Vol.
177,
May
31, 1991
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
MULTIPLE NADPH-CYTOCHROME IN HIGHER
FORMS I?450 PLANTS
105-112
OF REDUCTASE
Irene BENVENISTE*, Agnes LESOT: Marie-Paule HASENFRATZ*,GeorgKOCHS+and Francis DURST* *Laboratoire
d’Enzymologie
Cellulaire
Moleculaire
des
Institut
Plantes,
et Moleculaire, de
Strasbourg +Biologisches
Institut Pflanzen,
Received
February
II
der
Cedex,
28,
28
rue
Goethe,
67083
France
Universitbt,
Schanzlestrasse
de Biologic
Institut
Botanique,
Lehrstuhl
1, D-7800
fiir
Freiburg,
Biochemie
der
Germany
1991
of multiple forms of NADPH-cyt P450 SUMARY : We report on the presence This contrasts with the animal cyt reductase in microsomes from higher plants. P450 monooxygenases, where the numerous cyt P450 isoforms are reduced by a single form of reductase. Three NADPH-cyt c reductases have been resolved from Jerusalem artichoke tuber microsomes by chromatography on Reactive Red Agarose and Concanavalin A-Sepharose. Their molecular weights, determined by sodium dodecylsulfate-gel electrophoresis, are 80,000, 82,000 and 84,000. The three and are dependent upon FMN for catalytic proteins share common epitopes activity. They are highly selective for NADPH as electron donor, and allowed effective reconstitution of trans-cinnamic acid and 3,9-dihydroxypterocarpan 6a-hydroxylase activities with purified cyt P450 fractions from Heliantfius tuberosus and Glycine max, respectively. As such, they appear as true 0 1991 Academic Press, Inc. isoenzyme forms of NADPH-cyt P-450 reductase. In
eukaryotes,
multienzyme the
terminal P450
hemoprotein. also
becoming
have
have
recently
artichoke
observed,
microsomes,
with
specific
(8).
To elucidate
P450
reductase
the the
question
plant
two
of
microsomes,
three against
the
P450,
the
no isozymes source
from
(6),
attempted
to
the
NADPH to
the
is
well
of
cyt
P450
of
the
NADPH-cyt (5).
of isolate
P450
However,
we
Jerusalem cross-reacted
artichoke
isozymes
as
P45Os
which
purified of
are
serves
wound-induced
proteins
existence we have
from cyt
multiplicity
animal
the
which flavoprotein,
multiple
blots
of
monooxygenases
cyt
electrons
of
contrast,
Western raised
the
FMN containing
a particular
presence (7)
in
In
in
antibodies
FAD and
In plants,
(2-4). in
of
existence
11).
detected
P450-dependent
transfers
the
obvious been
an
which
many years
cyt
essentially
of
animals,
since
reductase
and
reductase, In
established
microsomal
consisting
oxidase,
NADPH-cyt
is
the
complexes
reductase the
NADPH-cyt the
three
proteins.
105
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
177, No. 1, 1991
This
communication describes,
characterization equivalents
BIOCHEMICAL
of
three
AND BIOPHYSICAL
for
the first
RESEARCH COMMUNICATIONS
time,
isoenzymes which are able to
from NADPHto cyt
P45Os. The activity
the separation transfer
and
reducing enzymes is
of the three dependent upon FMN, as generally described for the NADPH-cyt P450 reductase. Finally, evidence for multiple reductase forms in other plants, based on Western blot analysis, is presented. MATERIALSANDNETHODS : Emulgen 911 was a generous gift from Kao Chemical Co.. CHAPS ~3-~[3-cholamidopropyll-dimethyl~rmmonio~-l-propane sulfonate), Reactive Red 120-Agarose with diverse concentrations of dye, Concanavalin A-Sepharose and a-methylmannoside were from Sigma Chemical Co., Bio-beads SM-2 from Bio-Rad, DEAE Trisacryl from IBF and 2’5’ ADP-Sepharose 4B and molecular weight marker proteins from Pharmacia. Nitrocellulose (45 pm pore size) was from Schleicher and Schiill. Materials
Enzyme
assays : NADPH- or NADH-dependent reduction of cytochrome c was measured spectrophotometrically as described elsewhere (7). For kinetic studies, 14 concentrations ranging from 2.2 uM to 565 uM for NADPH, and from 4 uM to 12 mMfor NADH, were used. Reactivation of NADPH-cyt c reductase with FMN was achieved by including different concentrations of the cofactor directly into the spectrophotouetric assay, without preincubation with the purified reductases. SDS-polyacrylamide gel electrophoresis : The purity of the NADPH-cyt c reductase preparations was checked by SDS-polyacrylamide gel electrophoresis (7.5 or 12.5 % acrylamide, w/v), after Laemmli (9). Protein bands were stained by silver nitrate. Molecular weights were estimated using standard proteins (14-93 kilodaltons) electrophoresed under identical conditions. blot analysis : The microsomal proteins separated by SDSpolyacrylamide electroghoresis were electrotransferred onto gel nitrocellulose, after Towbin et al. (10). The remaining protein-binding sites on the nitrocellulose were blocked by 5% bovine serum albumin and 5% non-fat dry milk, in solution in phosphate-buffered saline containing 0.4% Tween 20. The rabbit anti-reductase serum was diluted 20,000 times. The specific antigen-antibody complexes were revealed by goat anti-rabbit immunoglobulins coupled with alkaline phosphatasle. BCIP (5-Bromo-4-chloro-3-indolyl phosphate) and NBT (Nitroblue tetrazolium), the substrates of the phosphatase were incubated at pH 9.6 in 0.1 M diethanolamine. Western.
of the multiple forms of NADPH-cyt c reductase : The NADPH-cyt c reductase was purified from 1-2 g microsomal protein from Jerusalem artichoke tuber bly chromatography on DEAE-Trisacryl M and 2’5’ ADP-Sepharose 4B columns, in the presence of Emulgen 911, as described previously (8). This preparation was then submitted to dye affinity chromatography, on a 20 ml Reactive Red 120 Agarose column (3.4 Wmoles dye/ml gel), equilibrated with 10 mM KPi, pH 7.4 containing 20% glycerol, 0.2% Emulgen 911, 1.5 mM 8-mercaptoethanol and 1mM EDTA. The fraction of reductase activity partially retained on the dye gel (form 1) was eluted by increasing salt cont.. The unbound reductase was applied onto a Concanavalin A-Sepharose column (15 ml) equilibrated with 10 mM Tris-HCil buffer, pH 7.2, containing 20% glycerol, 0.1% Emulgen 911, 1.5 mM8mercaptoethanol and 0.4M NaCl. This step resolved the reductase into two forms : form 2 was excluded from the lectin column, form 3 was retained and eluted by 0.6M a-methylmannoside. In order to replace Emulgen 911 by the zwitterionic detergent CHAPS, the enzymes were applied onto a small DEAETrisacryl column (5 ml), and eluted by a gradient of KCI (O-O.4 M) in the presence of CHAPS, and finally concentrated on carboxy methylcellulose. Isolation
106
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
: The cyt P450 isoform hydroxylating Reconstitution of monooxygenase activity cinnamic acid was purified by chromatography on DEAE-Trisacryl and on Hydroxylapatite (4). This preparation was totally free from any NADPH-cyt c reductase contamination. For reconstitution of monooxygenase activity, 26 pmoles cyt P450, in solution in 10mM NaPi buffer, pH7.4, 10% glycerol and 0.9% Emulgen 911, were mixed with different quantities of each reductase form. No phospholipids were added. The mixtures were treated by 100 ~1 Biobeads, equilibrated with 0.1 M NaPi buffer, pH 7.4, to remove excess detergent. These reconstituted systems were then incubated at 26OC for 1 hour in the presence of 10 NM FAD and FMN, 500 NM NADPH and 22 PM [‘“Cl cinnamate, and cinnamate hydroxylation measured as described (11). Pterocarpan hydroxylase activity reconstitution was as described in (3).
RESULTS Isolation
of
Affinity
the
chromatography
purification from
isoforms on
of NADPH-cyt
mammalian
Jerusalem
liver
were
2’5’
reductase
high
is
was
resolution
(Fig-l).
with
11,
step
yields
(form
that
molecular
from by this
in
fact
weight,
three
measured
SDS-polyacrylamide 2) and 84,000
the
protein
reductase
isolated
showed
after
for
a single c
specifically
apparent
82,000
efficient
NADPH-cyt
SDS-PAGE
proteins
(form
very
The
also
Their
standard
80,000
13) .
NADPH-cyt c reductase:
48 is
: this
(12,
microsomes
co-purified
comparison by electrophoresis,
ADP-Sepharose
P450
tuber
however
(81,
proteins
Jerusalem artichoke
microsomes
artichoke
procedure
of
(form
gel 3).
NADPH-cyt c reductase (forms 1, 2 and 3) Elution
>
form 1
) a-methyl mannoside
form 3
REACTIVEREDAGAROSE exclusion
I
NaCl
(forms 2 and 3) Elution
CONCANAVALIN A-SEPEAROSE exclusion 1 form 2 To
separate
summarized isoforms
these
above. for
It
lectins
Reactive
is
Form 1 was retained
while
form
2 was only
washing
of
form
form
the
we
based
on
has
been
deviced
differential
by a gel
grafted
and form
with
equilibration
with
to eliminate
any contaminating
gel,
we have
tested
gels
to
with
resolve
with
3.4
3 was readily
150 mM NaCl.
used
the
purification
affinities
scheme
of
the
three
dyes.
shown
retarded
column
1 was eluted also
have
the
and substrate-mimicking
Red-Agarose
(14).
2,
proteins,
form lower
107
NADP+ dependent Nmoles
dye per
excluded.
buffer to remove Two chromatographic
2. To avoid binding dye concentrations
enzymes
ml agarose,
After
abundant
loosely cycles
of form (0.2, 0.5
bound were
2 to the and 0.96
Vol.
177,
No.
1, 1991
BIOCHEMICAL
A
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B
” n 234.0 -80.0
1
2
3
4
21.5-m 14.4-a 1
2
Figure 1 A. SDS-polyacrylamide gel (12.5 Jerusalem artichoke NADPH-cyt P450 reductase 2’5”ADP-Sepharose. Lane 1 : molecular weight NADPH cyt c reductase. SDS-polyacrylamide gel (7.5%) e l& purxfied forms of NADPH-cyt P450 reductase. after
pmole
Lane
dye per
isoforms, Form Form
ml agarose)
form
2 was
not
specifically of
1 being
2 and
form
. These only
separated
retained
by
with
of
Kinetic
: We have
for
exhibited
typical
was
varied for
of NADH could
the
c
cyt
(2.5
Reactivation whereas occurs Activity (17). activity
is
not
profile
ba in
shows
the
three
bind
of on 2 :
of
any of
the
the
three
purified
kinetics The
when
apparent
enzymes.
since
be measured, present
in
the
by
reductase
c
the
in
vitro
progressive after
forms,
concentration
of
assay)
cyt
:
of
FAD results
forms
of
enzymes of
for
NADPH
NADPH was in
the
reactions
were
not
(up to 12 mM).
both
only
gel
non-enzymatically
Microsomal
:
nature
c reductase
the
used
NADPH-cyt
FAD and
(16). in
Loss enzyme
of FMN to the
restoration of the addition of increasing 108
three
the
(81,
by addition
The
c.
NADH reduced
presence
possesses
three
Km (=2O PM)
reductase
preparations
of the
cyt
Kinetics
and
c reductases.
affinity 50pM
bound
SDS-polyacrylamide
NADPH-cyt
of
3 was
a glycoprotein
the
apparent
chromatography.
form
indicating
presence
565uM.
while
shows
the
NADPH-cyt
reductase
can be restored
not
A-Sepharose
to the NADH concentrations
FHN of
NADH-cyt
of the
three
purified
characterized
readily Fig.2
to
respect by
reductase
the
FMN was
pM
with
in
three
did
column,
Fig.lB
Michaelis-Menten
presence saturable,
lectin
measured
2.2pM
the
however,
by Concanavalin
the
of the
NADPH,
from
identical
in dalton
electrophoresis
a-methylmannoside,
profile
reductase
gels,
reductase.
electrophoresis studies
scaled
profile chromatography x 10-s. Lane
retarded.
3 were
eluted
this
electrophoresis affinity
Lane 1 : NADPH-cyt P450 reductase on 2’5’ADP-Sepharose. Lane 2 : form 1 of the NADPH-cyt c 3 : form 2. Lane 4 : form 3 (glycoprotein). Proteins were
purification
reductase. silver-stained.
%) after
FMN of
(8,
15),
FMN, which
inactivation.
incubation
NADPH-cyt amounts
P450
medium
c reductase of FMN.
Vol.
177,
No.
1. 1991
02 O
BIOCHEMICAL
AND
BIOPHYSICAL
[FMijo&l) I0
w
RESEARCH
NADPH-cytc
COMMUNICATIONS
reductase
activity
(pkat)
Reactivation of the NADPH-cyt c reductase activity of the three riiluE2, isoforms as a function of the concentration of FMN added. NADPH-cyt c reductase activity is expressed in nkat reduced cyt c per ml isoform . form 2 ; (0-0) form 3. (0 -011 form 1 ; (t---t) piuure $ Reconstitution of cinnamic acid I-hydroxylase activity by the three NADPH-cyt P450 reductase isoforms. Reductases are expressed as enzyme units (pkat cyt c reduction activity). Cinnamic acid hydroxylase activity is expressed as pkat p-coumarate formed/nmole cyt P450. (o-o) form 1 ; (t---t) form 2 ; (o-0) form 3. Reconstitution
of
reductase
activity
hydroxylation
of
reconstituted
by
three
monooxygenase
forms
microsomes
and the
of
reductase
were
able
NADPH-cyt
P450 reductase.
activity from
showed
reductase
antibodies
raised
recognized
specifically
investigate
the
against existence
the
hemoprotein
isolated
three
electrons therefore,
deprived
the
activity
was
of
to
for
the
cyt
the
cell
isoenzyme
P450
cultures in
other
purified
(results plant
artichoke
these
flavoproteins
(7).
of
immunorelated
multiple
109
plant of
the
electron Ga-hydroxylase
cyt not
P450
(3)
isolated
shown).
microsomes NADPH-cyt They
the
catalysing
as isoforms physiological
to
of
from
3,9-dihydroxypterocarpan each
each
reductases
be considered
of
catalysing
Monooxygenase
All
of
isoforms
fraction
concentrations
of
soybean
the
prepared.
selectivity
by addition
elicitor-challenged for
no
in
P450
increasing
transfer
reconstitution
was achieved
Evidence
of
and may,
isoforms since
was
(Fig.3). to
hydroxylation
These
enriched
acid
addition
cinnamate
acceptor,
highly
cinnamic
: A cyt
activity
:
Polyclonal
P450
reductases
were
used
proteins
in
as tools other
to plant
Vol.
177,
No.
species, from the
1, 1991
BIOCHEMICAL
by Western
other
plant
detected
in
the
(molecular
Vicia potato
weights
maize
sa tiva
seedlings.
tuber,
avocado
Specific
between
80 and
and in
84 kDa), cell
were
suspension
were
and wheat
of
cross-
proteins
proteins
Veronica
to those
Three
CatAaranthus
Two immunoreactive
proteins
similar
antiserum.
embryos,
pear,
COMMUNICATIONS
proteins. mobility
reductase
from
RESEARCH
electrophoretic
anti-artichoke
microsomes
BIOPHYSICAL
on microsomal
with
reductases
with
wounded
analysis
microsomes,
artichoke
reacted culture,
blot
AND
revealed
cell
in
suspension
cultures.
DISCUSSION Three induced
microsomal
Jerusalem
artichoke
NADPH and very three
forms
other
triazine
NADPH-cyt
close
is
in
binding
mimic
Reactive
Red appeared
weak,
the
highest
commercially
available
chains
of forms
hindering
the
Alternatively, gel
also
elution
by
moiety,
of
animals
and
in
and
plants. 2
cannot
three
Furthermore,
monoclonal
antibodies
one
suppose
could of
efficient since
it
readily
of
has
been
cleaved
electrons purified
a single
to cyt
Cinnamic cyt
However,
at
a
share
native shown
at very
common
enzyme. reductases. that
low
the
binding
been
tested.
This reductase
Apparently,
with
the
selective
is
similar
an
first
other
since forms.
originate
from forms
were
of
lectins,
apparent
all
in
nature
domains
three
of the
c reductase,
Km for several
Therefore, proteolytic active
and
proteolytic degradation the hydrophobic domain, becomes
unable
to transfer
(18).
acid hydroxylase activity was reconstituted P450 fractions and each of the three reductases,
phospholipids.
dye.
existence This
to
with
excludes lacking
concentration,
P450 monooxygenases
with
all
the gel.
polypeptide
glycoproteic
the
would
However,
protease
the
immunogenic
isoforms
onto
ml of
and its
the
glucose.
cross-react
three
behaviour.
NADPH-cyt
c reductases
we prepared
P450
yet
the
interactions
with
stage,
the
like
immobilized
A-Sepharose
since
that
three
ionic
and/or
this
has not
NADPH-cyt
the
and
per
the
the pyridine
of
of
chromatographic
microsomal
excluded,
they that
NADPH-cyt
of
are
to
consistent
form
glycanes,
enzymes
site
for
Red, of
1 was bound
active
mannose
Reactive link
form folding
Concanavalin
are
be
of other
only
wound-
Surprisingly,
The affinity
from
hydrophobic
in
affinity
column.
result
observed
rich
apparent
dye concentration
the 3 with
a glycosylated
characteristic
cleavage
form
the
from
diphosphate
since
the
a-methylmannoside
example
NADPH .
in
of
oligosaccharide
1
of
explain
The interaction
All
access
differences could
matrix
forms
2 and 3 could
isolated
84 kD).
enzymes.
for of binding
the
NAD(P)+-depending
gel Lack
82 and
dye affinity
to
proteins with
been
of similar
(80,
on the
believed
have
spite
weights
differently
dyes, thus
tubers,
molecular
behaved
nucleotides,
c reductases
the
detergent 110
present
by mixing the without addition in
the
CYt
P450
Vol.
177,
No.
preparation
(1% Emulgen
reconstitution
of
concentration
911)
the
increased
corresponding
microsomal
reductase added
BIOCHEMICAL
1, 1991
The
three
challenged. P-450
reductase
For
proteins
mlcrosomes,
animal
reductase
is
reason More
to determ:ine with
different
cited
above
not if
the
origin
one
translational
of genes
maturation. subcellular
development,
multiple
will
was
Another
studied.
or/and
by
tissular
they
the
would
NADPH-cyt plants
Western rule
purified
cyt
higher
are
P450
in
acid
fatty epoxidation.
forms
P45Os,
these
will
and
a
spatial
these
by
and
this
remains be needed
our
isoforms
from
of
NADPH-cyt
organisms,
raised
result
localization,
of plants.
for productive interaction the other redox reactions
question
temporal
we
blotting
in
squalene
other
P450
that
low-represented
reductase
in
with
microsomes
microsomal
P-450
a
also
higher
and in
multiplicity: could
in
p-chloro-N-
laboratory
since
are equally competent Their involvement in
reductase
our
artichoke all
conserved
several
reductase
oxidations,
NADPH-cyt not
using
Finally, or
the
a single
heme oxygenation
hemoproteins. be
that
phenomenon.
P450.
to be the
in or
cyt
detected seem
P450-dependent
isoforms also
be
elicitorNADPH-cyt
Likewise, in
from
cyt
studies,
all
will
several of
have
evolved
detailed
concerns
terms
in
why plants has
not
admitted
and elongation,
situation
or
is
involved
desaturation
open.
it
rat
of
reconstitute
from
rabbit
with
of reductase
to
a general
be under-estimated
may
higher
P45Os was reconstituted
artichoke
microsomes
would forms
systems,
the
the
used.
between
is
hepatic
with
evaluation
multiple
cyt
from
able
(19).
of
conditions
purified
experiments
immunorelated in
also
as
avocado
10%
by using
interaction
P448
detergent
as a function
the
P450
cyt
Preliminary purified
this
were cyt
effective
rat
hydroxylase
improved
origins
as
about
under
After
the
linearly
reached
different
was
proteins,
Although
from
of purified
detected
be
Catalytic
contained
(20).
three were
immunoreactive
The
that
cinnamate
Two or
In
P45Os
reductase
reductases,
(3).
reductase
demethylase
reconstitute
should
with
phospholipids.
reached
isoforms
COMMUNICATIONS
lowering
increased
was not
cultures
reductase that
tested.
cell
system
methylaniline showed
This activity
reductase
trout
reconstituted liver
which
activity,
Ga-hydroxylase
and cyt
example,
rat
the
artichoke
soybean
the
complex,
and saturation
RESEARCH
efficiently
monooxygenase
since
3,9-dihydroxypterocarpan
BIOPHYSICAL
replaced
activity.
concentrations
flavoprotein
AND
findings encoded
different distribution,
enzymes
during
in plant
be investigated.
to Marie-France grateful technical assistance and to Monique Wehr for typing was supported by MRT grant nO154.85.
ACKNOULEDGNENTS: We are
Castaldi for this manuscript.
excellent This work
REFERENCES 1.
Guengerich, L.S. (1982)
by
post-
F.P., Dannan, G.A., Wright, Biochemistry 21, 6019-6030. 111
S.T.,
Martin,
M.V.
and Kaminsky,
Vol.
177,
2. 3. 4. 5. 6. 1.
8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Benveniste, I., Salaiin, J.P., Simon, A., Reichhart, D., and Durst, F. (1982) Plant Physiol. 70, 122-126. Kochs, G. and Grisebach, H. (1989) Arch. Biochem. Biophys. 273, 543-553. Gabriac, B., Werck-Reichhart, D., Teutsch, I-I., and Durst, F. (1991) Arch. Biochem. Biophys.,in press. Porter, T.D., Beck, T.W. and Kasper, C.B. (1990) Biochemistry 29, 9814-9818. Lesot, A., Benveniste, I., Hasenfratz, M.P. and Durst, F. (1990) Plant Cell Physiol. 31, 1177-1182. Benveniste, I., Lesot, A., Hasenfratz, M.P. and Durst, F. (1989) Biochem. J. 259, 847-853. Benveniste, I., Gabriac, 8. and Durst, F. (1986) Biochem. J. 235, 365-373. Laemmli, U.K. (1970) Nature 227, 680-685. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci USA 76, 4350-4354. Benvenist?, I., Salaun, J.P. and Durst, F. (1977) Phytochemistry 16, 69-73. Yasukochi, Y. and Masters, B.S.S. (1976) J. Biol. Chem. 251, 5337-5344. Shephard, E.A., Pike, S.F., Rabin, B.R. and Phillips, I.R. (1983) Anal. Biochem. 129, 430-433. Watson, D.H., Harvey, M.J. and Dean, P.D.G. (1978) Biochem. J. 173, 591-596. Iyanagi, T. and Mason, H. (1973) Biochemistry 12, 2297-2308. Jollie, D.R., Sligar, S.G. and Schuler, M. (1987) Plant Physiol., 85, 457-462 Vermilion, J.L. and Coon, M.J. (1978) J. Biol. Chem. 253, 8812-8819. Gunn, J.R. and Strobel, H.W. (1979) J. Biol. Chem. 254, 4177-4185. Williams, D.E., Becker, R.R., Potter, D.W., Guengerich, F.P. and Buhler D.R. (1983) Arch. Biochem. Biophys. 225, 55-65. O’Keefe, D.P. and Leto, K.J. (1989) Plant Physiol. 89, 1141-1149.
112
177,
May
31, 1991
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
MULTIPLE NADPH-CYTOCHROME IN HIGHER
FORMS I?450 PLANTS
105-112
OF REDUCTASE
Irene BENVENISTE*, Agnes LESOT: Marie-Paule HASENFRATZ*,GeorgKOCHS+and Francis DURST* *Laboratoire
d’Enzymologie
Cellulaire
Moleculaire
des
Institut
Plantes,
et Moleculaire, de
Strasbourg +Biologisches
Institut Pflanzen,
Received
February
II
der
Cedex,
28,
28
rue
Goethe,
67083
France
Universitbt,
Schanzlestrasse
de Biologic
Institut
Botanique,
Lehrstuhl
1, D-7800
fiir
Freiburg,
Biochemie
der
Germany
1991
of multiple forms of NADPH-cyt P450 SUMARY : We report on the presence This contrasts with the animal cyt reductase in microsomes from higher plants. P450 monooxygenases, where the numerous cyt P450 isoforms are reduced by a single form of reductase. Three NADPH-cyt c reductases have been resolved from Jerusalem artichoke tuber microsomes by chromatography on Reactive Red Agarose and Concanavalin A-Sepharose. Their molecular weights, determined by sodium dodecylsulfate-gel electrophoresis, are 80,000, 82,000 and 84,000. The three and are dependent upon FMN for catalytic proteins share common epitopes activity. They are highly selective for NADPH as electron donor, and allowed effective reconstitution of trans-cinnamic acid and 3,9-dihydroxypterocarpan 6a-hydroxylase activities with purified cyt P450 fractions from Heliantfius tuberosus and Glycine max, respectively. As such, they appear as true 0 1991 Academic Press, Inc. isoenzyme forms of NADPH-cyt P-450 reductase. In
eukaryotes,
multienzyme the
terminal P450
hemoprotein. also
becoming
have
have
recently
artichoke
observed,
microsomes,
with
specific
(8).
To elucidate
P450
reductase
the the
question
plant
two
of
microsomes,
three against
the
P450,
the
no isozymes source
from
(6),
attempted
to
the
NADPH to
the
is
well
of
cyt
P450
of
the
NADPH-cyt (5).
of isolate
P450
However,
we
Jerusalem cross-reacted
artichoke
isozymes
as
P45Os
which
purified of
are
serves
wound-induced
proteins
existence we have
from cyt
multiplicity
animal
the
which flavoprotein,
multiple
blots
of
monooxygenases
cyt
electrons
of
contrast,
Western raised
the
FMN containing
a particular
presence (7)
in
In
in
antibodies
FAD and
In plants,
(2-4). in
of
existence
11).
detected
P450-dependent
transfers
the
obvious been
an
which
many years
cyt
essentially
of
animals,
since
reductase
and
reductase, In
established
microsomal
consisting
oxidase,
NADPH-cyt
is
the
complexes
reductase the
NADPH-cyt the
three
proteins.
105
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
177, No. 1, 1991
This
communication describes,
characterization equivalents
BIOCHEMICAL
of
three
AND BIOPHYSICAL
for
the first
RESEARCH COMMUNICATIONS
time,
isoenzymes which are able to
from NADPHto cyt
P45Os. The activity
the separation transfer
and
reducing enzymes is
of the three dependent upon FMN, as generally described for the NADPH-cyt P450 reductase. Finally, evidence for multiple reductase forms in other plants, based on Western blot analysis, is presented. MATERIALSANDNETHODS : Emulgen 911 was a generous gift from Kao Chemical Co.. CHAPS ~3-~[3-cholamidopropyll-dimethyl~rmmonio~-l-propane sulfonate), Reactive Red 120-Agarose with diverse concentrations of dye, Concanavalin A-Sepharose and a-methylmannoside were from Sigma Chemical Co., Bio-beads SM-2 from Bio-Rad, DEAE Trisacryl from IBF and 2’5’ ADP-Sepharose 4B and molecular weight marker proteins from Pharmacia. Nitrocellulose (45 pm pore size) was from Schleicher and Schiill. Materials
Enzyme
assays : NADPH- or NADH-dependent reduction of cytochrome c was measured spectrophotometrically as described elsewhere (7). For kinetic studies, 14 concentrations ranging from 2.2 uM to 565 uM for NADPH, and from 4 uM to 12 mMfor NADH, were used. Reactivation of NADPH-cyt c reductase with FMN was achieved by including different concentrations of the cofactor directly into the spectrophotouetric assay, without preincubation with the purified reductases. SDS-polyacrylamide gel electrophoresis : The purity of the NADPH-cyt c reductase preparations was checked by SDS-polyacrylamide gel electrophoresis (7.5 or 12.5 % acrylamide, w/v), after Laemmli (9). Protein bands were stained by silver nitrate. Molecular weights were estimated using standard proteins (14-93 kilodaltons) electrophoresed under identical conditions. blot analysis : The microsomal proteins separated by SDSpolyacrylamide electroghoresis were electrotransferred onto gel nitrocellulose, after Towbin et al. (10). The remaining protein-binding sites on the nitrocellulose were blocked by 5% bovine serum albumin and 5% non-fat dry milk, in solution in phosphate-buffered saline containing 0.4% Tween 20. The rabbit anti-reductase serum was diluted 20,000 times. The specific antigen-antibody complexes were revealed by goat anti-rabbit immunoglobulins coupled with alkaline phosphatasle. BCIP (5-Bromo-4-chloro-3-indolyl phosphate) and NBT (Nitroblue tetrazolium), the substrates of the phosphatase were incubated at pH 9.6 in 0.1 M diethanolamine. Western.
of the multiple forms of NADPH-cyt c reductase : The NADPH-cyt c reductase was purified from 1-2 g microsomal protein from Jerusalem artichoke tuber bly chromatography on DEAE-Trisacryl M and 2’5’ ADP-Sepharose 4B columns, in the presence of Emulgen 911, as described previously (8). This preparation was then submitted to dye affinity chromatography, on a 20 ml Reactive Red 120 Agarose column (3.4 Wmoles dye/ml gel), equilibrated with 10 mM KPi, pH 7.4 containing 20% glycerol, 0.2% Emulgen 911, 1.5 mM 8-mercaptoethanol and 1mM EDTA. The fraction of reductase activity partially retained on the dye gel (form 1) was eluted by increasing salt cont.. The unbound reductase was applied onto a Concanavalin A-Sepharose column (15 ml) equilibrated with 10 mM Tris-HCil buffer, pH 7.2, containing 20% glycerol, 0.1% Emulgen 911, 1.5 mM8mercaptoethanol and 0.4M NaCl. This step resolved the reductase into two forms : form 2 was excluded from the lectin column, form 3 was retained and eluted by 0.6M a-methylmannoside. In order to replace Emulgen 911 by the zwitterionic detergent CHAPS, the enzymes were applied onto a small DEAETrisacryl column (5 ml), and eluted by a gradient of KCI (O-O.4 M) in the presence of CHAPS, and finally concentrated on carboxy methylcellulose. Isolation
106
Vol.
177,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
: The cyt P450 isoform hydroxylating Reconstitution of monooxygenase activity cinnamic acid was purified by chromatography on DEAE-Trisacryl and on Hydroxylapatite (4). This preparation was totally free from any NADPH-cyt c reductase contamination. For reconstitution of monooxygenase activity, 26 pmoles cyt P450, in solution in 10mM NaPi buffer, pH7.4, 10% glycerol and 0.9% Emulgen 911, were mixed with different quantities of each reductase form. No phospholipids were added. The mixtures were treated by 100 ~1 Biobeads, equilibrated with 0.1 M NaPi buffer, pH 7.4, to remove excess detergent. These reconstituted systems were then incubated at 26OC for 1 hour in the presence of 10 NM FAD and FMN, 500 NM NADPH and 22 PM [‘“Cl cinnamate, and cinnamate hydroxylation measured as described (11). Pterocarpan hydroxylase activity reconstitution was as described in (3).
RESULTS Isolation
of
Affinity
the
chromatography
purification from
isoforms on
of NADPH-cyt
mammalian
Jerusalem
liver
were
2’5’
reductase
high
is
was
resolution
(Fig-l).
with
11,
step
yields
(form
that
molecular
from by this
in
fact
weight,
three
measured
SDS-polyacrylamide 2) and 84,000
the
protein
reductase
isolated
showed
after
for
a single c
specifically
apparent
82,000
efficient
NADPH-cyt
SDS-PAGE
proteins
(form
very
The
also
Their
standard
80,000
13) .
NADPH-cyt c reductase:
48 is
: this
(12,
microsomes
co-purified
comparison by electrophoresis,
ADP-Sepharose
P450
tuber
however
(81,
proteins
Jerusalem artichoke
microsomes
artichoke
procedure
of
(form
gel 3).
NADPH-cyt c reductase (forms 1, 2 and 3) Elution
>
form 1
) a-methyl mannoside
form 3
REACTIVEREDAGAROSE exclusion
I
NaCl
(forms 2 and 3) Elution
CONCANAVALIN A-SEPEAROSE exclusion 1 form 2 To
separate
summarized isoforms
these
above. for
It
lectins
Reactive
is
Form 1 was retained
while
form
2 was only
washing
of
form
form
the
we
based
on
has
been
deviced
differential
by a gel
grafted
and form
with
equilibration
with
to eliminate
any contaminating
gel,
we have
tested
gels
to
with
resolve
with
3.4
3 was readily
150 mM NaCl.
used
the
purification
affinities
scheme
of
the
three
dyes.
shown
retarded
column
1 was eluted also
have
the
and substrate-mimicking
Red-Agarose
(14).
2,
proteins,
form lower
107
NADP+ dependent Nmoles
dye per
excluded.
buffer to remove Two chromatographic
2. To avoid binding dye concentrations
enzymes
ml agarose,
After
abundant
loosely cycles
of form (0.2, 0.5
bound were
2 to the and 0.96
Vol.
177,
No.
1, 1991
BIOCHEMICAL
A
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
B
” n 234.0 -80.0
1
2
3
4
21.5-m 14.4-a 1
2
Figure 1 A. SDS-polyacrylamide gel (12.5 Jerusalem artichoke NADPH-cyt P450 reductase 2’5”ADP-Sepharose. Lane 1 : molecular weight NADPH cyt c reductase. SDS-polyacrylamide gel (7.5%) e l& purxfied forms of NADPH-cyt P450 reductase. after
pmole
Lane
dye per
isoforms, Form Form
ml agarose)
form
2 was
not
specifically of
1 being
2 and
form
. These only
separated
retained
by
with
of
Kinetic
: We have
for
exhibited
typical
was
varied for
of NADH could
the
c
cyt
(2.5
Reactivation whereas occurs Activity (17). activity
is
not
profile
ba in
shows
the
three
bind
of on 2 :
of
any of
the
the
three
purified
kinetics The
when
apparent
enzymes.
since
be measured, present
in
the
by
reductase
c
the
in
vitro
progressive after
forms,
concentration
of
assay)
cyt
:
of
FAD results
forms
of
enzymes of
for
NADPH
NADPH was in
the
reactions
were
not
(up to 12 mM).
both
only
gel
non-enzymatically
Microsomal
:
nature
c reductase
the
used
NADPH-cyt
FAD and
(16). in
Loss enzyme
of FMN to the
restoration of the addition of increasing 108
three
the
(81,
by addition
The
c.
NADH reduced
presence
possesses
three
Km (=2O PM)
reductase
preparations
of the
cyt
Kinetics
and
c reductases.
affinity 50pM
bound
SDS-polyacrylamide
NADPH-cyt
of
3 was
a glycoprotein
the
apparent
chromatography.
form
indicating
presence
565uM.
while
shows
the
NADPH-cyt
reductase
can be restored
not
A-Sepharose
to the NADH concentrations
FHN of
NADH-cyt
of the
three
purified
characterized
readily Fig.2
to
respect by
reductase
the
FMN was
pM
with
in
three
did
column,
Fig.lB
Michaelis-Menten
presence saturable,
lectin
measured
2.2pM
the
however,
by Concanavalin
the
of the
NADPH,
from
identical
in dalton
electrophoresis
a-methylmannoside,
profile
reductase
gels,
reductase.
electrophoresis studies
scaled
profile chromatography x 10-s. Lane
retarded.
3 were
eluted
this
electrophoresis affinity
Lane 1 : NADPH-cyt P450 reductase on 2’5’ADP-Sepharose. Lane 2 : form 1 of the NADPH-cyt c 3 : form 2. Lane 4 : form 3 (glycoprotein). Proteins were
purification
reductase. silver-stained.
%) after
FMN of
(8,
15),
FMN, which
inactivation.
incubation
NADPH-cyt amounts
P450
medium
c reductase of FMN.
Vol.
177,
No.
1. 1991
02 O
BIOCHEMICAL
AND
BIOPHYSICAL
[FMijo&l) I0
w
RESEARCH
NADPH-cytc
COMMUNICATIONS
reductase
activity
(pkat)
Reactivation of the NADPH-cyt c reductase activity of the three riiluE2, isoforms as a function of the concentration of FMN added. NADPH-cyt c reductase activity is expressed in nkat reduced cyt c per ml isoform . form 2 ; (0-0) form 3. (0 -011 form 1 ; (t---t) piuure $ Reconstitution of cinnamic acid I-hydroxylase activity by the three NADPH-cyt P450 reductase isoforms. Reductases are expressed as enzyme units (pkat cyt c reduction activity). Cinnamic acid hydroxylase activity is expressed as pkat p-coumarate formed/nmole cyt P450. (o-o) form 1 ; (t---t) form 2 ; (o-0) form 3. Reconstitution
of
reductase
activity
hydroxylation
of
reconstituted
by
three
monooxygenase
forms
microsomes
and the
of
reductase
were
able
NADPH-cyt
P450 reductase.
activity from
showed
reductase
antibodies
raised
recognized
specifically
investigate
the
against existence
the
hemoprotein
isolated
three
electrons therefore,
deprived
the
activity
was
of
to
for
the
cyt
the
cell
isoenzyme
P450
cultures in
other
purified
(results plant
artichoke
these
flavoproteins
(7).
of
immunorelated
multiple
109
plant of
the
electron Ga-hydroxylase
cyt not
P450
(3)
isolated
shown).
microsomes NADPH-cyt They
the
catalysing
as isoforms physiological
to
of
from
3,9-dihydroxypterocarpan each
each
reductases
be considered
of
catalysing
Monooxygenase
All
of
isoforms
fraction
concentrations
of
soybean
the
prepared.
selectivity
by addition
elicitor-challenged for
no
in
P450
increasing
transfer
reconstitution
was achieved
Evidence
of
and may,
isoforms since
was
(Fig.3). to
hydroxylation
These
enriched
acid
addition
cinnamate
acceptor,
highly
cinnamic
: A cyt
activity
:
Polyclonal
P450
reductases
were
used
proteins
in
as tools other
to plant
Vol.
177,
No.
species, from the
1, 1991
BIOCHEMICAL
by Western
other
plant
detected
in
the
(molecular
Vicia potato
weights
maize
sa tiva
seedlings.
tuber,
avocado
Specific
between
80 and
and in
84 kDa), cell
were
suspension
were
and wheat
of
cross-
proteins
proteins
Veronica
to those
Three
CatAaranthus
Two immunoreactive
proteins
similar
antiserum.
embryos,
pear,
COMMUNICATIONS
proteins. mobility
reductase
from
RESEARCH
electrophoretic
anti-artichoke
microsomes
BIOPHYSICAL
on microsomal
with
reductases
with
wounded
analysis
microsomes,
artichoke
reacted culture,
blot
AND
revealed
cell
in
suspension
cultures.
DISCUSSION Three induced
microsomal
Jerusalem
artichoke
NADPH and very three
forms
other
triazine
NADPH-cyt
close
is
in
binding
mimic
Reactive
Red appeared
weak,
the
highest
commercially
available
chains
of forms
hindering
the
Alternatively, gel
also
elution
by
moiety,
of
animals
and
in
and
plants. 2
cannot
three
Furthermore,
monoclonal
antibodies
one
suppose
could of
efficient since
it
readily
of
has
been
cleaved
electrons purified
a single
to cyt
Cinnamic cyt
However,
at
a
share
native shown
at very
common
enzyme. reductases. that
low
the
binding
been
tested.
This reductase
Apparently,
with
the
selective
is
similar
an
first
other
since forms.
originate
from forms
were
of
lectins,
apparent
all
in
nature
domains
three
of the
c reductase,
Km for several
Therefore, proteolytic active
and
proteolytic degradation the hydrophobic domain, becomes
unable
to transfer
(18).
acid hydroxylase activity was reconstituted P450 fractions and each of the three reductases,
phospholipids.
dye.
existence This
to
with
excludes lacking
concentration,
P450 monooxygenases
with
all
the gel.
polypeptide
glycoproteic
the
would
However,
protease
the
immunogenic
isoforms
onto
ml of
and its
the
glucose.
cross-react
three
behaviour.
NADPH-cyt
c reductases
we prepared
P450
yet
the
interactions
with
stage,
the
like
immobilized
A-Sepharose
since
that
three
ionic
and/or
this
has not
NADPH-cyt
the
and
per
the
the pyridine
of
of
chromatographic
microsomal
excluded,
they that
NADPH-cyt
of
are
to
consistent
form
glycanes,
enzymes
site
for
Red, of
1 was bound
active
mannose
Reactive link
form folding
Concanavalin
are
be
of other
only
wound-
Surprisingly,
The affinity
from
hydrophobic
in
affinity
column.
result
observed
rich
apparent
dye concentration
the 3 with
a glycosylated
characteristic
cleavage
form
the
from
diphosphate
since
the
a-methylmannoside
example
NADPH .
in
of
oligosaccharide
1
of
explain
The interaction
All
access
differences could
matrix
forms
2 and 3 could
isolated
84 kD).
enzymes.
for of binding
the
NAD(P)+-depending
gel Lack
82 and
dye affinity
to
proteins with
been
of similar
(80,
on the
believed
have
spite
weights
differently
dyes, thus
tubers,
molecular
behaved
nucleotides,
c reductases
the
detergent 110
present
by mixing the without addition in
the
CYt
P450
Vol.
177,
No.
preparation
(1% Emulgen
reconstitution
of
concentration
911)
the
increased
corresponding
microsomal
reductase added
BIOCHEMICAL
1, 1991
The
three
challenged. P-450
reductase
For
proteins
mlcrosomes,
animal
reductase
is
reason More
to determ:ine with
different
cited
above
not if
the
origin
one
translational
of genes
maturation. subcellular
development,
multiple
will
was
Another
studied.
or/and
by
tissular
they
the
would
NADPH-cyt plants
Western rule
purified
cyt
higher
are
P450
in
acid
fatty epoxidation.
forms
P45Os,
these
will
and
a
spatial
these
by
and
this
remains be needed
our
isoforms
from
of
NADPH-cyt
organisms,
raised
result
localization,
of plants.
for productive interaction the other redox reactions
question
temporal
we
blotting
in
squalene
other
P450
that
low-represented
reductase
in
with
microsomes
microsomal
P-450
a
also
higher
and in
multiplicity: could
in
p-chloro-N-
laboratory
since
are equally competent Their involvement in
reductase
our
artichoke all
conserved
several
reductase
oxidations,
NADPH-cyt not
using
Finally, or
the
a single
heme oxygenation
hemoproteins. be
that
phenomenon.
P450.
to be the
in or
cyt
detected seem
P450-dependent
isoforms also
be
elicitorNADPH-cyt
Likewise, in
from
cyt
studies,
all
will
several of
have
evolved
detailed
concerns
terms
in
why plants has
not
admitted
and elongation,
situation
or
is
involved
desaturation
open.
it
rat
of
reconstitute
from
rabbit
with
of reductase
to
a general
be under-estimated
may
higher
P45Os was reconstituted
artichoke
microsomes
would forms
systems,
the
the
used.
between
is
hepatic
with
evaluation
multiple
cyt
from
able
(19).
of
conditions
purified
experiments
immunorelated in
also
as
avocado
10%
by using
interaction
P448
detergent
as a function
the
P450
cyt
Preliminary purified
this
were cyt
effective
rat
hydroxylase
improved
origins
as
about
under
After
the
linearly
reached
different
was
proteins,
Although
from
of purified
detected
be
Catalytic
contained
(20).
three were
immunoreactive
The
that
cinnamate
Two or
In
P45Os
reductase
reductases,
(3).
reductase
demethylase
reconstitute
should
with
phospholipids.
reached
isoforms
COMMUNICATIONS
lowering
increased
was not
cultures
reductase that
tested.
cell
system
methylaniline showed
This activity
reductase
trout
reconstituted liver
which
activity,
Ga-hydroxylase
and cyt
example,
rat
the
artichoke
soybean
the
complex,
and saturation
RESEARCH
efficiently
monooxygenase
since
3,9-dihydroxypterocarpan
BIOPHYSICAL
replaced
activity.
concentrations
flavoprotein
AND
findings encoded
different distribution,
enzymes
during
in plant
be investigated.
to Marie-France grateful technical assistance and to Monique Wehr for typing was supported by MRT grant nO154.85.
ACKNOULEDGNENTS: We are
Castaldi for this manuscript.
excellent This work
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