Phytoalexin synthesis in soybean: Purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 273, No. 2, September, pp. 543-553,1989

Phytoalexin Synthesis in Soybean: Purification and Reconstitution of Cytochrome P450 3,9-Dihydroxypterocarpan Ga-Hydroxylase and Separation from Cytochrome P450 Cinnamate 4-Hydroxylase GEORG KOCHS AND HANS GRISEBACH’ Biologisches Institut II der Universitiit, Lehrstuhl fiir Biochemie der Pjeanzen, Schiinzlestrasse 1, D-78W Freibwg, Federal Republic of Germany Received March 22,1989, and in revised form May 12,1989

Elicitor-challenged soybean (G&&e mux) cell cultures were used for detergent solubilization and purification of cytochrome P450 3,9-dihydroxypterocarpan Ga-hydroxylase (DGaH). D6aH was purified to electrophoretic homogeneity from such cells by a fivestep procedure. It could be separated from cytochrome P450 cinnamate 4-hydroxylase on hydroxyapatite. This is the first report on separation of two cytochrome P450 enzymes from a higher plant. On sodium dodecyl sulfate polyacrylamide gels D6aH migrated with a M, about 55,000. For reconstitution experiments soybean NADPH:cytochrome P450 (cytochrome c) reductase was purified to homogeneity. Reconstitution of D6aH in the presence of NADPH was dependent on cytochrome P450 DGaH, the reductase, and lipid. Dilauroylphosphatidylcholine gave higher D6aH activity than soybean lipids (asolectin). The reconstituted D6aH system showed a much higher temperature stability than the microsomal system. o 1989 Academic PEW., IIIC. P450 enzymes from higher plants has been accomplished in only a few cases. Cinnamate 4-hydroxylase was purified from the roots of Helianthus tuberosus (6). The 56kDa protein could be reconstituted with NADPH-cytochrome P450 (cytochrome c) reductase (6) and dilauroylphosphatidylcholine (7). Reconstitution of geraniol hydroxylase from V&a rosea (8, 9) and of digitonin 12-P-hydroxylase from cell cultures of Lligitalis lanuta (10) of unknown purity has also been reported. No enzyme activity could be assigned to a purified cytochrome P450 from Tulipagesneriana (11, 12). Separation of different P450 species from higher plants has not yet been reported. In soybean, the different sensitivities of 1To whom correspondence should be addressed. several cytochrome P450 enzymes toward 2Abbreviations used: Chaps, 3-(3-(cholamidoproa number of specific inhibitors indicate the pyl)dimethylammonio)-1-propanesulfonate; Cyt, cyexistence of several P450 species (13,14). tochrome; DGaH, 3,9-dihydroxypterocarpan Ga-hyIn this paper we report on detergent soldroxylase; Pmg, Phytuphthora megasperma f.sp. glyubilization, purification, and reconstitucinea; SDS, sodium dodecyl sulfate.

Accumulation of the phytoalexin glyceollin in soybean (Glycine mux L.) infected by Phytophthora megaspewnu f.sp. glycinea (Pmg)’ plays an important role in the resistance of soybean against this fungus (1,2). Biosynthesis of the pterocarpan derivate glyceollin I involves five cytochrome P450 enzymes. These include, besides cinnamate 4-hydroxylase (trans-cinnamate 4-monooxygenase; EC 1.14.13.11), the new enzymes isoflavone synthase (3), daidzein 2’-hydroxylase ((3), G. Kochs, unpublished), 3,9-dihydroxypterocarpan 6ahydroxylase (4), and a cyclase (5) (Fig. 1). In contrast to mammalian systems, purification and reconstitution of cytochrome

543

0003-9861189$3.06 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

544

KOCHS AND GRISEBACH

4-hydroxycinnamate

described (18). Six-day-old cultures with a conductivity of about 1.5 mS were induced for 24 h by addition of 30 mg Pmg elicitor per 400 ml of medium. Heatreleased Pmg elicitor (4.1% protein, 21% glucose equivalents) was prepared as described (19).

cinnamate

-f+-

HO

e

H Glyceollin I

1. Reactions in the biosynthesis of the soybean phytoalexin glyceollin I which involve Cyt P450 enzymes. (a) Cinnamate 4-hydroxylase; (b) isoflavone synthase; (c) daidzein 2’-hydroxylase; (d) 3,9-dihydroxypterocarpan Ga-hydroxylase; (e) cyclase. FIG.

tion of cytochrome P450 D6aH from elicitor-challenged soybean cell cultures and on its separation from cytochrome P450 cinnamate 4-hydroxylase. MATERIALS

The buffer solutions were degassed under vacuum, equilibrated with nitrogen, and again degassed. The following buffers were used: (A) 0.2 M Tris/HCl (pH 7.5) with 15% sucrose and 14 mM mercaptoethanol; (B) 50 mM potassium phosphate (pH 7.5) with 15% glycerol and 2.8 mM mercaptoethanol; (C) as B but pH 7.0; (D) 500 mM potassium phosphate (pH 7.0) with 15% glycerol, 0.1 mM EDTA, and 1 mM dithiothreitol; (E) as D but with 50 mM potassium phosphate; (F) as D but with 20 mM potassium phosphate; (G) 206 mM potassium phosphate (pH 7.0) with 15% glycerol, 1 mM EDTA, 1 M KCl, and 2.8 mM mercaptoethanol.

Analytical Methods Quantitative determination of cytochrome P450 and cytochrome 9 was carried out according to Omura and Sato (20,21) with a Shimadzu MPS-2000 spectrophotometer. The extinction coefficients used were 91 rnM-’ cm-’ (Adsdw .,) for cytochrome P450 .,) for cytochrome bt,. and 185 mM-’ cm-’ (A410425 SDS-polyacrylamide gel electrophoresis was carried out as described by Laemmli (22) with a 5% stacking gel and 10% separation gel. A Mini Protean II Cell from Bio-Rad (Munich) was used. Protein concentration was determined by a modified Lowry procedure (23) in the presence of SDS (24) with bovine serum albumin as standard and by the method of Bradford (35).

AND METHODS

Materials

Enzyme Assays

The (f)-3,9-dihydroxy-[6,11a-3H]pterocarpan used had been synthesized previously (4). The truns-[3i4H]cinnamic acid (2.07 GBq/mmol) was obtained from Amersham Buchler (Braunschweig). Chaps was synthesized as described (15) or was obtained from Serva (Heidelberg). Renex 690 was from Kao-Atlas Chemie (Essen). All other detergents were from Sigma (Munchen). Asolectin was purified according to published procedures (16,17). Bio-Beads SM-2 and hydroxyapatite were from Bio-Rad (Miinchen). Polyethylene glycol 6000 was from Merck (Darmstadt). All FPLC columns and column materials were obtained from Pharmacia (Freiburg).

3,9-Dihydroxypterocarpan Ga-hydroxylase was assayed in a total volume of 100 ~1 with 200 pmol (-c)dihydroxy [6,11a-3Hlpterocarpan (23,000 cpm) and about 30 pg microsomal protein for 15 min at 10°C as described (4). Cinnamate I-hydroxylase was assayed in a total volume of 100 ~1 with 1 nmol trons-[3-14C]einnamic acid (lo5 epm) and about 60 pg microsomal protein for 15 min at 10°C as described (25). NADPH-cytochrome P450 reductase was assayed with cytochrome c at 550 nm and 30°C (26). Cytochrome c oxidase was assayed as described (27).

Cell Cultures and Eli&n-s Cell suspension cultures of soybean (Glycine mox cv. Harosoy 63) were propagated in 400 ml medium as

Preparation of Microsomul Fraction All operations were carried out on ice or at 4°C. One kilogram of soybean cells was collected on a glass frit filter, aspirated dry, and frozen in liquid nitrogen.

CYTOCHROME P450 FROM SOYBEAN TABLE I ENZYMEINDUCTIONWITH Pmg ELICITOR Treatment of soybeancells

Cyt P450 (nmol/mg)

D6aH (pkat/kg)

Cinnamate I-hydroxylase Watki9

Control Pmg elicitor

0.013 0.166

0 7.8

1.3 37.5

Subsequently the cells were ground for 1 h in a mortar with 600 ml buffer A, 100 g Dowex l-x2 equilibrated in buffer A, and 100 g quartz sand. The extract was filtered through glass wool and centrifuged for 25 min at 10,OOOg.The microsomal fraction was prepared from the supernatant by Mgz+ precipitation (31) and centrifugation for 20 min at 40,OOOg. The pellet was suspended in buffer A. From 1 kgof cells about 70 ml microsomes containing 15 mg protein/ml was obtained.

545

a linear 0 to 1 M KC1 gradient in buffer C containing 0.25% Chaps. Step .IA: Hexyl-ugarose. The D6aH fraction from step 3A (20 ml) was loaded onto a column of hexylagarose (Pharmacia) (16 X 100 mm) which had been equilibrated with buffer D containing 0.1% Chaps. The column was washed with 40 ml buffer D containing 0.1% Chaps and 40 ml buffer E containing 0.1% Chaps. Subsequently D6aH was eluted with buffer F containing 0.05% Chaps and 0.3% Renex. Step 5A: Hydroxyapatite. The D6aH eluate from step 4A was loaded onto three hydroxyapatite columns (5 X 50 mm) which had been equilibrated with buffer F containing 5% glycerol, 0.05% Chaps, and 0.2% Renex. Each column was washed with 6 ml of the equilibration buffer. The columns were eluted with a linear gradient (24 ml) of O-40% buffer D and then with 4080% of buffer D (10 ml) containing 5% glycerol, 0.05% Chaps, and 0.2% Renex. step 3B: Polyethylene glycd preciipitation of NADPH: C!@P&i0 reductase. A 60% solution of polyethylene glycol in buffer C (without glycerol) was added with stirring to the KC1 eluate of step 2 (pool D) to produce

Enx yme PurQication All column chromatographic steps were carried out with a FPLC system (Pharmacia). Columns were operated at 5°C with the exception of Mono-Q and hydroxyapatite, which were operated at 20°C. Step 1: Solubilization To the microsomal suspension in buffer A (360 ml; 13.6 mg protein/ml) were added with stirring on ice aqueous solutionsof EDTA and Chaps to produce final concentrations of 2 and 15 mM, respectively. During 30 min of stirring the slurry was sonicated three times for 30 s (Sonorex Type Super RK 255, Bandelin, Berlin). The slurry was centrifuged for 90 min at 100,OOOg and the supernatant was filtered through glass wool. Step 2. w-Amirwoctyl-agarose. The solubilized fraction (360 ml) was diluted with 180 ml of a solution containing 2.8 rnM mercaptoethanol and 15% glycerol. This solution was loaded onto two columns (50 X 65 mm) of w-aminooctyl-agarose (Pharmacia) which had been equilibrated with buffer B. Each column was eluted at a flow rate of 3 ml/min with the following solutions: 100 ml buffer B containing 0.5% Chaps (pool A); 200 ml buffer B with 1.2% Chaps (pool B); 200 ml buffer B with 0.7% Renex and 0.05% Chaps (pool C); 200 ml buffer B with 0.2% Renex and 1 M KC1 (pool D). Step $A: S-Sepharose. The third eluate from the agarose column (pool C) was adjusted with 1 M KHzPOI to pH 7.0 and the solution was then applied to a S-Sepharose (Pharmacia) column (16 X 35 mm) which had been equilibrated with buffer C containing 0.3% Renex and 0.05% Chaps. The column was washed with 40 ml of buffer C containing 0.25% Chaps. Subsequently D6aH was eluted with 50 ml of

LOO Wavelength

500 inm)

FIG. 2. Difference spectra of Pmg elicitor-induced soybean cells (2 mg protein/ml). (a) baseline (oxidized versus oxidized microsomes); (b) oxidized versus reduced microsomes; the peak at 425 nm corresponds to cyt b5; (c) reduced versus reduced microsomes plus CO; the peak at 420 nm is due to inactive Cyt P420.

546

KOCHS AND GRISEBACH TABLE II SOLUBILIZATION OF MICROSOMAL MEMBRANE PROTEINS WITH DIFFERENT DETERGENTS

Detergent

CMC” (%I

Concentration (%)

Protein” bdml)

cyt P450 (nmol/ml)

None Chaps Na-cholate Triton X-100 Lubrol PX Renex 960 Emulgen 911 Octylglucoside Dodecylmaltoside

0.5 0.6 0.02 0.006 0.2 0.2 0.7 0.03

1 1.3 0.5 0.12 1 1 0.8 0.5

0.65 1.9 4.1 2.9 1.8 3.3 3.7 2.5 2.5

0 0.21 0.32 0.1 0.05 0.29 0.25 0.07 0.23

D6aH (pkat/ml)

Cinnamate I-hydroxylase (pkat/ml)

0 3.3 1.1 5.1 1.2 3.3 4.5 1.1 4.9

0 1.7 3.3 2.8 2.0 1.7 2.8 2.2 5.6

’ Critical micellar concentration. * All values in solubilized fraction: 400~1soybean microsomes and 2.8 mg protein were used for each solubilization experiment. a final concentration of 9% polyethylene glycol. After 45 min stirring, the precipitate was spun down at 100,OOOg for 45 min. The supernatant was brought to a concentration of 21% polyethylene glycol. After 45 min stirring the precipitate was spun down at 100,OOOgfor 90 min. The pellets were dissolved in buffer C containing 0.5% Chaps and 0.3% Renex. Step 4B: DEAE-Sepharose. The solution of the 21% polyethylene glycol pellet was applied to a DEAESepharose column (15 X 26 mm) which had been equilibrated with buffer C containing 0.2% Renex. The column was washed with the equilibration buffer. Subsequently protein was eluted with 30 ml of a O-O.5 M KC1 gradient in the same buffer. Step 5B: 2’,5’-ADP-agarose. Reductase-containing fractions from step 4B were loaded onto a 2’,5’-ADPagarose column (15 X 23 mm). The column was washed with 20 ml of buffer G containing 0.3% Renex and 20 ml of buffer C containing 0.2% Renex. Subsequently reductase was eluted with buffer C containing 0.2% Renex, 3 mM NADP+, and 2 mM Z-AMP. Step 6B: Mono Q. The reductase-containing fractions of step 5B (8 ml) were concentrated to 1.8 ml with an Amicon membrane YM 10. Change to buffer C containing 0.2% Renex was carried out on a Sephadex G-25 fine column (10 ml). Subsequently this solution was applied to a Mono-Q column (5 X 50 mm) which had been equilibrated with buffer C containing 0.2% Renex. After the column was washed with 8 ml of the equilibration buffer, enzyme was eluted with a linear 0 to 0.5 M KC1 gradient in the same buffer.

Recmtitution Enzymes

of Cytochrme P&i0

Dilauroylphosphatidylcholine or other lipids were dissolved (1 mg/ml) in 50 rnM potassium phosphate

buffer (pH 7.0) by sonication with a microtip sonicator (Branson Sonic Power Co., Danbury, CT). The column eluates of purified Cyt P450 (0.4 pmol) and of pure NADPH:Cyt P450 reductase (120 pkat) were mixed with 10 pl of the liposome suspension and the mixture was frozen in liquid nitrogen. Subsequently the frozen mixture was slowly thawed on ice and excess detergent was removed by five successive passages through a small column of Bio-Beads SM-2. This mixture was used for enzyme assays. The standard enzyme assays were used with addition of 10 PM FAD and 10 pM FMN. Assays were carried out for 30 min at 25°C.

Reactivation of NADPH:Cyt P450 Reductase with Flavins The purified reductase was incubated for 2 h at 4°C with the flavins. Unbound flavins were subsequently removed by ultrafiltration through a YM 10 membrane (Amicon). RESULTS

Induction of Cytochrme Cell Cultures

P450 in Soybean

To obtain a good source of cytochrome P450 enzymes for solubilization experiments the effect of various inducers on the activities of such enzymes in the microsomal fraction of cultured soybean cells was investigated. Clofibrate (1 mM), phenobarbital (0.8 mM), MnC& (20 mM), and ultraviolet light, which have been de-

:ROME P450 FROM

SOYBEAN

547

scribed as inducers of monooxygenases in plants (25,28), had no effects on the activities of DGaH, cinnamate 4-hydroxylase, and isoflavone synthase. Pectinase, which had been reported to potentiate the effect of the glucan elicitor from Pmg on the accumulation of glyceollin in soybean cell cultures (29), also had no significant effect on the activities of these three enzymes. With 40 ml of soybean cell suspension, yeast extract (125 mg/40 ml) increased the activities of D6aH and cinnamate 4-hydroxylase to about the same extent as Pmg elicitor (2 mg/40 ml). However, with a larger volume of cell suspension (400 ml) the results with yeast extract were variable and better results were obtained with Pmg elicitor. Routinely, 400 ml of a 6-dayold cell culture was treated with 30 mg Pmg elicitor for 24 h. The effect of Pmg elicitor on D6aH and cinnamate $-hydroxylase is shown in Table I. Difference spectra of microsomes from soybean cells challenged with Pmg elicitor are shown in Fig. 2. Such cells contained about 0.47 nmol cytochrome b5 and about 0.15 nmol cytochrome P450, per milligram of protein. The Cyt P450 content of osmotically stressed cells (30) was only about 0.09 nmol/mg protein. Solubilixation and Putification of Dihydroxypterocarpan Ga-Hydroxylase and NADPH:Cytochrome P&i0 Reductase A microsomal fraction from elicitorchallenged soybean cells obtained by MgClz precipitation (31) was used as enzyme source. Purification of this fraction on a discontinuous saccharose gradient did not result in an enrichment of D6aH activity (data not shown). A variety of detergents was used in solubilization experiments (Table II). All detergents totally inhibited D6aH and cinnamate 4-hydroxylase at concentrations below their critical micellar concentration. Chaps was selected for solubilization and purification of D6aH. Solubilization of various membrane-bound proteins by 1% Chaps is shown in Table III. Solubilization of mitochondrial cytochrome c oxidase by Chaps

548

KOCHS

AND

GRISEBACH

Microsomes 1. Solubilization I 2. *Aminooctyl-agarose

3A. S-Sepharose

38. Polyethylene glycol

J 4A. Hexyl-agarose

J48. DEAE-Bepharose

J5A. Hydroxyapatite

J58, 2’,5’-ADP-agarose 4 6B. MonoCl NADPH:Cyt P450 reductase

Dihydroxypterocarpan Ga-hydroxylase SCHEME 1. Purification

of Cyt P450 D6aH and NADPH:Cyt

was considerably lower than solubilization of proteins from the endoplasmic reticulum. The solubilized fraction also contained lower amounts of Cyt P4.20than the microsomes (data not shown). Although 75% of Cyt P450 of the microsomes was solubilized, the yield of D6aH activity was only 2-4 9%.

Purification of D6aH and of NADPH:Cyt P450 reductase was carried out according to Scheme 1. Chromatography of the solubilized fraction on w-aminooctyl-agarose separated the enzymes from phospholipids and pigments (pool B) and gave a highly enriched Cyt P450 fraction which contained the bulk of D6aH (pool C) as well as a reductase-containing fraction (pool D) (Fig. 3). Subsequent chromatography of

0 Tfme lmml

FIG. 3. Elution profile of microsomal solubilized fraction on w-aminooctyl-agarose. (0) D6aH activity; (A) NADPH:Cyt P450 reductase activity; (-) hemo nrotein (405 nm).

P450 reductase.

M

loI

150

200

250

300

Ttme Imud

FIG. 4. Elution profile of hydroxyapatite column. (0) D6aH activity; (Cl) cinnamate 4-hydroxylase activity; (-. -) phosphate buffer gradient; (-) hemo protein (405 nm).

549

CYTOCHROME P450 FROM SOYBEAN

1

2

34

s*l

6645\-362924zo-

.A -N78

-66

‘-45

FIG. 5. SDS Polyacrylamide gel of purified Cyt P450 D6aH from step 5A (lanes 1 (0.15 pg) and 2 (0.3 pg); silver stain) and of purified NADPH:Cyt P450 reductase from step 6B (lanes 3 (0.5 rg) and 4 (0.25 pg); Coomassie stain).

pool C on S-Sepharose and hexyl-agarose removed only proteins which were not bound to these matrices. Separation of D6aH from other proteins could not be achieved with a flat salt gradient or with a detergent gradient. Finally, chromatography on hydroxyapatite with a phosphate buffer gradient gave a baseline separation of D6aH from cinnamate 4-hydroxylase (Fig. 4). The fraction with the highest D6aH activity showed electrophoretic homogeneity on a SDS polyacrylamide gel with an M, 55,000 (Fig. 5). The estimated Cyt P4.50content (CO-difference spectrum) of this fraction was low but this fraction did not contain Cyt P420. The peak fraction of cinnamate 4-hydroxylase displayed sev-

eral proteins around 55,000 and at lower molecular weights on a SDS gel. Purification of D6aH is summarized in Table IV. The NADPH:Cyt P450 reductase was precipitated from pool D (Fig. 3) with 921% polyethylene glycol after the bulk of proteins had been precipitated with 9% polyethylene glycol. The precipitate of reductase contained some D6aH. The yield of this step was low because the pellet could not be completely redissolved in buffer. Subsequent chromatography on DEAESepharose separated the reductase from the low amount of D6aH. As has been shown previously (7, lo), affinity chromatography of the reductase on 2’,5’-ADPagarose and elution with 2’-AMP was an efficient purification step. This was followed by chromatography on Mono Q and gave an electrophoretically homogeneous reductase with an && 74,000 (Fig. 5). Purification of NADPH:Cyt P450 reductase is summarized in Table V. Optimal reductase activity was found in the presence of 10 pM each FMN and FAD. Reumstitution and Chara&mkation of D6aH For reconstitution experiments, purified D6aH (0.4 pmol Cyt P450), NADPH:Cyt P450 reductase, and liposome suspension were mixed. Excess detergent was removed with Bio-Beads SM-2. Since the reductase loses part of its flavin coenzymes during purification (‘7, lo), FAD and FMN

TABLE IV PURIFICATIONOF~~P~~~~,~-DIHYDROXYPTEROCARPAN~~-HYDROXYLASE

Purification stepa 1. 2.

Solubilisate w-Aminooctyl-agarose, pool c 3A. S-Sepharose 4A. Hexyl-agarose 5A. Hydroxyapatite a See scheme 1. *In the solubilized fraction.

Protein bd 2304 58 7.2 4.5 0.11

Cyt P450

(nmol) 594 62 4.6 4.2 0.07

Specific activity (&at&)

Purification (fold)

1.7

1

63.9 89 108 1300

38 52 64 765

Recovery (%I 100 (2”) 74 16 12 3.8

550

KOCHS

AND

GRISEBACH

TABLE

V

PURIFICATION OF NADPH:Cyt

Purification 1. 2. 3B. 4B. 5B. 6B.

Protein (mid

step”

Solubilisate o-Aminoocytl-agarose, pool D Polyethylene glycol precipitation DEAE-Sepharose 2,5’-ADP-agarose MonoQ

P450 REDUCTASE

Specific activity (mkat/kg)

Purification (fold)

2304

1.6

1

500

5.2

3.3

5.0 8.5 1587 2031

3.1 5.3 992 1270

172 46 0.24 0.15

Recovery (%) 100 70 23 11 10 8.4

a See Scheme 1.

were added to the D6aH assay. Dependence of D6aH on cytochrome P450 (Fig. 6), NADPHCyt P450 reductase (Fig. 7), and lipid (Fig. 8) was determined. Unexpectedly, dilauroylphosphatidylcholine gave a higher D6aH activity than soybean lipids (asolectin) (Fig. 8). The chain length of the fatty acids in the phospholipid proved to be important since the dimyristoy1 derivative (C,,) gave much lower D6aH activity than the dilauroyl derivative (C,,) (Fig. 8). Mixtures of dilauroylphosphatidylcholine with cholesterine and cardiolipin (5-20s) were not effective for reconstitution.

Cytochrome

P-GO

lpmoll

FIG. 6. Dependence of D6aH activity on Cyt P450 in the reconstituted enzyme assay. The assay contained 120 pkat NADPH:Cyt P450 reductase. Maximal enzyme activity corresponds to 0.033 pkat D6aH.

The temperature optimum for D6aH in the reconstituted system was 25°C and the optimal buffer concentration 50 mM potassium phosphate. Strong inhibition of enzyme activity was observed at higher ionic strength (Fig. 9). The pH optimum was 7.0 (Fig. 10). Addition of serum albumin (200 pg/lOO ~1) to the enzyme assay stimulated the reaction about twofold. The reconstituted enzyme system had a much higher temperature stability than the microsomal system (Table VI). At 25°C the half-life of D6aH in the microsomal system was about 20 min, whereas in the reconstituted system it rose to 340 min.

NADPH -Cytochrome

P-450

Reductase

lpkall

FIG. 7. Dependence of D6aH activity on NADPH: Cyt P450 reductase in the reconstituted enzyme assay. The assay contained 0.4 pmol cytochrome P450. Maximal D6aH activity corresponds to 0.06 pkat.

551

CYTOCHROME P450 FROM SOYBEAN

FIG. 8. Dependence of D6aH activity on lipids in the reconstituted enzyme assay. Maximal D6aH activity corresponds to 0.026 pkat.

PH

FIG. 10. Dependence of D6aH activity on pH. Kphosphate, 0.1 M (0); K-phosphate 0.25 M, (0). Maximal D6aH activity corresponds to 0.02 pkat.

DISCUSSION

Since soybean cell cultures challenged with Pmg elicitor contained relatively high activity of DGaH, microsomes obtained from such cells represented a good starting material for our investigation. Of the various detergents used for solubilization of DGaH, Chaps was selected for larger scale experiments for the following reasons: D6aH was solubilized with good specific activity, albeit with low yield (Table II); Chaps forms small micelles and is therefore suitable for purification; its high critical micellar concentration permits easy removal from protein solutions.

Buffer

Concentrot~on

ImMl

FIG. 9. Dependence of D6aH activity on ionic strength in the reconstituted enzyme assay (K-phosphate, pH 7.5). Maximal D6aH activity corresponds to 0.029 pkat.

Although Chaps has been described as “nondenaturing with respect to cytochrome P450” (15), the yield of solubilized D6aH was low compared to the yield of cytochrome P450 (Table III). Chaps seems to inhibit or denature D6aH irreversibly, since no enzyme activity could be detected in the pellet after Chaps treatment, and enzyme activity could not be restored by lowering the detergent concentration with Bio-Beads. Other effects which could cause the low activity of solubilized D6aH are self aggregation of the proteins and/or incomplete reconstitution of the enzyme system. In the purification procedure the w-aminooctyl-agarose step permitted separation of NADPH:cytochrome P450 reductase from the bulk of D6aH. Binding of D6aH to this matrix seems predominantly hydrophobic since the enzyme was eluted by a nonionic detergent. In contrast, the reductase bound tightly to this weak anion exchanger. According to their binding properties on ion exchangers, the reductase has an isoelectric point below pH 6 and D6aH has one above pH 8.5. Further purification of D6aH was therefore carried out on a cation exchanger (S-Sepharose) and that of the reductase on an anion exchanger (DEAE- and Q-Sepharose). In contrast to purification of animal cytochrome P450 enzymes, only low purification factors were achieved for D6aH on S-

552

KOCHS TABLE

AND

VI

TEMPERATURE STABILITY OF D6aH IN THE MICROSOMAL AND RECONSTITUTED SYSTEMS Half-life Temperature PC) 10 25 35

Microsomes 160 20 9

(mm) Reconstituted system 380 340 63

Note. Mixtures were kept at the indicated temperature for various lengths of time. Subsequently enzyme activity was assayed as described under Materials and Methods.

Sepharose and on a hydrophobic matrix (hexyl-agarose). Separation of DGAH from cinnamate 4hydroxylase on hydroxyapatite (Fig. 4) proves for the first time on a protein level the existence of different P450 species in a higher plant. Up to now different cytochrome P450 species were only postulated from induction and inhibition experiments (13, 14). Fractions between the peaks of D6aH and cinnamate 4-hydroxylase also contained proteins in the cytochrome P450 region (data not shown). Whether these proteins belong to other P450 enzymes is unknown. The activity of isoflavone synthase (3) was too low for reconstitution experiments. A homogeneous reductase had previously been obtained from wounded Jerusalem artichoke (H. tuberosus) (7) and from cell cultures of foxglove (D. Zunata) (10). Reductases with isoforms of unclear origin were reported from other plants (332). Reconstitution of cinnamate 4-hydroxylase from H. tuberosw (6) was also carried out with dilauroylphosphatidylcholine. The fact that the phase transition temperature of dilauroylphosphatidylcholine (O°C) is lower than that of the dimyristoyl derivative (23°C) could be the reason why the latter compound gave much lower D6aH activity (Fig. 8). It was suggested that in a reconstituted system with dimyristoylphosphatidylcho-

GRISEBACH

line vesicles, reduction of cytochrome P450 by reductase is a diffusion-limited reaction controlled by the viscosity of the phospholipid membrane (33). Results support the assumption of an electrostatic interaction between cytochrome P450 and reductase (34). The narrow optimum for buffer concentration at low ionic strength for D6aH (Fig. 9) and inhibition of the enzyme at higher ionic strength are consistent with this view. ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft (SFB 206) and Fonds der Chemischen Industrie. Georg Kochs thanks Dr. F. Durst and his colleagues (Strasbourg) for introduction into cytochrome P450 biochemistry. REFERENCES 1. EBEL, J., AND GRISEBACH, H. (1988) Trends Bti them. Sci. 13,23-27. 2. WALDMULLER, T., AND GRISEBACH, H. (1987) Plantu 172,424-430. 3. KOCHS, G., AND GRISEBACH, H. (1986) Eur. .I Biti che?rh 155,311-318. 4. HAGMANN, M. L., HELLER, W., AND GRISEBACH, H. (1984) Eur. J. Biochem 142,127-131. 5. WELLE, R., AND GRISEBACH, H. (1988) Arch. Biothem Biophgs. 263,191-198. 6. GABRIAC, B., BENVENISTE, I., AND DURST, F. (1985) C. R. Acad Sci. Paris 301,753-758. 7. BENVENISTE, I., GABRIAC, B., AND DURST, F. (1986) Biochem J. 235,365-373. 8. MADYASTHA, K. M., MEEHAN, T. D., AND COSCIA, C. J. (1976) Biochemists 15.1097-1102. 9. MADYASTHA, K. M., AND COSCIA, C. J. (1979) J. Biol Chem. 254,241s~2427. 10. PETERSEN, M., AND SEITZ, H. U. (1988) Btihem J. 252.537-543. 11. HIGASHI, K., IKEUCHI, K., KARASAKI, Y., AND OBARA, M. (1983) Biodem. Biophys. Res Cornmm. 115,46-52. 12. HIGASHI, K., IKEUCHI, K., OBARA, M., KARASAKI, Y., HIRONA, H., GOTOH, S., AND YOSUKE, K. (1985) Agric. Biol. C&m. 49,239s~2405. 13. KOCH& G. (1988) Doctoral thesis, Freiburg. 14. FRITSCH, H., KOCHS, G., HAGMANN, M. L., HELLER, W., JUNG, J., AND GRISEBACH, H. (1986) in Sixth International Congress of Pesticide Chemistry IUPAC, August 10-15, Ottawa. [Abstract 3B-161. 15. HJELMELAND, L. M. (1980) Proc Nat1 Acd Sci USA 77,6368-6370.

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