DTT
o1
10-0Os
104
1-
o-
ol
l
|
o40-
4~~
~~~~
~~~~~~~~~A
>
0
20
~~~~~~~A
cr
i0-6
0
10-
io-
Concentration CM)for SH
FIG. 4. Effect of DTT
crude extract of rice seeds
(0, 0),
as
GSH
(V), BSA (A, A),
substrate for ADH
and modified BSA
inactivator-, (0, A),
i03
10-2
io-1
10
I
Compounds,( mg/rn I)for
BSA
(B)
coleoptiles. (0, A, V, [1),
on
ADH inactivator from rice
those from yeast ADH
as
mol, and the BSA modified with iodoacetamide (see "Materials and Methods") contained 0.036 mol SH/mol. The scale
for DTT and GSH, and
mg/mi
data
using
substrate. The intact BSA contained 0.60 mol SH
on
the abscissa is in
the
groups/
molarity
for BSA.
Table IV. Effect of the ADH Inactivatorfrom Rice Coleoptiles on the SH
Content of Various Components
Each component was incubated with 104 units/ml inactivator for 4 h at
30°C and the loss of SH measured.
Residual SH Content
Component Exposed to ADH Inactivator
30
82
54
55
Yeast ADH, 0.52 mg/ml
BSA, 5 mg/ml
DTT, 0.1 mM
GSH, 0.1 mM
Table V. Effect of Removing 02 on ADH Inactivator
Yeast ADH (0.51 mg/ml) and rice coleoptile ADH inactivator (150
units/ml) were incubated as shown at 30°C for 4 h. For the anaerobic
treatment, ADH and the inactivator were separately placed in the two
compartments of a Thunberg tube. The tube was evacuated and filled with
N2 gas and the process repeated three times before mixing.
Sample
Residual ADH
Residual SH
ADH
ADH plus inactivator
ADH plus inactivator, N2
86
14
64
93
30
70
Activity
Table VI. Reversibility of SH Modification by the ADH Inactivator
Yeast ADH (0.53 mg/ml) and the ADH inactivator from rice coleoptiles
(150 units/ml) were incubated separately and together at 30°C for 4 h.
The reactions were stopped with TCA. The precipitate was dissolved in 8
M urea, 0.1 M Tris-HCL 0.05 M EDTA (pH 8.3), and the solution was
divided into two parts. The SH content of one part was immediately
measured ('before DTT treatment') and the other part was incubated with
50 mM DTT for 3 h and then mixed with TCA. The precipitate was
washed three times with TCA solution to remove DTT, dissolved in the
urea solution, and the SH content was determined ('after DTT treatment').
Sample
SH Content
A412/Mg protein
Inactivator
Before DTT treatment
0.06
After DTT treatment
0.08
ADH
Before DTT treatment
1.65
After DTT treatment
1.92
ADH plus inactivator
Before DTT treatment
0.28
After DTT treatment
1.81
substrate ADH and inactivator concentration. Thus the inactivators have the properties of enzymes. However the maize protein is
partially dialyzable (mol wt 10,000) rather heat resistant and
shows optimum activity at neutral pH, whereas the inactivator
from rice is retained on dialysis, is rapidly denatured at 100°C
and shows optimum activity at acidic pH. DTT strongly depresses
the activity of the inactivator from rice but is less effective in
maize; Lai and Scandalios (8) used 1 mm DTT in their assay
system, and at this concentration the rice inactivator is ineffective.
In this regard, the rice inactivator appears more similar to that
described from pea cotyledons (17).
The maize inactivator has been purified (8) and, from sucrose
density analysis of extracts, it was deduced to be a soluble cytosolic
component. However, when rice extracts prepared in the presence
of sucrose were centrifuged on sucrose density gradients, most of
-
electrophoresis. No loss of protein from the ADH band or approteolytic products was observed even after prolonged incubation (25 h at 30°C) when the ADH had lost 77%
of its original activity.
pearance of
DISCUSSION
In some respects the ADH inactivator from rice seedlings is
similar to that described by Scandalios and his coworkers from
maize (5, 7, 8). Neither is a general enzyme inactivator but both
attack ADH from several sources. Both act in a time and temperature dependent fashion and the reaction is dependent on
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746
Plant Physiol. Vol. 71, 1983
SHIMOMURA AND BEEVERS
E
.'
80
-60
,~40A
_
20
c'
C
A
0.1
The precise mechanism of interaction of inactivator and ADH
cannot be deduced from the results presented, but the requirement
for 02 suggests that this is the ultimate acceptor of reducing
equivalents from SH groups. The fact that aldolase, glutamate
dehydrogenase, and lactate dehydrogenase with essential cysteinyl
groups are not affected by the inactivator and that, when strongly
inactivated, the ADH is not reactivated by DTT unless the enzyme
is denatured, indicate that the reaction is more complex than
simple disulfide bond formation.
Several features of the inactivation process are similar to those
reported by Francis and Ballard (2) for cytosolic enzymes by a
microsomal fraction from rat liver. These authors showed a progressive loss of SH groups from enzymes which was 02 dependent
and observed the same responses to DTT that were shown by the
rice system. They suggest that the mixed function oxidase may be
involved in the formation of disulfide groups during inactivation.
In a previous paper, it was emphasized that ADH and potentially completely destructive levels of inactivator are present together in several organs of the rice seedling, and yet active ADH
can be extracted, suggesting that inactivator and ADH are separately compartmented within the cells (15). The finding that most
of the inactivator is bound to membranes (Figs. 1 and 2) while the
ADH is cytosolic, is clearly in support ofthis suggestion. However,
in the active fractions from the gradient, the inactivator had direct
access to added ADH. If the compartmentation in vivo is to be
effective, it must also be supposed that the inactivator is bound to
the inner surface of membranes, that is, not in contact with the
cytosolic ADH.
Acknowledgment-We thank Dr. D. E. Seaman, University of California Rice
Experiment Station, Biggs, CA for supplying the rice seeds.
LITERATURE CITED
0
0
2
4
6
8
Incubation Time (h)
FIG. 5. Time course of inactivation (A) and loss of SH groups from
ADH (B) during incubation with ADH inactivator from rice coleoptiles.
(A), 0.54 mg/ml yeast ADH plus 63 units/ml inactivator; (0), yeast ADH;
(0), inactivator.
the ADH inactivator was recovered within the gradient and was
membrane bound. The inactivator was not released from the
membranes by osmotic shock but was released by Triton X-100
and deoxycholate.
Again, the mechanism of action of the rice ADH inactivator is
different from that in maize. Scandalios and his coworkers (8)
suggested that the maize inactivator, while not a general proteinase, acted proteolytically on ADH. We found no evidence for
proteolysis when yeast ADH was treated with the inactivator from
rice. However, it was shown that, accompanying the loss of ADH
activity, there was a loss of SH groups, and the residual activity
was in proportion to the surviving SH in the ADH. Thus, the
inactivator appears to function by SH modification and it was
shown that the SH groups in DTT and reduced GSH, which
protect ADH from inactivation, are themselves modified by the
inactivator. Thus, the inactivator appears to act through SH
reactions, and the protectants could conceivably be effective by
offering competing substrates for the inactivator. The interactions
with SH compounds raise the possibility that these may be significant in regulating ADH levels in vivo.
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