Download with the Cell Wall and Evidence that Peroxidase Does

Plant Physiol. (1974) 54, 870-876
An Accounting of Horseradish Peroxidase Isozymes Associated
with the Cell Wall and Evidence that Peroxidase Does
Not Contain Hydroxyprolinet
Received for publication June 11, 1973 and in revised form July
15, 1974
EDWIN H. LIU2 AND DEREK T. A. LAMPORT
Michigan State University-Atomic Energy Commission Plant Research Laboratory, Michigan State University,
East Lansing, Michigan 48823
It is important to determine whether or not hydroxyproline
is
a component of an extracellular enzyme system such as
Isopyenic equilibrium centrifugation techniques were used peroxidase because this amino acid is a major constituent of
to determine whether any horseradish (Amoracia lapathifolia) extensin, the cell wall protein (5). In this investigation, the
peroxidase isozymes were associated with hydroxyproline con- technique of isopycnic equilibrium centrifugation is used to
taining moieties. Purified peroxidase, horseradish root extracts, re-examine the relationship between peroxidase and hydroxyand peroxidase isozymes released from horseradish root cell proline. If peroxidase contains hydroxyproline, then peaks of
walls were tested. In no case could any peak of peroxidase ac- peroxidase activity on cesium chloride gradients should cotivity be found to band with hydroxyproline.
sediment with peaks of hydroxyproline.
A fluorimetric method for measurement of peroxidase activSince peroxidase exists in multiple molecular forms in horseity was used to determine quantitatively the amount of total radish, it is important to determine both quantitatively the
peroxidase located on horseradish root cell walls. Twenty per amount of total peroxidase which is associated with the cell
cent of the total peroxidase is found in the cell wall fraction wall during biochemical extraction and qualitatively the actual
after extraction; 93 % of this cell wall associated peroxidase can isozymes found on the cell wall. It is also important to differbe removed by washing with 2 M NaCl. Some peroxidase entiate those peroxidase isozymes which are bound to the cell
isozymes released by salt washing are not found in the cyto- wall through ionic interactions from peroxidase isozymes which
plasmic extract. This indicates that not all of the ionically are tightly bound to the cell wall and are resistant to salt
bound peroxidase represents cytoplasmic contamination. The washing.
1.4% of the total peroxidase activity can thus be considered
tightly bound to the cell wall. Of this portion, 75% can be
MATERIALS AND METHODS
solubilized by treatment with a cellulase preparation. One
isozyme is released which was not present in the original
Commercially purified horseradish peroxidase was obtained
cytoplasmic extract.
from the Worthington Biochemical Company, Freehold, N. J.
Greenhouse-grown horseradish plants (Amoracia lapathifolia
Gilib. cv. Maliner Kren) were originally purchased as root
cuttings from the W. Atlee Burpee Co., Philadelphia, Pa.
Spectrophotometric Peroxidase Assay. o-Dianisidine in
methanol (0.1 ml of a 1.0% solution) is added to 2.9 ml of
0.05 M sodium phosphate buffer, pH 5.8, containing H,O, at
Peroxidase (EC 1. 11. 1.7) is a heme containing glycopro- a concentration of 9 x 10-' M. The reaction is started by adding
tein which utilizes H,02 to catalyze the oxidation of various 5 ,il of a peroxidase solution and measuring the increase in
hydrogen donors. This enzyme is found both in the cytoplasm absorbance at 460 nm. The reactions are run at 25 C. When
and in the cell wall of plants (1). The extracellular function of this assay is used, 1 unit of enzyme activity is defined as the
peroxidase is important, because this enzyme has been shown change in absorbance at 460 nm of 1 A unit per minute (1
to be responsible for lignin polymerization (5).
unit = 1 A4./ min).
Some horseradish peroxidase isozymes were initially reFluorimetric Peroxidase Assay. This assay monitors the
ported to contain hydroxyproline (12). Further work on the peroxidase catalyzed formation of the fluorescent comhorseradish peroxidase system indicates that the hydroxypro- pound, 2, 2'-dihydroxy-3, 3'-dimethoxybiphenyl-5, 5'-diacetic
line represented a contaminant fraction which could be puri- acid, from homovanillic acid (4). The reaction mixture confied away from the enzyme (13, 17). Other workers have re- sists of 2.9 ml of 0.05 M tris-HCl buffer, pH 8.5, containing
ported hydroxyproline-rich peroxidase in pea cell walls and 9 x 10-' M H,O, and 0.1 ml of 2.5 mg/ml solution of homotheir secretion from the cytoplasm in response to treatment vanillic acid in 0.05 M tris-HCl, pH 8.5. To this solution, 5 A1
with high levels of ethylene (9, 10).
of peroxidase are added, and the increase of fluorescence at
Rem of 425 nm is recorded with Xe. of 315 nm. To obtain standardized
results, a 0.1 mg/ml solution of quinine sulfate in
I
This work was supported by the United States Atomic Energy
0.1 N H,S04 is used to adjust an Aminco-Bowman spectrophoCommission under Contract AT(1 1-1)-1338.
2Present address: Department of Biology, University of South tofluorometer to 0.2 fluorescence unit (FU) before each use.
One unit of enzyme activity by this assay is defined as the.
Carolina, Columbia, S. C. 29208.
ABSTRACT
870
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol.
54, 1974
HORSERADISH PEROXIDASE ISOZYMES
change in fluorescence of 1 FU per minute (1 Unit = 1 FU/
min; Xe. 315, Xem 425).
Electrophoresis of Peroxidase Isozymes. Peroxidase isozymes were resolved by horizontal starch gel electrophoresis
followed by staining with benzidine-H202. The procedures
used have been described in detail elsewhere (1 1).
HydroXYproline Analysis. Colorimetric hydroxyproline analysis is accomplished by oxidation of hydroxyproline with sodium hypobromite followed by reaction with Ehrlich's reagent
(6). Color reaction at 560 nm is then compared to values on a
standard curve.
Cell Wail Preparation. Horseradish cell walls are prepared
by grinding horseradish root slices in a Waring Blendor for
30-sec intervals interspersed with cooling periods for a total
grinding time of 15 min in H20. Temperatures are not allowed
to rise over 8 C. The homogenate is then squeezed through
eight layers of cheesecloth and centrifuged at lOOOg for 20
min in H20. The wall fragments are then placed in a Biichner
funnel and washed with 8 liters of H,O.
Isopycnic Equilibrium Sedimentation of Peroxidases. Peroxidase was subjected to cesium chloride isopycnic centrifugation
according to methods described by Filner and Varner (3).
Three-ml CsCl gradients were employed with an average density of either 1.3 or 1.4. Beef liver catalase (150 enzyme units)
was added to each gradient as a marker. The gradients were
formed by centrifuging at 40,000 rpm for 72 hr in a Beckman 65B ultracentrifuge using the SW-65 swinging bucket
rotor. At the end of the centrifuge run, 3-drop fractions were
collected from the bottom of each tube. The refractive index of
every tenth fraction was recorded and plotted.
Hydroxyproline was assayed in every other fraction from the
CsCl gradient. Since the hydroxyproline assay is sensitive only
to the free amino acid, these fractions were first subjected to
acid hydrolysis. It was estimated that the volume of each fraction was 25 gl. Seventy-five ,ul of 8 N HCl were added to each
fraction, making the final concentration 6 N HCI. The tubes
were sealed and incubated for 18 hr at 105 C. The tubes were
opened and placed in a desiccator jar, where HCl was removed
by evacuation. The volume of these tubes was then brought up
to 1 ml with water, and hydroxyproline was assayed in the
usual manner.
Total peroxidase activity was determined in all the fractions
which were not used for hydroxyproline determination. A
spectrophotometric assay was employed, using o-dianisidine
as the hydrogen donor (12).
Catalase was assayed using an oxygen electrode to monitor
the rate of 02 production.
In order to resolve and estimate the buoyant densities of the
different peroxidase isozymes in CsCl gradients, 5-ll samples
of every other fraction in the gradient were placed on paper
wicks and these were loaded sequentially in a 12% starch gel.
The starch gel was subjected to electrophoresis and stained
for peroxidase activity using benzidine-H202. (This technique
allows the resolution of individual peroxidase isozymes in a
gradient and the estimation of their densities, since the peak
tube of distribution of any individual isozyme will have the
most intense benzidine color reaction.) Peak fractions for the
individual isozymes were determined by densitometer tracings
of photographs of the distribution of each particular isozyme
on the starch gel zymogram (8).
RESULTS
Lack of Association of Peroxidase and Hydroxyproline. In
our investigation with commercially purified horseradish peroxidase, we found that 80% of the hydroxyproline present was
attached to arabinose via an -glycosidic linkage.
871
Isopycnic equilibrium sedimentation centrifugation was used
to test the relation between hydroxyproline and peroxidase in
three horseradish systems: (a) a commercially prepared purified
horseradish peroxidase, (b) the supernatant fraction from a
homogenate of horseradish roots, and (c) peroxidase released
from cell walls by cellulase digestion. It was anticipated that
glycosylated hydroxyproline would probably band with peroxidase only if it were actually a constituent part of the enzyme.
A 20-mg sample of commercially purified horseradish peroxidase (Worthington HRP-HPOD-6FA) was subjected to isopycnic equilibrium centrifugation with CsCl; average p = 1.4.
Beef liver catalase was added as a marker. Three-drop fractions
were collected and total hydroxyproline, peroxidase, and catalase activity were determined across the gradient (Fig. 1).
Every other fraction was subjected to starch gel electrophoresis. Individual peroxidase isozymes were resolved, and the
peak tubes of their distribution were estimated by visual inspection and by densitometry (Fig. 2).
The hydroxyproline distribution does not coincide with peroxidase. The density of the hydroxyproline peak fraction is
1.372, while the density of the peak fraction of total peroxidase
is 1.342. The calculated density of the beef liver catalase is
.1I
;-v
.
*0
.3
£1
ILS
frtn., no.
FIG. 1. CsCl isopycnic equilibrium centrifugation of a 20-mg
sample of purified horseradish peroxidase (Worthington, HRPHPOD-6FA). The gradient is collected from the bottom in 120 three
drop fractions. Peroxidase is assayed spectrophotometrically with
o-dianisidine as hydrogen donor. One peroxidase unit = 1 A40/min.
Catalase is assayed by monitoring the production of oxygen with an
oxygen electrode. One catalase unit = 1 ,umole 02 produced/min.
Hydroxyproline is assayed colorimetrically after acid hydrolysis of
the fraction. Peroxidase (E
A); catalase (0
O); hydroxyproline (0
*).
O); density (0
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
qe2S*-M. ;a0,x.S:'.
872
Anode
::: ..:
... 5 :
....
.,.,... ;.......
*b....:- ...
: :
....
.:
...
:....
..
*.:.. :...:
:.i,... ..
::::... :.....
1
Jkl:
...
;:
4
Plant Physiol. Vol. 54, 1974
LIU AND LAMPORT
::
::
matter. The supernate after centrifugation was used as a source
of peroxidase. This was subjected to isopycnic equilibrium
centrifugation in a 3-ml CsCl gradient with an average density
of 1.33. At the end of the run, 3-drop fractions were collected
in 145 tubes. These tubes were assayed for total peroxidase,
marker catalase, and hydroxyproline.
Peroxidase, catalase, and hydroxyproline were assayed (Fig.
5). The position of the peak of total peroxidase activity is at
fraction 81, and the position of the peak of total catalase activity is at fraction 89. These positions coincide with the peak
fractions of peroxidase and catalase in a gradient of identical
construction which was used to measure the density of cytoplasmic peroxidase. Thus the average density of both the cytoplasmic and cell wall peroxidases are similar. The average
densities of these isozymes is 1.310. If the cell wall peroxidase
isozymes were distinct from cytoplasmic peroxidase by the
attachment of hydroxyproline-arabinosides or by cell wall
fragments, they would have greater average densities than the
cytoplasmic peroxidases.
FIG. 2. Resolution of peroxidase isozymes in a CsCl density
gradient of purified horseradish peroxidase (Worthington, HRPHPOD-6FA) by sequential starch gel electrophoresis. Five-,ul samples of alternate 3-drop fractions are subjected to starch gel electrophoresis. Odd numbered tubes from 25-119 are assayed in this
fashion. Notches are cut in the gel at the position of every fifth
sample. Peroxidase isozymes are visualized with benzidine H202.
There are two broad peaks of hydroxyproline-containing
components in the cellulase digest of horseradish cell walls.
.0
80+1;-
1.291. This demonstrates that most of the peroxidase does not
contain any hydroxyproline. There is some overlap between
hydroxyproline and peroxidase distributions. A zymogram of
the peroxidase isozymes in this gradient shows that the peak
of peroxidase activity plotted across the gradient actually represents a composite of several isozymes of different densities.
The most anodically migrating isozyme is the densest of all
the peroxidase isozymes, and the estimated density of this
isozyme, 1.379, is nearly identical to the density determined for
1.
\V
10
hydroxyproline.
An inspection of the overlap of peroxidase and hydroxyproline on the CsCl gradient shows that the peak of hydroxyproline begins at tube 34, while no peroxidase can be detected at
all until tube 60, at which point the hydroxvproline level is already 70% of maximum. For this reason it is unlikely that the
most anodically migrating isozyme is associated with the hydroxyproline peak.
A sample of the supernatant fraction of a horseradish root
homogenate was subjected to isopycnic equilibrium centrifugation in a 3-ml CsCl gradient with an average density of 1.33.
At the end of the run, 3-drop fractions were collected in 145
tubes. These tubes were assayed for peroxidase, marker catalase
and hydroxyproline. A plot of peroxidase, catalase and hydroxyproline was constructed across the gradient (Fig. 3).
Only one peak of hydroxyproline is seen, and it is near the
bottom of the gradient with an estimated density of 1.459.
This peak of hydroxyproline does not coincide with any peroxidase activity. The peroxidase activity shows a peak with a
density estimated to be 1.310. This is much lighter than the
average density of the purified horseradish peroxidase, but is
probable because the isozyme makeup of the two preparations
is different. A zymogram shows that the buoyant densities of
the cytoplasmic peroxidase isozymes differ, and the most anodically migrating isozyme is the densest (Fig. 4). However,
the density of none of these isozymes coincides with a peak of
hydroxyproline.
The supernate of a cellulase digestion of horseradish root
cell walls was reduced in volume by vacuum evaporation and
then centrifuged at 10,000g for 30 min to pellet particulate
,\
.2
0
a
It
in
.
.00
fracti. no.
FIG. 3. CsCl isopycnic equilibrium centrifugation of the supernatant from a horseradish root homogenate. The gradient is collected from the bottom in 145 three-drop fractions. Peroxidase is
assayed spectrophotometrically with o-dianisidine as hydrogen
donor. One peroxidase unit = 1 A460/min. Catalase is assayed by
monitoring the production of oxygen with an oxygen electrode. One
catalase unit = 1 ,umole 02 produced/min. Hydroxyproline is assayed colorimetrically after acid hydrolysis of the fraction. Peroxi); hydroxyproline (0
O);
dase (A
A); catalase (0
*).
density (0
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
Plant
1-W~ ~ .; ,_.
Physiol. Vol. 54, 1974
HORSERADISH PEROXIDASE ISOZYMES
Anode
g. . . . . :
L
I> Sto~~~~,i "'>. ; . . . . .
.
..
.'],
X~~~~~~.
X
FIG. 4. Resolution of peroxidase isozymes in a CsCl density
gradient of the supernatant of a horseradish root homogenate by
sequential starch gel electrophoresis. Five-,ul samples of alternate
3-drop fractions are subjected to starch gel electrophoresis. Odd
numbered tubes from 45-139 are assayed in this fashion. Notches
are cut in the gel at the position of every fifth sample applied to the
gel. Peroxidase isozymes are visualized with benzidine-H202.
Neither of these two peaks of hydroxyproline-containing material coincide with the peak of peroxidase released from cellulase treatment of horseradish walls, nor do they coincide with
any of the resolved peroxidase isozymes (Fig. 6). The densities
of these two peaks of hydroxyproline-containing material are
1.357 and 1.267.
It is clear that nearly all the hydroxyproline-containing
material released by cellulase treatment of cell walls is not related to peroxidase. Furthermore, the density of peroxidase
from the cell wall is identical to that of cytoplasmic peroxidases, which do not contain hydroxyproline. Therefore, it is
unlikely that the peroxidase from cell walls contains any
hydroxyproline-arabinosides or any dense groups which should
make them distinct from cytoplasmic peroxidase.
Peroxidase Isozymes Bound to Horseradish Root Cell Wails.
The usual sepctrophotometric substrates for peroxidase such
as benzidine and o-dianisidine are useful only for assaying the
soluble enzyme and do not produce reliable results when used
to measure the peroxidatic activity of particulate, insoluble
cell walls. However, the homovanillic acid assay for peroxidase
overcomes the shortcomings of spectrophotometric substrates
in measuring the activity of cell walls. Since this assay is
fluorimetric and measures light production at a given wavelength, particulate materials such as cell walls do not interfere
greatly in the monitoring of this reaction. The biphenyl reaction product also does not appear to bind to the cell walls.
This assay allows the continuous monitoring of fluorescence
production when insoluble cell walls are used as a source of
peroxidase. From these data, initial velocities can be calculated, and the peroxidase activity bound to cell walls can be
determined.
The peroxidase activity of the pooled filtrate and supernatants were determined fluorimetrically. The peroxidase activity of the cell walls were also determined fluorimetrically,
and the specific activity was reported as units/mg dry weight.
Twenty per cent of the total peroxidase found in horseradish
roots is associated with the cell wall fraction (Table I).
As a precaution, the water-washed cell walls were further
washed with 8 liters of distilled water. No change in the per-
873
oxidase specific activity of these walls could be detected. These
walls were then incubated in 750 ml of 2 M NaCl for 2 hr at
4 C with stirring. The slurry was squeezed through eight layers
of cheesecloth, washed again with water, and labeled "saltwashed walls." Peroxidase in the salt-washed walls and the
filtrate from salt washing was assayed with homovanillic acid.
The specific activity of peroxidase on cell walls dropped from
49.3 X 10` units/mg dry weight of wall. 92.6% of the activity
found in water-washed walls can be released by treatment with
2 M NaCl (Table II). Thus 93% of the peroxidase found on
cell walls is ionically bound. Other experiments with salt washing up to 10 M LiCl showed no further release of peroxidase
activity.
Does this peroxidase which can be removed from the cell
walls by salt washing represent cytoplasmic peroxidase which
has become attached during the homogenization period? Because of the charge characteristics of cell walls, which at the
extraction pH of 4.6 primarily represents the carboxyl groups
of uronic acids, this is a possibility. However, a zymogram of
the peroxidases released from cell walls by salt washing (Fig. 7)
does not have the same relative distribution of isozymes as does
2i
I
.1
a
I3
I
301
S
I
fraction
no.
FIG. 5. CsCl isopycnic equilibrium centrifugation of the incubation medium of a cellulase digestion of salt washed horseradish
root cell walls. The gradient is collected from the bottom in 145
three-drop fractions. Peroxidase is assayed spectrophotometrically
with o-dianisidine as hydrogen donor. One peroxidase unit = 1
A4w/min. Catalase is assayed by monitoring the production of oxygen with an oxygen electrode. One catalase unit = 1 ujmole 02
produced/min. Hydroxyproline is assayed colorimetrically after
acid hydrolysis of the fraction. Peroxidase (A -A); catalase
0 ); hydroxyproline (0
(0
0).
O); density (
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
.|!~ ~ . o.s
874
An
;Bi '. .::.
.
.......
LIU AND LAMPORT
on
. .,,.f,
5
Table I. Peroxidase Activity, in Supernatant anid Cell Wall
Fractionis of a Horseradish Root Homnogenate
The assay was made fluorimetrically with homovanillic acid.
Volume
Dry XVt
m
tPtl
g
1275
41.0
Peroxidase
Specific Total
Per- Total
PerActivity
oxidase
oxidase
6.34 units/ml
49.3 X 10-3
units/mg
dry wt
u
itnits
%
8087
2020
80
20
Table II. Peroxidase Activity Released from Horseradish Cell
Walls by Treatmenit with 2 mt NaCl
The assay was made fluorimetrically with homovanillic acid.
V'olume Dry Wit
mnl
H20-washed
Peroxidase
DISCUSSION
There appears to be no correlation between the buoyant
density of horseradish peroxidase and any hydroxyproline containing moiety, either in purified peroxidase, the supernate of
a root homogenate, or the peroxidase isozymes released from
cell walls by cellulase treatment. It is, therefore, unlikely that
any of the peroxidase isozymes contain any hydroxyproline.
These results confirm the work of Shannon's group on cytoplasmic peroxidase isozymes, where hydroxyproline was shown
to be a contaminant (13, 17). Work by Welinder et al. (14-16)
on the complete amino acid sequencing of a horseradish peroxidase isozyme also demonstrates the absence of hydroxyproline.
The most anodically migrating peroxidase isozyme is also
the densest, which suggests that this isozyme contains the greatest percentage of carbohydrate. However, in the purified per-
oxidase preparation, the peak of hydroxyproline extends over
areas of the gradient where absolutely no peroxidase is detected, so it is unlikely that the hydroxyproline present can be
accounted for by this isozyme.
Anode
Toital
Unit
|Activity
Peroxidase Recovery
oxidase Released
49.3 X 10-3
2020
Specific
The peroxidase which was released from cell walls by the
cellulase treatment is soluble since 97% of the peroxidatic activity in the incubation medium is found in the supernatant
fraction after centrifugation for 60 min at 100,000g. A zymogram of the peroxidase isozymes which are released from cell
walls by cellulase treatment shows one isozyme which has no
counterpart in the horseradish root homogenate (Fig. 9).
2-
g
41.0
cell walls
Salt wash
Salt-washed
cell walls
resistant to salt washing can be obtained
treatment (Table III).
FIG. 6. Resolution of peroxidase isozymes in a CsCl density
gradient of the incubation medium of cellulase treated horseradish
root cell walls by sequential starch gel electrophoresis. Five-1ul
samples of alternate 3-drop fractions are subjected to starch gel
electrophoresis. Odd numbered tubes from 45-139 are assayed in
this fashion Notches are cut in the gel at the position of every
fifth sample applied to the gel. Peroxidase isozymes are visualized
with benzidine-H202
Fraction
are
400 ml of a 0.5% solution of a cellulase preparation (Trichoderma viride, Worthing Biochemicals). Peroxidase was released
in the incubation medium (Fig. 8). After 24 hr incubation at 30
C, the specific activity in the medium of cellulase-treated cell
walls rose to 37.2 units/ml when assayed spectrophotometrically with o-dianisidine as the hydrogen donor. This compares
to a final peroxidase activity of 2.3 units/ml in the medium of a
control treatment which did not contain added cellulase.
Seventy-five per cent of the peroxidase activity remaining on
salt-washed horseradish cell walls can be released by cellulase
6
7
Supernatant
Cell wall
cell walls and
by treating the salt-washed walls with cellulase. Salt-washed
horseradish cell wall (11.25 g [dry weight]) were incubated in
04
Fraction
Plant Physiol. Vol. 54, 1974
units/mg
750
41.0
dry wt
2.64 units/ml 1980
3.66 X 10-3 150
4-
93
--
7
-
106
units/mg
dry wt
an equivalent amount of enzyme from the tissue homogenate.
In fact, there are two isozymes found in the peroxidase released
from cell walls that have no counterparts in the cytoplasmic
peroxidases. The peroxidase isozyme pattern of either sample is
not altered either by incubation in 2 M NaCl or its removal by
dialysis.
Identification of the peroxidase isozymes which
5
are
bound
1
2
3
4
FIG. 7. Peroxidase isozymes associated with horseradish root cell
walls. Samples are adjusted to identical specific activities using the
spectrophotometric o-dianisidine assay. Peroxidase activity of
samples is 390 units/ml. 1 and 2: Supernatant fraction from a
2 M NaCl washing of horseradish root cell walls; 3 and 4: supernatant from a homogenate of horseradish root tissue slices.
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Copyright © 1974 American Society of Plant Biologists. All rights reserved.
16-'_.:X.Q,_._
875
HORSERADISH PEROXIDASE ISOZYMES
Plant Physiol. Vol. 54, 1974
Anode
M-
..s
...i ....
.........
,.............
....
..:::
2-
)_
@
v7
c
>
4
.:
J
:.
C :
t_
@b
:_S
s F
7 -- '.e
.:.
......
.*
..
:
.
1
2
FIG. 9. Peroxidase isozymes released from salt-washed horseradish root cell walls by treatment with cellulase. 1: Supematant
fraction from a homogenate of horseradish root tissue; 2: peroxidase in the incubation medium of a 24-hr cellulase digestion of 2
M NaCl-washed horseradish root cell walls.
how,
3
20
10
Incubatio
FIG. 8. Release of peroxidase into the incubation medium of
cellulase treated horseradish root cell walls (0
O). 11.25 g of
walls incubated in 400 ml of 0.05 M acetate buffer, pH 5.5, con0). 11.25 g of walls incubated in
taining 2 g of cellulase (0
400 ml of 0.05 M acetate buffer, pH 5.5, containing no cellulase.
cellulase treatment, the sum of peroxidase found in the supernate and remaining on walls is 108% of the peroxidase activity
on walls before treatment. These calculations show that the
fluorescent homovanillic acid peroxidase assay is a valid
method of determining the peroxidatic activity of cell walls to
within 10%.
The estimation that 20% of the total horseradish peroxidase
is associated with the cell wall includes, of course, all the cytoplasmic peroxidase which has become attached to the cell wall
during the homogenization period. This does not, however,
represent a general contamination of cellular peroxidase. In
summary, 80% of the total peroxidase of horseradish roots is
found in cytoplasmic extracts, 18.6% is found ionically associated with the cell wall, and 1.4% is tightly bound to the cell
wall.
Peroxidase Released from 2 u NaCl-washed Cell Walls
by Treatmenzt with Cellulase for 24 Hr at 30 C
The assay was made fluorimetrically with homovanillic acid.
Table
III.
Fraction
V'olume Dry WVt
ml
Salt-washed
cell walls
Cellulase supernatant
Cellulasetreated cell
walls
400
Peroxidase
Specific
Activity
Ttl
Per-
Peroxidase Recovery
oiaeReleased
g
%
11.25 3.66 X 10-3 41.3
units/mg
dry wt
84.3 X 10-3 33.7
units/ml
4.52 2.44 X 10-3 11.0
units/mg
I
dry wt
75.41
J
108
The density of peroxidase released from cell walls by cellulase treatment coincides with the density of cytoplasmic peroxidases. It is unlikely that these wall isozymes differ from their
cytoplasmic counterparts by the covalent addition of any
hydroxyproline containing other cell wall related fragments.
After salt washing, the total activity found in the walls and
supernatant fraction amounts to 106% of the activity calculated to be on the cell walls before the washing step. After
LITERATURE CITED
1. DEJO-NG. D. W. 1967. An inv-estigation of the role of plant peroxidase in cell
wall dev-elopment by the histochemical method. J. Histochem. Cytochem.
15: 335-346.
2. DEJONG, D. W., E. F. JANSEN, AND A. C. OLSON. 1967. Oxidoreductive and
hydrolytic enzyme patterns in plant cell suspension cells. Exp. Cell Res.
47: 139-156.
3. FILNER, P. AND J. E. VARN-ER. 1967. A test for de novo synthesis of enzymes:
density labeling with H2018 of barley amylase induced by gibberellic acid.
Proc. Nat. Acad. Sei. U.S.A. 58: 1520-1526.
4. GuILBAULT, GEORGE C.. P. BRIGNAC, AND M. ZINIMER. 1968. Homovanillic
acid as a fluorimetric substrate for oxidative enzymes. Anal. Chem. 40:
190-196.
5. HARKIN-, J. M. AND J. R. OBST. 1973. Lignification in trees: Indication of
exclusiv-e peroxidase participation. Science 180: 296-298.
6. LANIPORT, D. T. A. 1963. Oxygen fixation into hydroxyproline of plant
cell wall protein. J. Biol. Chem. 238: 1438-1440.
7. LANIPORT, D. T. A. 1969. The isolation and partial characterization of
hydroxyproline-rich glycopeptides obtained by enzymic degradation of
primary cell walls. Biochemistry 8: 1955-1961.
8. QUAIL, P. H. AND J. E. VAR-NER. 1971. Combined gradient-gel electrophoresis
procedures for determining buoyant densities or sedimentation coefficients
of all multiple forms of an enzyme simultaneously. Anal. Biochem. 39:
344-355.
9. RIDGE, I. AN-D D. OSBORNE. 1970. Hydroxyproline and peroxidases in cell
walls of Pi.sum saticum: regulation by ethylene. J. Exp. Bot. 21: 843856.
10. RIDGE, I. AND D. J. OSBORNE. 1971. Role of peroxidase when hydroxyprolinerich protein in plant cell walls is increased by ethylene. Nature New Biol.
229: 205-208.
11. SCANnDALIOS, J. G. 1969. Genetic control of multiple molecular forms of
enzymes in plants. Biochem. Genet. 3: 37-79.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1974 American Society of Plant Biologists. All rights reserved.
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LIU AND LAMPORT
12. SHANTNON, L. M., E. KAY, AND J. LEW. 1966. Peroxidase isozymes form
horseradish roots. I. Isolation and physical properties. J. Biol. Chem. 241:
2166-2172.
13. SHILi, J. H. C., L. 'M. SHANNON\, E. KAY, AND J. Y. LEW. 1971. Peroxidase
isoenzymes from horseradish roots. IN. Structural relationships. J. Biol.
Chem. 246: 4546-4551.
14. WELINDER, K. G., L. B. SMIILLIE, AND G. R. SCHONBAU.M. 1972. Amino acid
sequence studies of horseradish peroxidase. I. Tryptic peptides. Can. J.
Biechem1. 50: 44-62.
Plant Physiol. Vol. 54, 1974
15. WELINDER, K. G. AND L. B. SMIsLLIE. 1972. Amino acid sequence stu(dies
of horseradish peroxidase. II. Tlhermolytic peptides. Can. J. Bioclhem.
50: 63-90.
16. WELINDER, K. G. 1973. Amino acicl sequence studies of lhorser-adish
peroxidase. Tryptic glycopeptide containing two histi(dine residuies and a
disulfide bridge. FEBS Lett. 30: 243-245.
17. WHITELAND, J., E. KAY, J. LEW, AND L. M. SHA-NNON. 1971. PreparatiVe
column electrophloresis in apparattus using Seplhadlex G-25. Anal. Bicclhem.
40: 287-291.
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 1974 American Society of Plant Biologists. All rights reserved.