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K M Madyastha and C J Cosciacharacterization.Catharanthus roseus. Purification andc(P-450) reductase from the higher plant,Detergent-solubilized NADPH-cytochrome:
1979, 254:2419-2427.J. Biol. Chem.
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THE JOURNAI. OF BOLOGCAL CHEMI STRY
Vol. 254, No. 7, k.we of April 10, pp. 2419-2427. 1979
Prrnted in 1l.S A.
Detergent-solubilized NADPH-Cytochrome c (P-450) Reductase from
the Higher Plant, Catharanthus roseus
PURIFICATION AND CHARACTERIZATION*
(Received for publication, August 7, 1978)
K. Madhava Madyastha and Carmine J. Coscia
From the E. A. Doisy Department of Biochem istry, St. Louis University Scho ol of Medicine, St. Louis, Missou ri 63104
A detergent-solubilized NADPH-cytochrome c (P-
450) reductase from etiolated 5-day-old Catharanthus
roseus seedlings has been purified 120-fold by a com-
bination of ion exchange chromatography and gel fil-
tration. In another approach, the reductase was re-
solved by aff ini ty chromatography on 2,5-ADP-Seph-
arose 4B to afford in 36 yield, a 745-fold purified
reductase which was capable of reconstituting geraniol
hydroxylation act ivi ty in the presence of partially pu-
rified cytochrome P-450 and lipid. The purified reduc-
tase was devoid of P-450- and bs-type heme proteins,
NADH-cytochrome c reductase and DT-diaphorase.
Upon sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis, enzyme obtained by both methods was
resolved into two polypeptide bands of 78,000 and
63,000 daltons. However, the 78,000-dalton polypept ide
was enriched in reductase preparations isolated by
aff in ity chromatography. Exogenous FMN stimulated
reductase act ivi ty. The affinity-chromatographed re-
ductase exhibited a characteristic flavoprotein visible
spectrum and contained 0.76 mol of FMN and 0.37 mol
of FAD/mol of enzyme.
Upon thin layer isoelectric focusing of the reductase,
a p1 of 5.3 was observed. The purified reductase trans-
ferred electrons to ferricyanide and 2,6-dichlorophen-
olindophenol, and exhibited menadione-mediated
NADPH oxidase but not adrenaline oxidation act ivi ty.
The apparent K, for NADPH was determined to be 5.7
pM
and that for cytochrome c to be 7.8
pM.
The reductase
was insensitive to antimycin A, dicoumarol, superoxide
dismutase, and the alkaloid, catharanthine, while being
inhibited by NADP+ (competitive ly, Ki = 18 PM), p-chlo-
romercuribenzoic acid, and cetylt rimethy l ammonium
bromide.
In higher plants, NADPH-dependent oxidoreductases,
which transfer electrons to cytochrome c, have been found to
be associated with microsomes as well as other types of
membranes (l-9). This enzyme has been utilized as a micro-
somal marker in sucrose density gradient centrifugation of
homogenates from castor bean endosperm (l), sweet potato
root (2), and artichoke tubers (3). Subcellular localization
studies with cell-free extracts from Catharanthus
roseus
seed-
lings however revealed the presence of NADPH-dependent
cytochrome c reductases in vacuolar and microsomal mem-
brane fractions (4). Microsomes of Beta vulgaris petioles (5),
* Th is work was supported by National Scie nce Foundation Grant
BMS-75-15241. The cost s of public ation of this article were defrayed
in part by the payment of page charges. Th is ar ticle must therefore
be hereby marked advertisement in accorda nce with 18 USC.
Section 1734 solely to indicate this fact.
cotton hypocotyls (6), Marah macrocarpus seed endosperm
(7), and pea seedlings (8) also exhibit this acti vity. The reduc-
tase from cauliflower buds has been studied and its NADPH-
dependent reduction of cytochrome P-450 under anaerobic
conditions was shown to conform to firs t order kinetics (9).
Despite the relative stabili ty and ease of assay of such plant
reductases, their particulate nature has hampered purification
and characterization. In fact, there have been but few reports
of the solubilization of membrane-bound plant enzymes.
The paucity of data on the plant reductases contrasts with
the plethora of information gained for their mammalian coun-
terparts, the microsomal NADPH-cytochrome c reductases.
First studied in a highly purified form 15 years ago (lo),
virtually homogeneous preparations of soluhilized liver micro-
somal NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4)
have now been obtained. If initial solubilization is accom-
plished with detergents rather than steapsin or proteases, the
flavoprotein obtained ef ficiently transfers electrons to cyto-
chrome P-450 (11-13) which is regarded as its biological
electron acceptor. The adrenal cortex mitochondrial NADPH-
adrenodoxin reductase is also a flavoprotein component of a
cytochrome P-450-dependent monooxygenase, but it can only
transfer electrons to cytochrome c or P-450 via the iron-sulfur
protein, adrenodoxin (14).
Recently, we have succeeded in solubilizing, resolving, and
reconstituting the components of a plant cytochrome P-450-
enzyme complex which hydroxylates the C-10 methyl group
of the monoterpene alcohols, geraniol and nerol (15). We now
wish to report the further purification and characterization of
the detergent-solubilized, NADPH-cytochrome c (P-450) re-
ductase, from the higher plant, C. roseus. In the experiments
to be described, a 3,000 to 20,000 x g pellet was used as the
source of enzyme. In addition to mitochondria and other
organelles, the fraction contains vacuolar membranes in which
the monoterpene hydroxylase is localized (4). This membrane
fraction also has bs-type cytochromes as evidenced by oxidized
versus reduced difference spectra. I t is possible, consistent
with the endomemhrane concept, that the hydroxylase-asso-
ciated vacuolar membranes represent a differentiated form of
endoplasmic reticulum.
EXPERIMENTAL PROCEDURES
Preparation of Cell-free Extracts-C. roseus (L) G. Don seeds
were germinated in the dark on a bed of moist vermiculite covered
with filter paper and maintaine d at 30-32C. After 5 days, seed coats
were removed and the seedlings were washed with distilled water. All
subsequ ent operations were carried out at 0-4C.
Seed lings (100 g) were gently ground in a cold mortar for about 2
min in 2 volumes of 0.1 M Tris-HC l buffer, pH 7.6, contain ing 0.4 M
sucrose , 10 mM KCl, 10 mM MgC12,lO mM EDT A, 5 mM meta bisulfite,
and 1 mM dithiothre itol. The slurry was mixed with 3.3 g of washed
polyclar AT and squeezed through two layers of silk . The filtrate was
2419
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2420
Plant NADPH-Cytochrome c Reductase
centrifuged at 3,000 X g for 20 min and the supernatant sedimen ted
at 20,000 x g for 20 min. The 20,000 x g pellet was suspended in 0.1
M Tris-HC l, pH 7.8, contain ing 1 mrvr dithiothre itol, 1
m M
EDTA, and
15% glycerol (w/v). The suspensio n (2.5 to 3.5 mg/ml) was sonicate d
for four 20-s intervals in a Branson ultrason ic disintegrator (model
W, 14OD) at maximum output. Sodium cholate (10% aqueous solution)
was added dropwise with stirring to give a final cholate to protein
ratio of 1:2 and the mixture was stirred for an addition al 30 min. A fter
centrifugation at 106,000 X g for 1 h, the supernatant (with 65 to 70%
of the original NADPH -cytochrome c reductase activity) was applied
to a DEA E-cellu lose column (1.25 x 12 cm) previously equilibrated
with 0.1
M
Tris-HC l, pH 7.8, contain ing 1 mrvr dithiothreito l, 0.005%
sodium cholate , 0.1 mM EDT A, and 15% glycerol (w/v). After w ashing
the column with 100 ml of the equilibratio n buffer, the reductase (6
to 8 mg) was eluted from the column with 100 ml of the equilibration
buffer contain ing 0.25
M
KCl.
Fractions from the DEAE-cellulose column which contained the
NADPH -cytochrome c reductase were concentrated to 10 ml by
ultrafiltration through an Amic on PM-10 membrane. The reductase
from 10 to 12 such different preparations were pooled (70 to 75 mg)
and further concentrated to 30 ml. After dialys is for 6 to 8 h against
30 volumes of 0.05
M
Tris-HCl, pH 7.8, contaming 0.1
I T I M
EDTA, 1
I T I M dithiothreito l, 0.0055; sodium cholate , and 5% glycero l, the re-
ductase fraction was treated with calcium phosphate gel (280 mg of
dry weight, in 4.0 mlj with stirring. The mixture was stirred for an
additional 20 min and centrifuged at 10,000
x
g for 10 min. The
supernatant was discarded. The reductase was eluted from the pellet
by stirring for 30 min with 15 ml of 0.1 M potassium phosphate buffer,
pH 7.8. The mixture was centrifuged and the supernatant was set
aside. The pellet was again eluted with 15 ml of the same buffer and
the combined 0.1 M phosphate buffer eluates (30 ml) were concen-
trated in an Amic on ultrafiltration cell with a PM-IO filter. Th e
concentrated reductase fraction (10 to 13 mg) was dialyzed 6 to 8 h
against 30 volumes of 0.05
M
Tris-HCl, pH 7.8, containing 0.05
M
KCl,
0.005% sodium cholate, and 0.1
I I IM
dithiothreitot (Buffer A). The
enzyme was then applied to a DEAE-Se phadex A-50 column (0.9 x
15 cm) previously equilibrated with Buffer A . After washing with 50
ml of the same buffer, the column was eluted with 50 ml of Buffer A
containing first 0.15
M
KC1 and then 0.25
M
KC1 (Fig. 1). The 0.25
M
KC1 &ate which contained at1 the reductase activity was concen-
trated to 5.0 ml (2.5 to 3.0 mg) by membrane uttrafillration and then
subjecte d to chromatography on Sephadex G-200. The column (0.9
x 39 cm) was equilibrated with 0.05
M
potassium phosphate buffer,
pH 7.8, contain ing 0.1 mM dithiothreito l and the reductase was eluted
from the column using the same buffer. Fractions of 2.5 ml were
collected. Early fractions which contained most of the reductase
activity (Fig. 2, Fractions 6, 7, and 8) were pooled (0.6 to 0.65 mg),
concentrated by membrane ultrafiltration, and stored at -70C. A t
this temperature, the enzyme did not lose appreciab le activity for a
period of 9 months,
Affinity Chromatography on 2,5-ADP-Se pharose 4B-It was
also poss ible to isoiate the reductase by affinity chromatography
following the procedure of Yasuko chi and Masters (13). The 20,000
x g pellet (418 mg) from 5-day-old etiolated C. roseus s eedlin gs (750
g) was solubilized using sodium cholate and subjected to DEAE-
cellulose column chromatography as described above. The reductase
fraction (43 mg) was concentrated by ultrafiltration and dialyzed for
6 to 8 h agains t 30 volumes of 10 I T I M potass ium phosphate buffer, pH
7.7 contain ing 20% glycerol, 0.02 m M EDTA, and 0.2 I I IM dithiothreitol
(Buffer B). It was then applied to a 2,5-ADP-Se pharose 4B column
(1.75 x 7 cm) previously equilibrated with Buffer B containin g 0.1%
Renex 690 (Fig. 3). The c olumn was eluted with 60 ml of equilibration
buffer and then with 75 ml of 200 mrvr potassiu m phosphate buffer,
pH 7.7, contain ing 20% glycerol, 0.1% Renex 690, 0.4 mM EDT A, and
0.2
m M
dithiothre itol. Reductas e activity was not detected i n these
eluates. After the column was again washed with 25 ml of equilibration
buffer, the reductase was eluted with 70 ml of a linear concentra tion
gradient of 2-AMP from 0 to 5
m M
in equilibratio n buffer. The
reductase fraction was then applied to a small DEAE-cellulose column
(1.75 x 7 cm) previously equilibrated with 0.1 M Tris-HCt, pH 7.8,
containing 0.1 m M dithiothreitol, 0.1 m M EDTA, and 15% glycerol,
Lower concen trations of glycerol were used to facilitat e positive
adsorption of the reductase to the calci um phosphate gel (11).
The abbreviations used are: 2,5-ADP-Sep harose 4B, Sepharose
4B-bound N-(6.aminohexyl)adenosine 2,5-bispho sphate; DCPIP,
2,6-dichlorophenolindophenol; SDS, sodium dodecyl sulfate.
The column was washed well with 100 ml of the equilibration buffer
and the reductase was etuted with 10 ml of equilibratio n buffer
contain ing 0.3 M KCl. Th is fraction was concentrated by ultrafiltration
using an Amic on PM-10 membrane and stored at -70C. The puri-
fication of the reductase by affinity chromatography was also carried
out in the presence of FMN and FAD, following essen tially the same
procedure as that outlined in Tab le II, except that all buffers used
contained 1
PM
FMN and 1
pM
FAD. T he reductase was then
subjected to DEAE -cellulose chromatography as described earlier
without adding FMN and FAD to the eluting buffers. It was then
dialyzed for 8 to 10 h agains t 50 volumes of 0.1
M
Tris-HC l, pH 7.8,
containing 0.1
m M
dithiothreitol, 0.1
m M
EDTA, and 15% glycerol.
Aliqu ots of this preparation were used for flavin estima tions and
reconstitution studies.
Isolatio n of Partially Purified Cytochrome P-450-The cyto-
chrome P-450 fraction used in reconstitution studies were obtained
by DEA F,-cellulose chromatography of the solubilize d membrane
fraction. The cholate solubilized 20,000 x g pellet as described above
was subjecte d to DEA F,-cellulose chromatography in the presence of
nonion ic detergent Renex 690. The column (1.75 x 15 cm) was
equilibrated with 0.1
M
Tris-HC l, pH 7.8, contain ing 15% glycerol, 1
mM dithiothreito l, 0.1
m M
EDTA, 0.05
M
KCl, and 0.2% Renex 690
successively eluted w ith 100 ml of equilibration buffer alone and equal
volumes of the same buffer contain ing 0.3, and then 0.5 M KCl. The
elution profile of cytochrome P-450 and NADPH -cytochrome c re-
ductase is shown in Fig. 6. The early fractions (Fractions 9, 10, and
11) contained measurable amounts of cytochrome P450. Since these
fractions also exhibited tow levels of reductase activity, they were
pooled and passed through a small 2,5-ADP-Sepharose 4B column
(1 x 3 cm). Cytochrome P-450 eluting from the column was devoid of
reductase activity, but contained low levels of cytochrome b-,.
Reco nstitution of Geraniol Hydroxylase Actiuity-Th ese experi-
ments were conducte d as described previously (15). The reductase
purified by affinity chromatography and the partially purified P-450
heme protein fraction from DEA E-cellu lose chromatography were
used in these studies. The crude lipid fraction was obtained by
chloroform:metha nol (2:1, v/v) extraction of the 20,000 x g pellet
which was subseque ntly taken in 0.02 M Tris-HCl, pH 7.8, containing
1 mM EDT A and the mixture was sonicate d before use.
Thin Layer Iso electric Focusing -Glass plates (20 x 10 cm) were
coated with a suspe nsion of Sephadex G-75 superfine (7.0 to 7.5 g/
100 ml of water) co ntaining approximately 1R (w/v) ampholytes (pH
range, 2 to lo), 0.1% lysine, and 0.1% arginine. The plate contain ing
carrier gel (final thicknes s of 0.5 to 0.7 mm) was placed on a precooled
(0-5C) metal block of a Desaga TLC double chamber and the protein
sample (0.8 to 1.5 mg) was applied as a narrow band across the midd le
of the plate. An electrode strip soaked in 1
M
H:aPOd w as placed at the
anodic side and another strip soaked in 1
M
NaOH at the catho dic
side. The gel was focused at 200 V for 90 min and then raised to 400
V for 4 h. At the end of the focusin g period, the gel bed was sectioned
into l-cm ba nds (a l-cm band was cut starting from the cathode end)
and each band was eluted with 5.0 ml of 0.1
M
phosphate buffer, pH
7.7, contain ing 0.1 mM dithiothreitot and 5% glycerol. The ampholyte
was removed by repetitive pressure dialys is usin g an Amic on PM-10
filter. To obtain the pH profile at the end of the focusin g period, a
portion of each band was suspend ed in deionized water and the pH of
the suspension was determined.
Disc Gel Electrophoresis-Polyacrylamide disc gel electrophoresis
was performed in the presence of sodium dodecyl sulfate as described
by Laemm li (16). Before applying to the stacking get, protein samp les
were hea ted for 2 min at 100C in 0.065
M
Tris-HCl, pH 6.8, containing
2% sodium dodecyl sulfate, 5% mercaptoetha nol, 10% glycol, and
0.001% bromphenol blue. The separating gel (10.5 X 0.5 cm) containe d
7.5% acrylamide. The electropho resis was carried out at room tem-
perature at 2 mA/g el during stacking and 3 mA/ge l during separation.
Proteins were detected by staining with Coomassie blue R250 and
destained using 7% acetic acid. For estimation of molecular weight by
SDS-polyacrylamide disc gel electropho resis, the protein standards,
phosphorylase a (92,500), bovine serum album in (68,500), catala se
(SO,OOO), DNase (31,000), and ovalbumin (43,000) were used. Poly-
acrylamide disc gel electropho resis of NADPH -cytochrome c reduc-
tase under nondenaturing cond itions was carried out according to the
procedure of Davis (17). Paralle l gels were stained for protein with
Coom assie blue R250 and activity with NADPH -neotetrazolium as
reported by Fan and Masters (18).
Enzyme Assays-T he NADPH -cytochrome c reductase activity
was measured at 550 nm as previously reported (10-13) except that
0.2
M
potass ium phosphate buffer, pH 7.6, was used and the assay
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Plant NADPH-Cytochrome c Reductase
2421
was conducte d at room temperature. During early stages of purifica-
tion of reductase, 100 pM KCN had to be included in the assay
mixture. The spe cific activity is expressed as nanom oles of cyto-
chrome c reduced per min per mg of protein, utilizing the extinction
coeffic ient for reduced minu s oxidized cytochrome c of 21 x lo3 M-
cm- (10). One unit of reductase is defined as the amount of enzyme
catalyzing the reduction of 1 nmol of cytochrome c/min under the
conditions described. The assay system for both ferricyanide and
DCPIP reduction was the same as that for cytochrome c (10-13).
Addition of KCN was not essential. Reduction of ferricyanide and
DCPIP were measured using extinction coeff icients of 1020 Mm'
cm-
(19) and 21 X 10: M- cm- (20), respectively.
DT-diapho rase activity was determined at room temperature by
measuring the NADP H-dependent, menadione -mediated reduction
of cytochrome c spectropho tometrically at 550 nm. The reaction
mixture contained 0.05 M potass ium phosphate buffer (pH 7.6), 5 ITIM
KCN, 2 mM cetyltrimethyl ammon ium bromide, 0.05 mM cytochrome
c, 0.1 mM menadion e, 0.1 mM NADPH , and enzyme in a total volume
of 1 ml. The reaction was initiated by the addition of menadione. In
the presence of 1 to 2 InM cetyltrimethyl ammon ium bromide, the
NADPH-cytochrome c reductase is completely inhibited, whereas
DT-diapho rase activity is stimula ted (21, 22).
NADPH and NADH oxidase activitie s were determined at neutral
(7.4) and acid ic (5.5) pH spectropho tometrically followin g the de-
crease in absorption at 340 nm at room temperature (23). The assay
mixture containe d 30 mM potassiu m phosphate buffer, pH 7.4 or 5.5;
1 mM KCN was added where indic ated. The reaction was initiated by
the addition of 0.1 mM NADPH or NADH. Corrections were made
for nonenzymatic breakdown of NADH and NADPH . The menadi-
one-dependent NADPH oxidase activity was measured following the
procedure of Yam ashita and Sato (24).
Adrena line oxidation was assayed at room temperature by the rate
of adrenochrome formation at 480 nm using an t of 4020 M- cm-
(25). The reaction mixture contained , in a total volume of 1.0 ml, 500
pM adrenaline, 100 pM EDT A, 100 PM NADPH , 0.15 M potass ium
phosphate buffer (pH 8.5), and enzyme (30 to 850 pg of protein) (25).
FMN and FAD content was determined according to the method
of Faeder and Siege l (26), whereas P-450- and &-type heme protein
concen trations were assayed by difference spectra a s previously de-
scribed (15).
Protein was usually determined by the Lowry method (27); when
compo nents which interfere with the Lowry estima tion were present,
the procedure of Dulley and Grieve (28) was used.
All spectropho tometric assays were performed in a Heath spectro-
photometer, model EU-701-51, at room temperature using cuvettes
of l-cm light path. The spectrum of the purified reductase was taken
with an Aminco-Chance DW-2 split beam spectrophotometer.
Materials-DCPIP, 1,4-DL-dithiothreitol, Id-adrenaline, cyto-
chrome c (horse heart, type III), NADPH, NADH, NADP, DEAE-
cellulose (medium mesh, capaci ty, 0.94 meq/g), sodium cholate, FAD,
FMN, dicoumarol, and cetylt rimethyl ammonium bromide were pur-
chased from Sigma. Sephadex G-200, DEAE-Sephadex A-50, and
Sephadex G-75 (superfine) 2,5-ADP-Sepharose 4B were obtained
from Pharmacia. Calcium phosphate gel was the product of Bio-Rad.
Ampholytes (pHisoly tes, pH 2 to 10) were obtained from Brinkmann.
Menadione was purchased from General Biochemicals. Polyclar AT
(polyvinylpyrrolidone) was a gif t from GAF. Superoxide dismutase
from bovine erythrocytes was obtained from Miles Laboratories. 2.
AMP was purchased from P-L Biochem icals.
RESULTS
Purification of the NADPH-Cytochrome c Reductase-Ta-
ble I summarizes the initial protocol adopted to puri fy the C.
roseus cytochrome c reductase. Cholate solubilization of the
reductase and subsequent DEAE-cellulose chromatography
has been described (15). The chromatographic mobi lity of the
C. roseus reductase on this ion exchange column was similar
to that o f the hepatic microsomal enzyme (29). Reductase
activity /mg of protein in the 20,000 x g pellet was about a of
that of hepatic microsomes of untreated rabbits, whereas the
apparent cytochrome P-450 concentration was F,of that of
the mammalian system (30). In general, plant microsomes
appear to have lower levels of cytochromes P-450 and b, and
of NADPH and NADH-cytochrome c reductase activities
than normal rabbit liver microsomes (9, 30).
The DEAE-cellulose eluate was adsorbed on calcium phos-
phate gel and the reductase was eluted with 0.1 M phosphate
buffer, pH 7.7. This step eliminated residual amounts of P-
450- and &-type heme proteins present in the reductase
fract ion from the DEAE-cellulose column (15). The eluate
was concentrated by ultrafiltration and then subjected to ion
exchange and gel exclusion chromatography (Table I) . Figs. 1
and 2 reveal the chromatographic behavior of the reductase
on columns of DEAE-Sephadex A-50 and Sephadex G-200.
The appearance of the reductase as a sharp peak soon after
the void volume of the latter column suggested aggregation of
the enzyme. While this procedure afforded a highly purified
preparation on the basis of SDS-polyacrylamide gel electro-
phoresis, it had a low flavin content and specific activity and
did not hydroxylate geraniol well in reconstitution experi-
ments (see below). For these reasons, another approach to the
purification of this enzyme was undertaken utilizing bioaffin-
ity chromatography as described by Yasukochi and Masters
(13).
Aff ini ty Chromatography of Partially Purified Reduc-
tase-Table II represents a typical purification scheme for
the C. roseus NADPH-cytochrome c reductase by aff ini ty
chromatography. The cholate-solubilized 20,000 x g pellet
was subjected to DEAE-cellulose chromatography as de-
TABLE I
Purifica tion of NADPH -cytochrome c reductase from C. roseus
Reduc tase was assayed by measuring the rate of cytochrome c
reduction (AASSO ,,p, at room temperature. The reaction mixture con-
tained in a final volume of 1.0 ml, 0.2 M potass ium phosphate buffer
(pH 7.6), 0.05 mM cytochrome c, 0.1 mM NADPH, and reductase (3 to
100 pg of protein). During the early stages of purificatio n, 0.1 mM
KCN was included in the assay mixture. The reaction was initiated
by the addition of NADPH and followed for 2 to 3 min. Stimulation
of activity by flavins was determined by adding 5 nmol of FAD or
FMN to the assay mixture. Spe cific activity values given are those
before FAD or FMN were added.
Preparation
Total
p0-
tein
Specific
activity
Yield
20,000 X g
pellet
Sodium cholate solubilized
DEAE -cellulose column eluate
Calcium phosphate gel
DEAE-Sephadex A-50 column
eluate
Sephadex G-200 column eluate
624
301
71
13
3
0.6
nmol/min/
m ?
96
12 100
16 67
119 112
267 45
586 25
1429 12
-0.05 M KCI--0.15 M KCI
t-O.25 M KCI
-700
FRACTION NUMBER
FIG. 1. Elutio n p rofile of solubilize d NADPH -cytochrome c reduc-
tase from a DEAF,-Sephadex A-50 colum n. Column size, 0.9
x
15 cm;
fraction size, 5 ml. . . . . , NADPH cytochrome c reductase activity.
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2422 Plant NADPH-Cytochrome c Reductase
FRACTION NUMBER
FIG. 2. Elution profile of solubilized NADPH-cytochrome c reduc-
tase from a Sephadex G-200 column, Column size, 0.9 x 30 cm;
fraction size, 2.5 ml. 0- - -0, NADPH-cytochrome c reductase
activity.
TABLE
II
Purification of C. roseus NADPH-cytochrome c reductase by
aff in ity chromatography on 2,5-ADP-Sepharose 4B
Preparation Total
protein
Specific ac-
tivity
Yield
20,000 X g pellet
Solubilized 20,000 x pellet
DEAE-cellulose column eluate
2,5-ADP-Senharose 4B
w
wol/min/mg
418
0.023
100
204 0.03 64
43
0.19
86
0.2 17.146 36
a After concentration by ultrafiltration.
After removal of 2-AMP and Renex 690 by DEAE-cellulose
column chromatography.
K
1
;
El
.c
E
-z
c
I
-A B-
C
1
4
.0
FRACTION NUMBER
FIG. 3. Aff in ity chromatography of C. roseus NADPH-cytochrome
c reductase on a 2,5-ADP-Sepharose 4B column. Column size, 1.75
x
7 cm; fraction size, 5 ml. 0, NADPH-cytochrome c reductase
activity.
scribed earlier (15). The reductase fraction was then applied
to 2,5-ADP-Sepharose 4B column pre-equilibrated with
Buffer B containing 0.1 Renex 690. As shown in Fig. 3, most
of the reductase (about 95 ) is adsorbed onto the column.
The reductase eluted as a sharp peak when the elution buffer
contained approximately 1 InM 2-AMP. Most o f the Renex
690 and 2-AMP were removed by subjecting the reductase to
DEAE-cellulose chromatography as described under Exper-
imental Procedures. The specifi c activity o f the reductase
was 17 pmol/min/mg of protein with an overall yield o f 36
(Table II). The yield can be significantly increased (54 ) if
the same purification procedure is carried out with buffers
containing 1
pM
FMN and 1
pM
FAD.
Electrophoresis of Reductase Preparations-Upon sub-
jecting the reductase fraction eluted from the Sephadex G-200
to SDS-polyacrylamide disc gel electrophoresis with phos-
phorylase a, bovine serum albumin, catalase, DNase, and
ovalbumin, the estimated molecular weight of the two major
polypeptide bands were found to be 63,000 and 78,000 (Fig. 4).
Of the two, the 63,000-dalton band was predominant in most
preparations. When the same Sephadex G-200 eluate was
subjected to polyacrylamide disc gel electrophoresis under
nondenaturing conditions and stained with NADPH-neotetra-
zolium, two pink bands were observed (Fig. 4) which corre-
sponded in RF to two bands stained by Coomassie blue (not
shown). SDS-polyacrylamide disc gel electrophoresis of the
aff in ity chromatographed reductase also gave two polypeptide
bands of comparable intensity corresponding to molecular
weights of 78,000 and 63,000 (Fig. 4). This preparation also
exhibited two major bands stained by Coomassie blue and
two NADPH neotetrazolium posi tive bands under nondena-
turing conditions.
Flavoprotein Nature of the Reductase-Table II reveals
that, after DEAE-Sephadex A-50 chromatography, reductase
act ivi ty could be stimulated by addition of FMN. Exogenous
FAD had little eff ect . In fac t addition of FMN to DEAE-
Sephadex A-50 and Sephadex G-200 eluates stimulated
NADPH-DCPIP reductase act ivi ty to almost the same extent
FIG. 4. Sodium dodecyl sulfate and native polyacrylamide disc gel
electrophoresis. A, SDS-polyacrylamide gel of 35 pg of Sephadex G-
200 eluate; B, SDS-polyacrylamide gel of 19 gg of reductase obtained
by aff ini ty chromatography. The direction of migration was from top
to bottom and the lowest band is tracking dye . The gels were stained
with 0.3 Coomassie brilliant blue in water/acetic acid/methanol (5:
1:5) and destained in 7.5 acetic acid. Gel C represents native poly-
acrylamide gel electrophoresis carried out with Sephadex G-200 eluate
(35 pg) and stained with NADPH-neotetrazolium.
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as that of NADPH-cytochrome c reductase, whereas ferricy-
lose chromatography of the solubilized 20,000
x
g pellet
anide reduction was much less affected. On the other hand,
carried out in the presence of nonionic detergent Renex 690
the NADPH-cytochrome c reductase purified by aff in ity chro-
(Fig. 6) gave a better separation of cytochrome P-450 from
matography (Table II) was only stimulated to the extent of
NADPH-cytochrome c reductase and a more active prepara-
35 upon addition of FMN (Table III). The effect of exoge-
tion than our previous procedure (15). Most of the reductase
nous FMN becomes almost insignificant (7 to 10 ) when the
present in the cytochrome P-450 fraction was removed upon
purification of the reductase as described (Table II) was passing it through a small 2,5-ADP-Sepharose 4B column.
carried out in the presence of 1 PM FMN and 1 FM FAD. It
The partially purified cytochrome P-450 still contained low
appears that under these conditions, the dissociation of FMN levels of &,-type cytochromes.
prosthetic groups does not occur. The reductase purified by
Reconstitution of Geraniol Hydroxylase Activity-This
aff in ity chromatography (Table II ) was found to contain 0.37
was carried out using affinity-chromatographed NADPH-cy-
nmol of FAD and 0.76 nmol of FMN/78,000 ng of protein by
tochrome c (P-450) reductase (specific acti vity, 17 pmol/min/
fluorimetric analysis . Nearly 10 times lower values for FAD
mg of protein), partially purified cytochrome P-450, and a
and FMN were obtained if the fluor imetric estimat ions were
crude lipid fraction. The lipid was obtained by chloroform:
carried out with reductase purified by ion exchange-gel filtra-
methanol (2:1, v/ v) extraction of a 20,000 X g pellet. This
tion methods (Table I). This suggests that during DEAE-
fract ion was taken up in 0.02
M
Tris-HCl, pH 7.8, containing
Sephadex and Sephadex G-200 column chromatography, some
1 mM EDTA. and sonicated. The linid solution. which was
enzyme molecules lose their flavin prosthetic groups. A similar
prepared just prior to use, on thin-layer chromatographic
phenomenon has been observed in the case of NADPH cyto-
analysis showed significant amounts of phosphatidylethanol-
chrome P-450 reductase isolated from yeast microsomes (31). amine and phosphatidylcholine. As demonstrated in Table
Furthermore, solutions of high ionic strength can cause dis- IV, maximum geraniol hydroxylase acti vity was observed
sociation of flavin and it has been reported that liver micro-
when cytochrome P-450 fraction, reductase, and lipid were
somal cytochrome P-450 reductase loses its FMN but not its combined. The reductase alone was comnletelv devoid of anv
FAD prosthet ic group upon treatment with KBr or ammo- activi ty, whereas cytochrome P-450 fract ion alone showed
nium sulfate (24, 32). minimal act ivi tv. When linid fraction or reductase was deleted
The oxidized spectrum of the reductase purified by aff ini ty from the complete incubation system, a significant decrease
chromatography is given in Fig. 5. Absorption maxima occurs
in hydroxylase activ ity was observed.
at 452 and 375 nm with a shoulder at about 472 nm.
Isoelectric Focusing Experiments-Reductase prepara-
Preparation of Cytochrome P-450 Fraction-DEAE-cellu- tions at different stages of purification were subjected to thin
layer isoelectric focusing using carrier gels prepared from
TABLE III
Sephadex G-75 containing ampholytes having a pH range of
Effect of flauins on reductase activity using different electron
2 to 10. The calcium phosphate gel eluate had an isoelectric
acceptors
point (PI) of 4.8 with a 2-fold increase in specific activit y (Fig.
Five nano moles of FMN and 5 nmol of FAD are added to the assay
7). However, upon polyacrylamide disc-gel electrophoresis,
mixture. See Table I and Experimental Procedures for incuba tion
this fraction was resolved into several major protein bands. A
conditions.
shi ft in the p1 from 4.8 to 5.3 was observed when the reductase
% stimulation
from the DEAE-Sephadex A-50 column was subjected to thin
NADPH- NADPH-
layer isoelectric focusing (Fig. 7). A 3-fold increase in the
cytochrome c
DCPIP
NADPH-K:jFe(CN)e
specif ic activ ity of reductase also occurred.
Preparation
reductase reductase
reductase
-
Irrespective of the source of reductase, isoelectric focused
FMN FAD FMN FAD FMN FAD
preparations were stimulated by FMN. Since the enzyme
DEAE-Sepha- 74 8 76 7 22 11
act ivi ty of the isoelectric-focused DEAE-Sephadex eluate was
dex A-50
enhanced to a greater extent than that o f the similarly treated
eluate
calcium phosphate gel eluate, the shi ft in p1 from 4.8 to 5.3
may reflect conversion of holoenzyme to apoenzyme with
Sephadex G- 130 10 129 8 55
I
200 eluate
release of FMN. Under isoelectric focusing conditions, puti-
Plant NADPH-Cytochrome c Reductase
2423
2,5-ADP- 35 4
r
)
r I I I
Sepharose
kO.OSM KCI+0.3M KCI-kO.SM KCI--C(
4B column
35.0
eluate
30.0
I I
I r ,
2.0 -
25.0 ;
i
0 10 20 30
40
FRACTION NUMBER
FIG. 6. Elutio n p rofile of solubilize d NADPH -cytochrome c reduc-
FIG. 5. Oxidized spectrum of the reductase purified by affinity
tase from a DEAE-cellulose column in the presence of 0.2% Renex
chromatography. Spe cific activity of the reductase was 17 pmol/m in/
690. Column size, 1.75 X 15 cm; fraction size, 7 ml. 0, NADPH-
mg of protein when cytochrome c was used as the acceptor.
cytochrome c reductase activity.
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Plant NADPH-Cytochrome c Reductase
TABLE IV
TABLE V
Geraniol hydroxylation in a reconstitute d system from C. roseus
The assay mixture contained 150 pmol of Tris-HCl, 5 nmol of
FMN, 1.5 pmol of dithiothre itol [1-Hlgeraniol(450,OOO cpm, 11 nmol),
and 0.5 pmol of NADPH in a total volume of 1.5 ml. Variable fractions
used were 9 pmol of P-450, 4.3 units of affinity-chromatographed
reductase, and 0.2 mg of total lipid (CHClz:MeOH, 2:1, extract). The
mixture was incubate d at 35C for 30 min.
Enzyme activitie s with different electron acce ptors at various
stages ofpurification
NADPH -DCPIP, NADP H-ferricyanide, and NADH -cytochrome
c reductase activities were assayed as described in Table I with the
following modifications. In DCPIP-reductase reaction mixtures, 40
nmol of DCPIP was substituted for cytochrome c and the decrease in
absorbance at 600 nm was measured. In ferricyanide reductase assay
mixtures cytochrome c was replaced with 210 nmol of ferricyanide
and the decrease in absorbance at 420 nm was determined. Ferricya-
nide was omitted from the blank. In NADH-cytochrome c reductase
assays, 0.1 mM NADH was substituted for NADPH. DT-diaphorase
activity was assayed as describe d under Experimental Procedures.
The NADPH -cytochrome c reductase activity of these fractions are
given in Table I.
Geraniol hydroxyl-
ation
Cytochrome P-450
Reductase
Lipid
Reduc tase + cytochrome P-450
Cytochrome P-450 + lipid
Cvtochrome P-450 + reductase + lipid
nnd/nin/nmol
P-450
0.069
0
0
0.166
0.104
0.53
60
r
i
-1
1
0.0
5.0
0 I
cz
LO.0
5.0
0
FRACTION NUMBER
FIG. 7. Thin layer isoelectric focusing of the NADPH-cytochrome
c reductase f rom DEAE-Sephadex A-50 column eluates (0.85 mg of
protein)
(A)
and calcium phosphate gel-treated
(1.5
mg of protein)
(B) preparations. In the absenc e of FMN the spe cific activity of the
latter (B) fraction was 0.78 pmol/min/m g of protein, whereas upon
addition of 5 PM FMN it increase d to 1.50 pmol/min /mg of protein.
daredoxin reductase dissociates into FAD and apoenzy&e
(33). Alternately, since both of these preparations contain
DT-diaphorase at different relative concentrations with re-
spect to the reductase (Table V), the shi ft in p1 may reflect
the change in the proportion of diaphorase present.
Electron Transfer Capability of Solubilized Reductase
Preparations-Highly purified liver microsomal cytochrome
P-450 reductase can catalyze the reduction of cytochrome c,
ferricyanide, and DCPIP at comparable rates (11-13). Al-
though the plant reductase preparation exhibited the same
ratio of activity for ferricyanide
versus
DCPIP throughout
the purification, an appreciable change in relative activi ties
Specific activity
Preparation NADPH- NADPH- Ratio
NADH-
DCPIP
KJWCN),
(A) (B)
ATfB
cyto- Dl-dia-
chrome
phorase
c
nmol/min/mg
DEAE -cellulose 70 290 0.24 117 620
column eluate
Calcium phos- 121 530 0.23 46 46
phate eluate
DEAE-Sepha- 274 1200 0.23 0 16
dex A-50 col-
umn eluate
Sephadex G-200 245 1120 0.22 0 0
column eluate
between cytochrome c and the two former electron acceptors
occurred during Sephadex G-200 chromatography (compare
Tables I and V). DT-diaphorase can be assayed in the pres-
ence of NADPH-cytochrome c reductase by selectively in-
hibiting the latter with cetylt rimethy l ammonium bromide
and measuring menadione-mediated residual cytochrome c
reduction (21, 22). In this manner DT-diaphorase was found
to be present at various stages of the purification with com-
plete removal occurring upon Sephadex G-200 column chro-
matography (Table V). At this step, the NADPH-cytochrome
c reductase was enriched 3- to 4-fold, whereas the ferricyanide
and DCPIP activit ies remained unchanged. Such a variation
in reductase act ivi ty supports the possibility that the DT-
diaphorase contributes signifi cantly to the overall reduction
of the latter two electron acceptors, ferricyanide and DCPIP.
It has been demonstrated that DT-diaphorase can transfer
electrons to ferricyanide and DCPIP (21). On the basis of
constant A/B ratios in Table V, it appears that the diaphorase
exhibits the same relative rate for both electron acceptors as
elicited by the NADPH-cytochrome c reductase. Conn and
co-workers have reported the presence of NADPH-DCPIP
diaphorase act ivi ty in sorghum microsomes (34).
The NADH-cytochrome c reductase, which has a lo-fold
higher specific act ivi ty than the NADPH dependent reductase
in the 20,000 x g membrane fraction, was completely elimi-
nated during the purification (Table V). Menadione-mediated
NADPH oxidase ac tiv ity was also found in C. roseus prepa-
rations. Yamashita and Sato have demonstrated that this
enzyme is identical with NADPH-cytochrome c reductase
(24).
Aust et al. (25) have reported that rat liver microsomes
oxidize adrenaline to adrenochrome utilizing superoxide an-
ions generated by NADPH-cytochrome c reductase. Co-puri-
fication of adrenaline oxidation and NADPH-cytochrome c
reductase act ivi ty was demonstrated. In contrast the crude
20,000
x
g pellet as well as partially and highly purified
reductase fractions from C. roseus were incapable of catalyzing
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Plant NADPH-Cytochrome c Reductase
2425
FIG. 8. Lineweaver-Burk plots of the
AAs5,,
change associated with increasing
concentra tion of NADPH (A) and cyto-
chrome c (B). The reaction mixtures
contained , in a final volume of 1.0 ml, 0.2
M potass ium phosphate buffer (pH 7.6),
0.05 mM cytochrome c (A) or 0.05 mM
NADPH (B), reductase (7 pg of protein
DEAE-Sep hadex A-50 column eluate),
and varying concen trations of NADPH
(A, 5 to 100 PM) or cytochrome c (B, 5 to
50 PM). Assay s were carried out at room
temperature.
-0 176
TABLE VI
Effect of inhibitors on NADPH-cytochrome c reductase and DT-
diaphorase activities
See Tab le I and Experimental Procedures for incuba tion condi-
tions. T he source of reductase in these assays was Sephadex G-200
column eluates.
76activity
Concentration
NADPH-
cvto-
DT-
chknne diaphorase
c reductase
rnM
Antimycin A
1o-:
100
10- 100
p-Chloromercuribenzoic acid
lo-
18
2 x lo-~ 0
Catharanthine 1 96
Cetyltrimethyl ammon ium 0.6 14
bromide 0
100
Dicouma rol 5 :, 1o- 100
0.1 0
Superoxide dismutase 240 units 106
100
n At this concentration dicoumarol inhibited menadione-mediated
NADPH-oxidase 12%.
NADPH-dependent oxidation of adrenaline. If control assays
were run with phenobarbital-induced rabbit liver microsomes
and solubilized microsomes, significant adrenaline oxidation
activ ity was observed.
While crude membrane fractions (20,000
x
g pellet) ex-
hibited both NADPH oxidase (1.96 nmol/min/mg of protein
at pH 7.4 and 5.9 nmol/min/mg of protein at pH 5.5) and
NADH oxidase activities (3.96 nmol/min/mg of protein at pH
7.4 and 1.97 nmol/min/mg of protein at pH 5.5), both activities
were lost upon purification. This oxidation act ivi ty was in-
hibited by KCN (100 at pH 7.4, 75 at pH 5.5).
Characterization of the Purified Reductase-Kinetic anal-
ysis revealed the reductase possessed an apparent K, of 5.7
PM for NADPH (Fig. 8), a value close to that found for
cauliflower microsomal NADPH-cytochrome c reductase (9)
and the enzyme from kidney microsomes (18). The apparent
K,,, for cytochrome c was 7.8
pM
(Fig. 8), whereas the highest
specifi c activit y of the reductase observed was 22 pmol/min/
mg of protein in the presence of 5 pM FMN.
Phillips and Langdon (35) have observed that mammalian
NADPH-cytochrome c reductase act ivi ty is dependent on
ionic strength. A similar eff ect was observed with the plant
reductase with phosphate buffer. An increase in specific activ-
ity occurred with increasing ionic strength which was com-
3 The failur e to observe activity, however, could also be due to the
small amount of purified reductase (60 pg) used in oxidase assays. The
oxidase activity of the purified reductase may represent only a sma ll
percentage of the NADPH -cytochrome c reductase rate.
0.6
I
i I [NADPH. vM]
-o.iza
l/[Cyi c. uhl]
1 I I I
-18
50
100
NADPH
2QJM
50pM
100pM
NADP+, pM
FIG. 9. A Dixon plo t exhibiting the compe titive inhibitio n of
NADP at fixed concentrations of NADPH as indicated. The reaction
mixtures containe d, in a final volume of 1.0 ml, 0.2 M potassiu m
phosphate buffer (pH 7.6), 0.05 mM cytochrome c, NADPH as indi-
cated, 10 to 100 PM NAD P, and 7 pg of protein of Sephadex G-200
column eluate. Assay s were carried out at room temperature.
parable to that observed for the mammalian enzyme under
the same conditions (35).
Inhibition of NADPH-Cytochrome c Reductase-Although
the 20,000 x g pellet contains most of the plants mitochon-
drial membranes (4), all preparations of the NADPH-cyto-
chrome c reductase were insensitive to antimycin A (Table
VI). As in the case of the mammalian reductase (lo), p-
chloromercuribenzoic acid is a potent inhibitor of the plant
enzyme. Catharanthine, an end product alkaloid which is a
noncompetitive inhibitor of the hydroxylase, K, = 1 ITIM (36),
had no eff ect on reductase act ivi ty. Fig. 9 demonstrates the
competitive nature of NADP+ inhibition of the plant reduc-
tase (K; = 18
pM)
again analogous to the mammalian enzyme.
The K, of NADP for the kidney microsomal reductase is 4.4
PM
(18), whereas that of a microsomal preparation from arti-
choke tuber was reported to be 24
PM
(3). Dicoumarol is a
potent inhibitor of hepatic microsomal NADPH-DT-diapho-
rase, whereas it has little eff ect on the NADPH-cytochrome
c reductase of that tissue (22). Table III reveals that the plant
enzymes again have comparable properties. Superoxide dis-
mutase at high concentrations does not aff ect cytochrome P-
450-dependent cinnamate hydroxylation in sorghum (34) and
this correlates well with the current interpretation of data for
the animal sys tem. As shown in Table VI superoxide dismu-
tase does not affect the C. roseus reductase.
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2426 Plant NADPH- Cytochrome c Reductase
DISCUSSION
The C. roseusNADPH-cytochrome c (P-450) reductase
bears a resemblance to the mammalian microsomal enzyme
with respect to its substrate spec ific ity, isoelectric point, and
sensitiv ity to known reductase inhibitors. Evidence for the
flavoprotein character of the plant enzyme preparation was
obtained by fluorimetric flav in analyses, FMN stimulation
data, and the oxidized spectrum of the purified enzyme (Fig.
5). It is known that the cytochrome P-450 reductase in hepatic
microsomes contains equimolar quantities of FMN and FAD.
\The aff in ity chromatographed plant reductase contains con-
siderably lower levels of FAD (0.37 mol/mol of protein),
whereas the concentration of FMN (0.76 mol/mol of protein)
is comparable to that o f mammalian enzyme. Preincubation
of homogeneous mammalian microsomal NADPH-cyto-
chrome c reductase with FAD or FMN did not enhance its
act ivi ty (37). However, FMN-depleted NADPH-cytochrome
c reductase from hepatic microsomes .was restored to almost
full act ivi ty upon reconstitution with FMN. Addition of FAD
or FMN to the apoenzyme of mammalian menadione-depend-
ent NADPH oxidase restored only 20 of its original value
(24).
On the other hand, it has recently been reported by Aoyama
et al. (31) that the NADPH-cytochrome P-450 reductase of
yeast microsomes which has been purified to apparent ho-
mogeneity contains both FAD and FMN in the range of 5 to
7 nmol/mg of protein. These values were significantly lower
than expected, calculated on the basis of the apparent molec-
ular weight of the reductase. They attribute this to the dis-
sociation of flavin prosthetic group from the enzyme during
the course of purification. Another interesting observation
made by the same group (31) was that the dissociation of
FAD prosthetic group from the enzyme is a unique character-
istic of the cholate-solubilized preparation. It is possible that
the cholate solubilization of C. roseus NADPH-cytochrome c
reductase also leads to lower levels o f FAD. It has been
observed that activity lost due to dissociation of FMN can be
restored by addition of FMN, whereas dissociation of FAD
always results in a loss of act ivi ty which cannot be recovered
by preincubation with FMN or FAD (38). The purest plant
reductase preparations had speci fic activities of about one-
third the value of the homogeneous mammalian microsomal
enzyme (Table II), possibly due to an irreversible dissociation
of FAD.
Elevation of the ionic strength of a solution of reductase
will result in its dissociation to apoenzyme (32, 38). However,
the plant enzyme was exposed to solutions of comparable
ionic strength on the DEAE-cellulose and DEAE-Sephadex
A-50 columns and lower ionic strength solutions on the Seph-
adex G-200, yet the latter chromatography afforded an enzyme
preparation whose activ ity was most enhanced by FMN, while
the DEAE-cellulose column eluate exhibited no significant
stimulation of cytochrome c, DCPIP, and ferricyanide reduc-
tion. Thus it appears the state of purity is also a factor in the
dissociation of the FMN prosthetic group for the plant en-
zyme. A characteristic of the plant enzyme preparation was
its variable relative specif ic activi ties with different electron
acceptors. The highly purified mammalian microsomal
NADPH-dependent reductase will catalyze electron transfer
to cytochrome c, ferricyanide, and DCPIP at comparable
rates. Not only was the DCPIP act ivi ty relatively low for the
plant preparation but, while the rates of reduction of the latter
two electron acceptors remained constant during the purifi -
cation (Table V), they varied with the cytochrome c speci fic
act ivi ty (Table I). The results suggested the presence of at
least two NADPH-dependent reductases and this was con-
firmed by subsequent experiments in which DT-diaphorase
act ivi ty was detected. The fac t that the DT-diaphorase was
removed during Sephadex G-200 column chromatography can
explain the 3-fold enrichment of NADPH-cytochrome c re-
ductase in this step without concomitant change in DCPIP or
ferricyanide reductase activity.
The plant reductase purified by ion exchange-gel filtration
techniques also has different act ivi ty towards the three diffe r-
ent electron acceptors tested (Table III) in the presence of
exogenously added FMN and FAD. As shown in Table III ,
ferricyanide reduction by plant enzyme is not stimulated to
the same extent as the reduction of cytochrome c and DCPIP
upon addition of FMN. This raises the question of whether
FMN is required for ferricyanide reduction. In fac t Vermilion
and Coon (38) have shown that FMN-depleted enzyme can
reduce ferricyanide, whereas FMN is necessary for the trans-
fer of electrons to cytochrome P-450, cytochrome c and
DCPIP. The fact that the Sephadex G-200 eluate which is
devoid of DT-diaphorase does show some stimulation of fer-
ricyanide reduction with FMN (Table II I) suggests a diffe r-
ence in the specific ity of the flav in groups of the plant and
mammalian enzymes. It was also observed that the plant
reductase differs from the mammalian enzyme in its inability
to oxidize adrenaline. This raises the question of whether the
plant reductase can generate superoxide anion. The aff ini ty-
chromatographed plant reductase could reconst itute geraniol
hydroxylation act ivi ty when combined with partially purified
cytochrome P-450 and a crude lipid fract ion (Table IV). When
the reductase or the cytochrome P-450 fract ion was deleted
from the reaction mixture, a significant loss in hydroxylase
act ivi ty was observed. The aforementioned factors taken to-
gether suggest that the NADPH-cytochrome c reductase of
C.
roseus
can be identified as NADPH-cytochrome P-450
reductase.
The detergent-solubilized mammalian cytochrome P-450
reductase has been shown to have a molecular weight of about
78,000 (ll-13), whereas proteolyt ical ly solubilized reductase
has an estimated molecular weight of about 68,000 (18, 39).
The C.
roseus
NADPH-cytochrome c reductase purified by
ion exchange, gel filtration methods on SDS-polyacrylamide
disc gel electrophoresis revealed the presence of two major
polypeptide bands corresponding to molecular weights of
78,000 and 63,000, out of which the one corresponding to
63,000 was predominant. This reductase preparation (specific
act ivi ty, 1.4 pmol/min/mg of protein (Table I)) did not satis-
facto rily reconstitute geraniol hydroxylation act ivi ty in the
presence of partially purified cytochrome P-450 and lipid.
Thus it is quite possible that the band corresponding to 63,000
is the plant counterpart of the proteolytica lly solubilized
hepatic microsomal NADPH-cytochrome c reductase having
the molecular weight of 68,000. The same reductase prepara-
tion on polyacrylamide gel electrophoresis carried out under
nondenaturing conditions and stained with NADPH-neotetra-
zolium gave two pink bands, indicating the presence of two
reductases. It has been observed by Fan and Masters (18) that
proteolytically solubilized reductase is also capable of reducing
neotetrazolium in the presence of NADPH. The reductase
purified by aff in ity chromatography on 2,5-ADP-Sepharose
4B has a signif icantly higher specifi c act ivi ty (Table II). Such
a purified reductase on SDS-polyacrylamde disc gel electro-
phoresis gave one major polypeptide band corresponding to
molecular weight of 78,000 and another band corresponding
to 63,000.
It is interesting to note that although the purified reductase
from C. roseus s devoid of NADH-cytochrome c reductase
act ivi ty, the membrane-bound monooxygenase-catalyzed hy-
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Plant NADPH-Cytochrome c Reductase 2427
droxylation of monoterpene can be stimulat ed by NADH (15).
357, 1037-1038
This synergistic effect is consistent with a growing body of
22. Huang, M., West, S. B., and Lu, A. Y. H. (1977) Bioche m. Biophys.
evidence which indicates interaction between various electron
Res. Commun. 74, 1355-1361
transport systems in endo plasm ic reticulum (40-42).
23. van Berkel, T. J. C., and Kruijt, J. K. (1977) Arch. Bioche m.
Biophys. 179,8-14
L.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Achnow ledgrnents-W e wish to thank Charles Morrow and John
24. Yam ashita, H. N., and Sato, R. (1970) J. &o&em . (Tokyo) 67,
199-210
McFarlane for their assist ance .
25. Aust, S. D., Roerig, D. L., and Pederson, T. C. (1972) Bioche m.
REFERENCES
Biophys. Res. Commun. 47,1133-1137
26. Faeder, E. J., and Siegel, L. M. (1973) Anal. Bioche m. 53, 332-
1. Lord, J. M., Kagawa, T., Moore, T. S., and Beevers, H. (1973) J. 336
Cell Biol. 57, 659-667
27. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Rand all, R. J.
0 Tanaka, Y., Kojim a, M., and Uritani, I. (1974) Plant Cell Physio l.
(1951) J. Bio l. Chem. 193,265-275
l&843-854
28. Dulley, J. R., and Grieve, P. A. (1975) Anal. Bioche m. 64, 136-
Benveniste, I., Salaun, J., and Durst, F. (1977) Phytochemistry
141
16,69-73
29. Lu, A. Y. H., and Levin, W. (1974) Biochim. Biophys. Acta 344,
Madyastha, K. M., Ridgway, J. E., Dwyer, J. G., and Cosc ia, C. J.
205-240
(1977) J . Cell Biol. 72,302-313
30. Ichikawa, Y., and Mason, H. S. (1973) in Oxidases and Related
Martin, E. M., and Morton, R. K. (1955) Nature 176, 113-114
Redox Systems (King, T. E., Mason, H. S., and Morrison, M.,
Frear, D. S., Swanso n, H. R., and Tanaka, F. S. (1969) Phyto-
eds) Vol. 2, pp. 605-625, University Park Pres s, Baltimore .
chemistry 8, 2157-2169
31. Aoyama, Y., Yoshid a, Y., Kubota, S., Kumaoka, H., and Furum-
Hasson, E. P., and West, C. A. (1976) Plant Physiol. 58,479-484
ichi, A. (1978) Arch. Biochem. Biophys. 185,362-369
Ishimaru, I., and Yamazaki, I. (1977) J. Biol. Chem. 252, 199-204
32. Coon, M. J., Haugen, D. A., Guengerich, F. P., Verm ilion, J. L.,
Rich, P. R., and Bendall, D. S. (1975) Eur. J. Biochem. 55, 333-
and Dean, W. L. (1976) i n The Structural Ba sis of Membrane
341
Function (Hatefi, Y., and Djavadi-Ohaniance , L., eds) pp. 409-
Williams, C. H., Jr., and Kamin, H. (1962) J . Biol. Chem. 237,
427, Academic Press, New York
587-595 33. Tsai, R. L., Gunsalus, I. C., and Dus, K. (1971) Biochem. Biophys.
Verm ilion, J. L., and Coon, M. J. (1974) Bioche m. Biophys. Res.
Res. Commun. 45, 1300-1306
Commun. 60, 1315-1322
34. Potts, J. R. M., WekIych, R., and Conn, E. E. (1974) J. Biol .
Dignam, J. D., and Strobel, H. W. (1975) Biochem. Biophys. Res.
Chem. 249, 5019-5026
Commun. 63,845-852
35. Ph illips , A. H., and Langdon, R. G. (1962) J. Biol. Chem. 237,
Yasuko chi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 251,
2652-2660
5337-5344
36. McFarlane, J., Madyastha, K. M., and Cosc ia, C. J. (1975) Bio-
Omura, T., Sanders, E., Estabrook, R. W., Cooper, D. Y., and
them. Biophys. Res. Commun. 66, 1263-1269
Rosen thal, 0. (1966) Arch. Biochem . Biophys. 117,660-673
37. Dignam, J. D., and Strobel, H. W. (1977) Bioche mistry 16, 1116-
Madyastha, K. M., Meehan, T. D., and Coscia, C. J. (1976)
1123
Biochem istry 15, 1097-1102
38. Verm ilion, J. L., and Coon, M. J. (1976) in Flavins and Flavo-
Laem mli, U. K. (1970) Nature 227,680-685
proteins (Singer, T. P., ed) pp. 674-678, Elsevier Scientific
Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427
Publishing Co., New York
Fan, L. L., and Masters, B. S. S. (1974) Arch . Bioche m. Biophys.
39. Iyanagi, T., and Mason, H. S. (1973) Biochemistry 12, 2297-2308
i65, 665-671
40. Jansson, I., and Schenkman, J. B. (1977) Arch. Biochem. Biophys.
Schellenb erg, K. H., and Hellerman, L. (1958) J. Biol. C hem. 231,
178,89-107
547-556 41. Oshino, N., and Omura, T. (1973) Arch. Bioche m. Biophys. 157,
Steyn-Parve, E. P., and Beinert, H. (1958) J. Biol . Che m. 233,
395-404
843-852
42. Sasame, H. A., Thorgeirsson, S. S., Mitchell, J. R., and Gillette,
Lind, C., and Ernster, L. (1976) Hoppe-Seylers 2. Physio l. Chem.
J. R. (1974) Life Sci. 14,35-46
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