j. biol. chem.-1979-madyastha-2419-27.pdf

8/11/2019 J. Biol. Chem.-1979-Madyastha-2419-27.pdf

1/10

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.

http://www.jbc.org/content/254/7/2419.citationAccess the most updated version of this articleat

.SitesJBC AffinityFind articles, minireviews, Reflections and Classics on similar topics on the

Alerts:

When a correction for this article is postedWhen this article is cited

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/254/7/2419.citation.full.html#ref-list-1This article cites 0 references, 0 of which can be accessed free at

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom
http://www.jbc.org/content/254/7/2419.citationhttp://www.jbc.org/content/254/7/2419.citationhttp://affinity.jbc.org/http://affinity.jbc.org/http://affinity.jbc.org/http://affinity.jbc.org/http://affinity.jbc.org/http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;254/7/2419&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/7/2419.citationhttp://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=254/7/2419&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/7/2419.citationhttp://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=254/7/2419&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/7/2419.citationhttp://www.jbc.org/cgi/alerts/etochttp://www.jbc.org/content/254/7/2419.citation.full.html#ref-list-1http://www.jbc.org/content/254/7/2419.citation.full.html#ref-list-1http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/content/254/7/2419.citation.full.html#ref-list-1http://www.jbc.org/cgi/alerts/etochttp://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=254/7/2419&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/7/2419.citationhttp://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;254/7/2419&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/254/7/2419.citationhttp://affinity.jbc.org/http://affinity.jbc.org/http://www.jbc.org/content/254/7/2419.citationhttp://affinity.jbc.org/


2/10

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

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/


3/10

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

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


4/10


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.

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


5/10

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.

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


6/10

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-


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.

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


7/10


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

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


8/10


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.

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


9/10

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-

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom


10/10

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

bygueston

Septem

ber11

,2

014

http://www.jbc.org

/

Downlo

adedfrom

j. biol. chem.-1979-madyastha-2419-27.pdf

Documents