Download 363 Isoelectric focusing of indoleacetic acid degrading enzymes

Isoelectric
acid
focusing
degrading
of
indoleacetic
enzymes
from
pea
roots.
C.A.
van
Botanisch
der
Mast
Utrecht
Laboratorium,
SUMMARY
In
homogenates
IAA. It
protein
neutral
as
at
complex
least
of
3
other
commonly displayed by
two
an
possesses
At least
pH.
only
of pea roots
consists
one
which
isoelectric
peroxidases
the
is
protein complex
enzymes
of
point
are
9.5
and
with
present
only
similar
a
the
intimately
has
degradingprotein. Probably
IAA
for
responsible
probably
are
net
small
a
This
charge
of molecular
range
these
degradation of
associated.
at
weight
enzymes are also bound
to membranes.
1.
INTRODUCTION
Several studies
IAA-oxidase from
on
Sequeira &
gel chromatography.
distinct fractions
Cronenberger
Janssen
roots.
one
of
capable
al.
et
(1969)
found
found
same
can
with small
niques
offer
only
one
enzyme
group
dilferences in
The
two
roots.
with
pea
of peas in which
indicating
different types of enzyme molecules
One type is
roots.
the presence
can
Use
be
showed
of
gel
that
the
tech-
specific
to a
another
column
of
presence
IAA in
degrading
chromato-
compounds
of other
assigned
indicating
enzyme
But
resolution of
inadequate.
results
capable
peroxidative
a
is
activity.
the
of IAA
already
gives
of
peak
because
weight
(1969)
support, i.e. polyvinylpyrrolidone,
of pea
one
degradation
author
present
to
separation
molecular
is necessary before the
enzyme.
presence
working
in roots
other, possibly
of
of tobacco
fraction when
one
with
performed
enzyme in both fractions.
Most authors ascribe
graphy
the
to
the
homogenates
enzyme fractions
two
fraction could be converted
of the
only
have been
plants
(1966) reported
1AA in
degrading
(1966)
of
roots
Mineo
two
homogenates
while the other
type is
non-
peroxidative.
Another
difficulty
IAA-oxidase
peroxidase
causal
as
In the
activity
electric
is
present
pH
point
Acta Bot. Neerl.
ubiquitous
is
difficult
presence of
study
use
gradient
is
is reached.
June
to
has
et
a
peak
prove with
are
on
of IAA
1967).
The various
a
peak
degradation.
and
But
proof
of
a
is
assayed.
been made of isoelectric
al.
Identification of
the fact that
gel chromatography
Under
established in which
1970
peroxidases.
commonly founded
homogenates
(Vesterberg
19(3),
is
coincidental with
when crude
electrophoresis
a
is the
peroxidase
relationship
impossible
current
a
proteins
peroxidases
focusing,
influence
are
of
move
easily
a
the
form of
applied
until their iso-
resolved
by
this
363
C.
when
technique
column of
Sephadex
MATERIAL
AND
Seeds of Pisum
earlier
Me
10
MgCl2 .
instructions
in
the
vessel
stepped
from
The
buffer
out
of
compound
7.5%. Eight gels
ml
duration of
tained
at
gradient
was
3/4
as
some
were
4 ml
The
focusing
The
applying
in
15
the
this
of 1
cm
gels
an
were
but
ml
these
In
disturban-
The column
obtained.
were
addition of this
first
into
method
30
during
gel
the
experiments
omitted later
the
cm
higher
was
were
the
at
20%
and
potential
25
cm
were
was
a
main-
pH
cathode
measured
406 M 5, in
nr
glass
sucrose.
of the
The
V/cm.
membrane.
and immersed in 1 ml
50 ppm IAA and 2 ppm
in
or
sucrose
was
gel. Drifting
resulting pH’s
of 1
3
used
from the electrode solu-
is level with the
pieces
as
influence
no
acrylamide
carrier
which
was
excess
minutes
as
Ampholine carrier, 40%
overnight
residual IAA
the
to
of the
Ingold microelectrode,
extension,
out
the
diameter of 0.5
the
a run
an
cut
avoid
according
and 2.6 mA
per
potential,
with
mA.
gradient.
low,
separated
containing
V/cm
carried
and the
the
and
buffer, pH 5.0, containing
was
a
16-17 hours in
12
bridge, through
incubation
is
current
was
and
cm
gels
water
1
polymerisation
In the
This step
containing
on
was
at
instances the
then removed
cold
determined
room.
the
by
p-coumaric
The
pieces
addition of
Salkowski’s reagent.
Gel
364
of distilled
intervals
of Me Ilvaine’s
acid.
of
300 V, in the
at
to
1/4
to
maintained
pH-ranges used,
kept
to
Ampho-
in the different fractions.
performed
was
the lower electrode. After
at
which the salt
In
ml
layered
negligible
was
gels
was
solution
pH
persulphate.
length
V, resulting
250
taken
in the
a
Usually 3/4
simultaneously.
and the enzymes
of the
of 2-2
the
added
was
was
all
at
was
steadily
region
to
described earlier in Me Ilvaine’s buffer.
(1968).
by
hours
case
in
Stockholm-Bromma,
detectable. The concentration of
was
with
20-24
out
contained
according
temperature
Fractions
as
focusing
Fawcett
removed
was
which
7.4,
upper electrode
carrier
Mitchell (1967).
by
The
in the acid
pump.
of the
with ammonium
persulphate
by
and
carried
was
27000 g supernatant
decreased
the
as
tested
was
was
current
taken
was
isoelectric
carried
The
a
been
described
as
grown
performed
was
in the former
eliminated variations of
(1968)
recommended
of the
focusing
the
V;
of IAA
were
(LKB-produkter,
solution.
voltage
peristaltic
a
Polyacrylamide
of Wrigley
tion
enzymes
roots
buffer, pH
focusing
4.5 ml
lighter
buffering capacity
this
have
volume of 110 ml. The concentration of the
coagulating compounds
virtually
of the
the
the kathode
degradation
the
As
a
700
to
up
drained with
was
isoelectric
%. Usually
containing
experiments
ces
1
Tris-HCI
manufacturer
3-10 and 7-10. The
pH
latter
the
in
or
The duration of the
2°C.
at
i. e.
pH 5.0,
column with
was
of the
1969). Homogenation
Liquid
from
a
line carrier
degrading
Gele Krombek”
“Vlijmsche
cv.
Mast
Ilvaine’s buffer,
Sweden)
containing
Fractions obtained from
gel.
IAA
MAST
METHODS
sativum
der
(van
mM
G-100,
polyacrylamide
VAN DER
this method.
analyzed by
2.
in
performed
A.
chromatography
on
Sephadex
G-100
was
performed
in
Acta Bot. Neerl.
columns with
19(3),
June
1970
ISOELECTRIC
FOCUSING
dimensions of 35
OF lAA DEGRADING ENZYMES
1.9
X
tions after the
passage
of
destilled
focused
polyacrylamide
on
with
mentioned.
out
on
retained
strength
proteins
35
were
effected
was
The isolation of membrane-bound IAA
in
a
The
model L 50
Spinco
pellet
teflon
resuspended
was
C-25,
1.9
with
degrading
with the aid of
a
up
1
ml
and
acidic cation
elution
strength
was
0.06.
buffer
same
in
sucrose
weakly
The
ionic
the
40%
perThe
by raising
obtained.
48000 RPM
at
a
cm.
and
were
and
enzymes
during
glass cylinder
1
carried
was
hour
and
a
in
out
50.
rotor
tightly fitting
piston.
Peroxidases
Total
Dubin
3.
centrifuge
6.5
pH
0.3. Fractions of 5 ml
to
carrier
X
taken
lyophilized,
CM-Sephadex
buffer,
NaH 2 P0 4-Na0H
removal of the
the ionic
as
gels
carried
was
Ampholine
obtained. The first 8 frac-
were
were
the
The column dimensions
exchanger.
formed
containing
water
exchange
Ion
Fractions of 5 ml
cm.
of the void volume
were
located with benzidine
phosphate
determined
was
reducing
as
according
to
agent.
the
method
of
Ames
&
(1960).
RESULTS
isoelectric
Liquid
rier with
One
Fig.
1.
a
range
peak
Liquid
carrier
anode
the
pH
of IAA
isoelectric
of pea
3-10
focusing
a
root
gives
degrading
establishing
of
gradient
figure.
Peroxidases
Bot. Need.
are
activity
pea
of
found
IAA is localised
19(3),
June
1970
supernatant using
the results
root
pH
solution. The last 6 fractions
gradation of
Acta
focusing
of
is
represented
apparent with
supernatant during
3-10
was
consisting
used.
anl.E.P.
Ampholine
an
isoelectric
20 hours.
An
The first 6 fractions
of the cathode
in all the fractions
around
an
between
car-
in fig. I.
solution
pH
3
are
and
point
Ampholine
contain
the
not shown in
pH
11. The de-
of 9.5.
365
A.
C.
2.
Fig.
isoelectric
Liquid
size
foregoing figure.
of
is the
9.5,
one
broad.
same as
one
but the broadness
mask
of such
was
of 8.4
of
is
this
Nearly
ding
all
an
were
enzymes
showed IAA
of these
the
enzymes
pea
with
366
Sephadex
on
of 5.6.
was
performed
broader,
than the
of
20
fraction
gradual
I.E.P.
an
of
presence
only
in
in
a
in
at
pH
fig.
only
was
a
der
the
Mast
the
ones
of
only
pH
in
of 8.4.
1970)
and
one
Peroxidase
containing
3-10
pH
due
a
to
at
root
a
pH
by
9.5
gradient
9.7
were
subsequently
distribution of enzyme
Acta
pro-
activity,
the electrode
reduced
was
degrading
to
ribosomes
peak
focused
pea
pH 7-10, only
of IAA.
probably
gra-
slope
of
gradient
5.6 all the fractions between this
and
the
activity
are
pH
to
was
present.
1.2 and 6.5. When IAA
pH
membranes and
pH
If
I.
region coagulated compounds
This is
a
fractions with
of IAA
peak
with
enzymes
3-10 and the
a
gradient
is rather
peak
although
given
one
pH
degradation
second
a
In this
supernatant
(van
the
was
become
not
situated between
activity
G-100,
around
this
and
degrapH
9.5
continuous release
from the membranes and the concomitant
G-25
Sephadex
indicating
MAST
during
7-10
even more
degrading
and refocused in
focusing
20 hours and all the
ed
is
gradient
is still localised
IAA
focusing
gradient
a
for the
hours,
Removal of
root
pH
gradient. The
pH gradient
in all fractions outside the
found
abolished this
When
in
degrading activity.
kathode.
totally
I.E.P.
phosphate
of the
of the
could be found. This all indicates that
responsible
instead of 20
found with
of the
of other
gradual
more
solutions. If the duration of
10 hours
did
pooled
activity
apparent
was
presence
peak
even
were
slope
does not increase
If, however,
first focused
9.7
to
peak
complex
however,
is
gradient
a
supernatant
same
{fig. 2)
peak
gradual slope
the
different I.E.P. ’s.
dient of 7-10
tein
the
to
of the
The
side
degradation of IAA
1. The
of
gradient
a
acid
DER
for IAA conversion.
proteinresponsible
This could
slightly
pH
in the
depicted in fig.
of 9.5. Due
(I.E.P.)
the
supernatant in
been omitted at the
have
than the
a
of pea root
focusing
hours. Several fractions
VAN
migration
towards
ultracentrifugation
was
of
then
apparent.
3-10
pH
pooled,
during
concentrated
gel-chromatograph-
molecules
Bot. Neerl.
was
the
19(3),
same
June
1970
ISOELECTRIC
Fig.
3. Ion
FOCUSING
OF
lAA
exchange study
0.06
strength
with
membrane-bound
tein
molecules.
strength.
No
DEGRADING
of IAA
counterions.
as
while
enzymes,
The
at
degrading enzymes
sodium
elution
influence
ENZYMES
the retained
of this
retained
of the flow rate
on
pH
6.5 on
The first
peak
peak
CM-Sephadex
of
peak
is
activity
consists of free IAA
can
be
the retention
effected
was
C-25 at ionic
largely
to
the ionic
by raising
detected
due
degrading pro-
although
this
pa-
2
rameter in the
as
various
experiments
found after addition of KC1
present in the
form
can
Ion
high
peaks
of the void
organelles
because
of
on
a
consists
but
only
/hour.
i.e.
1969),
no
enzyme is
its lower molecular
degrading
removal
IAA
degrading activity
in
activity
of these
of these
this
cell
not
to
molecules from this first
peak.
retention of all IAA
the column. Ion
due
is
peak
most
at
this
weight
counterions
way
of
retained
orcinol
is
activity
possibly
Further
the
degrading
lowering
elution with
enzymes,
even
strength
0.06 did
not
the ionic
destilled
the column.
by
Most of the
enzymes
due
the
to
relatively
strength
of the
free
to
a
of the
enzyme
strength by carrying
water
resulted
membrane-bound
rise
as
The
disappeared.
in
retained
total
molecules, by
of isolated membranes with
give
These
buffer,
eluant
test.
the ionic
0.005 resulted in total removal
and
the
membrane-bound
to
of this
rise
gives
elutes after the passage
pH
not
pH. Lowering
exchange chromatography
tion buffer of ionic
6.5,
confirmed with the
removed in this
elution buffer from 0.06
the
therefore
are
structures
molecules
homogenation
pH
at
as
peak
of membranes and ribosomes.
mostly
and
was
3. The first
fig.
charge
I.E.P.,
of ribosomes
IAA
the
Mast
der
(van
ml/cm
C-25 with sodium
shown in
negative
of their lower
charge
as
volume and
after
out
urea
weight peak
CM-Sephadex
activity
have
The presence
weak
or
between 8-25
then be detected.
exchange
to two
molecular
varied
peak
an
of
eluIAA
degrading activity.
Acta Bot. Need.
19(3), June
1970
367
C.
Fig.
4.
Polyacrylamide isoelectric focusing
tion with
were
adjusted,
fraction
3
of 9.3
pH
a
after
portions only
Separation
the small
with
The
in each
the IAA
present.
gel.
Due
were
dient of
Due
Pea
two
I.E.P.
itself in this
always
present
16
pH,
time. It
in the
pH
net
hours
associated
fraction of
pH
a
6
3
and
of IAA
charges
of this
protein
5 have
reached
occur.
The
around
enzymes
to
the enzyme band
elute from the
of
can
be inferred from the
can
first focused
was
in
liquid
given
seen
faster
fraction of
9.0 similar results
in
several
although
fig.
moving
pH
a
following
of
gradient
to
took
pH 7,6
place
in
and
a
5,
gra-
in fig. 4.
from
9.3.
were
raising
isolated and divided into 3 portions.
7.5% polyacrylamide
enzymes in
be
con-
indicate
colour achieved
column after
of the enzyme molecules in the 3
focusing
with the
initial
or
are
charge
the
I.E.P.
0.3.
types
was
of 9.3
3%
on
their
acrylamide
denotes that the blue
hatching
faster
in the various
pH
areas
In this
to the
does not
to
portions
5, respectively.
is confined
MAST
The frac-
The
portions.
with citric acid after addition of the carrier
varying
of the
after
net
DER
degrading enzymes.
pH 7,
adjusted
stippled
the
enzyme
7-10. The results
adjustment
to
VAN
to red.
supernatant
Refocusing
the
enzymes
The
%.
degrading
root
adjusted
pH
to
7.5
IAA
divided into
degradation
varying
of the elution buffer
3-10. The fraction with
These
and
carrier,
The
to the
degrading
turns
quickly
IAA
strength
respectively.
368
are
was
The association of the
pH
liquid focused
isolated
addition of the
amount of diffusion while
benzidine
experiments:
of
was
of the two types of
molecules
enzyme
employed
retained
the ionic
the
peroxidases
moving protein
centration
(see fig. I)
A.
gels
the
have
pH
4 that
protein.
portions
has
degradation
Two
other
established
of IAA
peroxidases
experiment
the
but in the
with
fractions with
Acta Bot.
after
yet reached their
gradient
the
Repeating
found,
not
Neerl.
19(3),
pH
June
is
are
the
9.5
1970
OF
ISOELECTRIC FOCUSING
Fig.
5.
lAA
DEGRADING
isoelectric
Polyacrylamide
focusing
gelchromatographed on Sephadex
addition
dases
of the
possessing
concentration
the
±
carrier,
pH
0.1.
of 9.4
gradient.
AH the
except
branes
as
positive charge
7.5
%.
The
degradation of
at
this
of the
8
this
pH
pH
fractions
of the
pH
pH
at the
with fraction
peroxidases
degrading protein. The
the various fractions is indicated
are
same
the
3th
6
8
cm
in
the
of the
due to the
the
large
same
benzidine
the
gels
an
amount of
was
I.E.P.
mem-
range of molecular
same
protein
in
shading. Hatching
to red
turns
measuring
around
of the
differences in
acrylamide
for
various
gels
The
only peroxi-
The
used
5.0.
was
that
figure.
relative concentration
achieved with
buffer
was
point
present in
by corresponding
colour
in
supernatant
root
pea
implying,
fraction
to
of
elution
to 6.5,
shown
are
IAA is localised in the
gel corresponding
indicates areas where the blue
first
The
gel corresponding
here. Three other
the IAA
of the
G-100.
pH 7-10, raises
range
The variation
in the
present
weight
a
was
ENZYMES
within
a
few
minutes.
and 9.7 the slowest
moving protein
3
migration
the
% acrylamide
In the
next
on
polyacrylamide
with
Only peroxidases
ones
being displaced
peroxidase
when the
absent. In
pea
root
with
gels
supernatant
was
concentration of
a
degrading protein
was
twice
with
gels
corresponding
Acta Bot. Neerl.
an
from the
cut
into
fraction 6
19(3),
June
freeze-drying.
positive charge
gel
I.E.P. of 9.4.
were
to
after
a
1970
at
The results
pH
6.5
are
are
shown in
shown,
the
towards the anode. All the fractions
Only
pieces
this
protein
showed
and incubated with
showed
IAA
fast.
as
gelchromatographed
G-100. The first 8 fractions after the passage of the void volume
Sephadex
focused
experiments
was
of the IAA
fig.
5.
negative
display
degradation
IAA.
on
were
a
of IAA
However, the gel
degradation throughout
the
gel
till
369
A
C.
the
13th
These
transition
deviating
between
result in
is
proteins
this
the
the
solution and
degrading
the
membranes in
until
anode
may then
enzymes
this
reach the
they
electrode
I.E.P.
This
protein
dative
at
fig.
solution. The
account
for the
but
which
all
one
to
an
tive
protein
to
the
the
other
was
possible,
and of
the
(Margolis
&
travelled twice
at a
not
a
Mast
as
fig.
is
as
It
compound.
was
and
of
apparently
non-peroxi-
a
showed the
presence
fraction
assumed that this
also
were
proved
now
located
because
in
the
was
was
non-
after removal of all
of
peroxidase capable
a
fast
least have the
separation
in
degrading IAA,
as
be
of
separately
move
should then be
degrading
enzymes
sodium ions.
an
equal
size
is
present.
At
the
IAA
At
is
gel
a
50
was
and
indicating
found
at
the
3%
two
the
of
way.
7.5%
150
interference
outside
still
happen
nm
band
enzyme
of the
separation
never
same
concentration of
nm
latter concentration
But
It
amount
which should
polyacrylamide gels,
were
performed with
was
distributed over each molecule in the
in the
and nega-
positive
exchanger
and elution
7.5% polyacrylamide,
the
by
did
types
peroxidase
the presence of non-peroxidative IAA
degrading
band.
seen
was
ratio of
ion
associated
intimately
are
concentrations of
7.5% acrylamide.
that in all the fractions
present with
evidence
on an
strength
increasing
1967).
as
same
when all the IAA
ionic
band, indicating
can
enzyme molecules
pore diameter of this
degradation
is
of
difference in
Kenrick
5 it
protein
1970)
of IAA
peroxidative
that both types of enzymes have
not occur
mean
and the free IAA
370
at
zero
of
molecules in this
From
two
I.E.P.’s
possessing
degradation
a
peroxidases
case, even
at
course,
did
enzyme
enzyme
IAA
the
concentration of
occur
positive
of
small
acrylamide
gel
types
negative charges
separation
a
not
gradient
positive
only
ones
far-fetched. When both enzyme types
should
they
exchanger
very shallow
when
present. But
are
In that paper another enzyme
this
to
assumption
seems not
remains
Also
complex
remain valid. The
to
polyvinylpyrrolidone
on
This indicates that
This
possible.
promotes the
there still remains
I.E.P. of 9.5
bound
peroxidases
the
one
4.
fig.
charges.
only
But in fig. 4 is shown that
seems
identical with
1969).
adsorb
not
This
peak.
two more
this conclusion
enzyme molecules, i.e.
The conclusion that both
to
protein
of IAA,
5.
Mast
der
peroxidases
shown in
IAA
degradation
chromatography
(van
peroxidase,
other
in
two
mentioned that did
adsorbed
of this
complex
least
as
one,
of the latter
for the
probably
are
depicted
1 and 2 it could be apparent that
figs.
degrade
peroxidases
consists of
in
given
not
9.0 and 8.3
a
carrier
of the
presence
towards
MAST
gel.
responsible
around
these do
other
a
the
the
to
move
DER
DISCUSSION
even
as
will
release of bound IAA
From the results
of
due
probably
structures
zone
subsequent
4.
This is
cm.
fraction.
VAN
an
I.E.P.
presented
degrading
enzyme
of 9.4.
indicating
In
a
displaying degradation
previous
that both
paper
(van
of
der
the membrane-bound
molecules consist of the
Acta Bot.
two
Need.
enzyme
19(3),
types
June
1970
ISOELECTRIC
lAA
FOCUSING OF
mentioned. The
DEGRADING
range of molecular
large
found after gel
enzymes
continuous release of
chromatography
indications for
It
can
of
range
larger
the
as
degrading
weights.
IAA
This
three
nevertheless
by
in this
the
cover
also attached
are
peroxidases
a
factor
peroxidases
stimu-
2-3
shows
by gel
a
to
not ca-
they
as
process
with
protein
identical
a
show the
these molecules
peroxidases
specific
to
during
found in
are
of discrimination between these groups of enzymes
possibility
due
state
be communicated later.
ribosome-bound
of several
overlap
free
phenomenon
participate
the
degrading
other, basic, peroxidases
these
or
IAA
probably
the
to
degrading protein, i.e.
Possibly
of IAA
degradation
communicated).
IAA
Membrane-bound
structures.
this
from fig. 5 that
seen
molecular
of
pable
late
also be
phenomenon
this
free
is
Sephadex
Further evidence will
electro-focusing experiments.
same
of the
weight
on
membrane-bound molecules
Several
chromatography.
ENZYMES
be
(to
the
im-
chromato-
graphy.
Due
the fact that the IAA
to
lose
Galston
was
in
or
due
starch,
from
peroxidases
to
intact
cellular
dases found in this material
to
other anodic cell
ments
A
dases achieved
of the
two
positive
It is
material
IAA
the
in
case
anodic
to
gel
This is
due
ion
exchange
differences in the
charge
to
open
that
peroxi-
membranes
or
from
peas.
the
to
restricted
peroxi-
diffusion
structure.
and
electrophoresis,
Mast
question,
the
1969) probably
of these enzymes. The
enzyme molecules
non-peroxidative
phenols, although
der
&
present
was
electro-focusing experi-
degrading protein
gel.
cellu-
Siegel
They presumed
that the
possible
on
I and fig. 3 shows the better resolution of
the
polyvinylpyrrolidone (van
by
indications.
faulty
phases,
two
performed
types of enzymes composing the IAA degrading protein
inseparable by
of the
when
in fact cathodic but attached
polyacrylamide
protein imposed by
As the
be
fig.
as
of Pisum sativum.
roots
This is also
structures.
between
on
introduce
can
organelles.
are
of short duration for the
comparison
such
present in
are
enzymes
duration,
noted that acetocarmine
(1967) already
anodic
among
this
strips
acetate
degrading
of short
electrophoresis experiments
still
to
seems
separation
can
to
on
be accounted for
not
that
assumption
this
seem
achieved
is
compound
adsorption
due
to
poly-
valid.
ACKNOWLEDGEMENT
gratefully acknowledges
The author
the technical
assistance
of Miss
M.
A. T. Gebbink.
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