plant biochemistry of xenobiotics: isolation and properties of soybean o- and n-glucosyl and o- and...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 287, No. 2, June, pp. 341-350, 1991

Plant Biochemistry of Xenobiotics: Isolation and Properties of Soybean 0- and N-Glucosyl and 0- and IV-Malonyltransferases for Chlorinated Phenols and Anilines

Heinrich Sandermann, Jr.,l Rudolf Schmitt,2 Heidrun Eckey, and Tobias Bauknecht GSF-Institut fiir Biochemische Pflanzenpathologie, D-8042 Neuherberg, Germany

Received November 19, 1990, and in revised form February 1, 1991

0-Glucosyltransferase (0-GT), 0-malonyltransferase (O-MAT), N-glucosyltransferase (N-GT), and N-malonyltransferase (N-MAT) activities have been detected in cultured soybean cells, using pentachlorophenol and 3,4- dichloroaniline as xenobiotic standard substrates. The 0-GT was purified - lOOO-fold, and the N-MAT - 70- fold. There was an extensive copurification of 0-GT and O-MAT. The following functional molecular weight values were obtained, 47 kDA (0-GT), 48 kDA (O-MAT) 43 kDa (N-GT), and 48 kDa (N-MAT). 0-GT and N-MAT appeared to be monomeric polypeptides with isoelectric pointsof -4.8 and -6.1, respectively. The 0-GT, N-GT, and N-MAT activities had marked substrate specificities for chlorinated aromatic xenobiotics and thus illustrate the existence of plant isoenzymes with specificity for xenobiotics. Cc’ 1991 Academic Press, Inc.

Terrestrial vegetation is exposed to and can act as a sink for pesticides and numerous additional xenobiotics. These foreign chemicals are in many cases metabolized in plants by transformation and conjugation reactions and then compartmentalized in the plant tissue (l-3). Knowledge on the participating plant enzymes is generally very limited. Two relatively well-characterized enzyme activities are glutathione S-transferase isoenzymes with specificity for certain herbicides (4-6) and a wheat ester- ase isoenzyme specific for the plasticizer chemical, bis(ethylhexyl)phthalate (7).

’ To whom correspondence should be addressed at GSF-Miinchen, Institut fur Biochemische Pflanzenpathologie, Ingolstadter Landstrasse 1, D-8042 Neuherberg, Germany. FAX: (49-89) 3187-3322.

’ Present address: Bethesda Research Laboratories GmbH, Diesel- strasse 5, D-7514 Eggenstein 1, Germany.

000%9861/91 $3.00 Copyright @ 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

The present report deals with novel conjugation enzymes for chlorinated phenols and chlorinated anilines. These chemicals are frequent environmental contami- nants (8). We have previously isolated and structurally elucidated the 0-P-D-glucosyl and the (0-malonyl)-O-P- D-glucosyl conjugates of pentachlorophenol (9) and the IV-malonyl and N-glucosyl conjugates of 4-chloroaniline and 3,4-dichloroaniline (10). The structures of these metabolites are shown in Fig. 1. The plant enzymes respon- sible for formation of these conjugates have been detected in crude extracts of soybean and wheat (9, 11). There are previous reports on a soybean N-glucosyl transferase for chlorinated anilines (12) and a peanut N-malonyl transferase for 3,4-dichloroaniline (13). The formation of xenobiotic /3-D-glucosides in intact plant systems is well known (1, 2, B), and a crude 0-glucosyltransferase preparation from mung bean was found to utilize monohalo- genated phenols (14). However, only plant O-glucosyl- transferases acting on natural substances have so far been highly purified and characterized (15, 16). As part of a program aimed at the characterization of plant enzymes for xenobiotics, the isolation and some properties of enzyme preparations leading to the conjugates in Fig. 1 are now reported. Three of these transferases showed specificity for chlorinated substrates. Some of the present results have been briefly presented in abstract form (11, 17, 18).

MATERIALS AND METHODS

Materials The general materials used and the chemical synthesis of the reference

PCP-@-D-glucoside” were as previously described (9). [U-%]PCP (27

3 Abbreviations used: PCP, pentachlorophenol; DCA, 3,4-dichloroaniline; 2,4-D, 2&dichlorophenoxyacetic acid, 0-GT, O-glucosyltransferase; N-GT, N-glucosyltransferase; O-MAT, O-malonyltransferase; N- MAT, N-malonyltransferase; SDS, sodium dodecyl sulfate.

441

342

OH

Cl

SANDERMANN ET AL

Cl

(4)

FIG. 1. Chemical structures of (1) the fl-D-glucopyranosyl and (2) the (G-malonyl)./-D-glucopyranosyl derivatives of pentachlorophenol and (3) the N-fl-D-glucopyranosyl and (4) the N-malonyl derivatives of 3,4- dichloroaniline.

Ci/mol) was purchased from CEA (Gif-sur-Yvette. France). 2,6-Di-t- butyl-[U-‘4C]phenol (12.2 Ci/mol), 2,4-dichloro-[U-i4C]phenol (15 Ci/ mol), 2,4,6-trichloro-[U-i4C]phenol (87 Ci/mol), [4-i4C]cholesterol (58 Ci/mol), and D-cu-[5-methyl-3H]tocopherol (13 Ci/mmol). 2-Nitro- [Ui4C]phenol (21.6 Ci/mol) and 4-nitro-[U”C]phenol (22.4 Ci/mol) were provided by Dr. K. Figge, NATEC, (Hamburg, Germany).

UDP-[U-‘4C]glucose (296 Ci/mol) and [2-‘4C]malonyl-CoA (58 Ci/ mol) were obtained from Amersham-Buchler (Braunschweig, Germany). 4Chloro-[U”C]aniline (19 Ci/mol), 3,4-dichloro-[Ui4C]aniline (6.12 Ci/ mol), and 2,4,5-trichloro-[U-i4C]aniline (6.12 Ci/mol), and 2,4,5-trichloro-[U-“Claniline (14.82 Ci/mol) were purchased from Pathfinder Laboratories Inc. (St. Louis, MO).

Reference proteins were from Serva (Heidelberg, Germany) and Sigma (Munich, Germany). The following chromatographic materials were purchased from Pharmacia (Freiburg, Germany): DEAE-Sepharose Fast- Flow-GB, phenyl-Sepharose CL-4B, Polybuffer-Exchanger 94, Sephacryl S-200, and Mono-Q-HR 5/5. Matrex Gel Red A was from Amicon (Wit- ten, Germany).

Herbicides were generally obtained from Riedel-de-Haen (Seelze, Hannover, Germany). Chloramben was kindly donated by Union Carbide (New York, NY) while picloram was purchased from Promochem (Wesel, Germany). Ot.her chemicals used as substrates were from Fluka (Neu- Ulm, Germany) or Merck (Darmstadt, Germany). Nonradioactive acyl- CoA esters were from Sigma. PCP-P-D-[U-i”C]glucoside was obtained by pooling ethyl acetate extracts of thin layer chromatography (TLC) product spots from O-glucosyltransferase assays. Aliquots (2.7 nCi) were brought to dryness in Eppendorf tubes and used for the O-MAT assay procedure described below.

General Procedures

Protein was determined by a modified Lowry procedure (19) after precipitation of protein with trichloroacetic acid in the presence of de- oxycholate (20). Bovine serum albumin was used as a standard. Gel permeation chromatography was performed with Dextran Blue as a void volume marker and the following marker proteins: bovine serum albumin (67 kDa), ovalbumin (45 kDa), carboanhydrase (30 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (22 kDa), P-lactoglobulin (18.4 kDa),

myoglobin (17 kDa), and cytochrome c (13 kDa). Thin-layer chromatography was performed on precoated silica gel G plates (Merck, No. 5715) using the following solvent systems: (A) ethyl acetate/acetic acid/ water (63/l/2, v/v/v), (B) same solvents (8/1/l, v/v/v), (C) benzene/ dioxane/acetic acid (90/25/4, v/v/v). (D) ethyl acetate/propanol-2/water (66/23/11, v/v/v), and (E) butanol-l/acetic acid/water (60/15/25, v/v/ v; upper phase). Descending paper chromatography was performed with solvent system (F) butanol-l/acetic acid/water, 4/l/5 (v/v/v; upper phase).

Enzyme products were identified by cochromatography with the previously described metabolites and defined reference compounds (9, 10).

Radioactivity on TLC plates was detected and quantified by use of a linear TLC analyzer (Berthold LB 2842, Wildbad, Germany). Gel electrophoresis was either carried out under nondenaturing conditions (21) using 10% (w/v) polyacrylamide in glass tubes of 5 mm inner diameter, or in the presence of 0.1% (w/v) SDS on 10 or 13% polyacrylamide slab gels (22). Proteins in gels were visualized with Coomassie brilliant blue R-250 (23) or with a silver stain procedure (24). Isoelectric focusing was carried out on IEF-PAG plates according to the manufacturer’s appli- cation note (LKB, Freiburg, Germany). Protein solutions were desalted using Spectropor dialysis tubing (Serva) or by chromatography over PD.10 prepacked Sephadex G-25 columns (Pharmacia). Protein solutions were concentrated by ion-exchange chromatography (DEAE-Se- phacel), by ammonium sulfate precipitation (80% (w/v) saturation), or by centrifugation (4OOOg, l-2 h, 2°C) through Amicon Centricon- filter tubes.

The following buffer systems were used: 1. 200 mM Tris-HCl (pH 7.5), 2 mM MgCl,, 2 mM dithiothreitol; 2. 20 mM Tris-HCl (pH 7.5), 2 mM MgCl,, 2 mM dithiothreitol, 1

mM phenylmethylsulfonyl fluoride; 3. 100 mM potassium phosphate (pH 6.5); 4. 25 mM histidine, 2 mM dithiothreitol, 2 mM MgCl (pH 6.2); 5. 20 mM imidazole, 2 mM dithiothreitol, 2 mM MgClz (pH 6.8) 10%

(v/v) ethylene glycol.

O-Glucosyltransferase Assay

The standard assay mixt,ure (final volume, 200 ~1) contained 200 mM Tris-HCl, (pH 7.5), 2 mM dithiothreitol, 2 mM MgCl,, 20 mM UDP- glucose, 0.125 mM [U-i4C]PCP (0.15 &i, added in 10 ~1 2-methoxy- ethanol). Unless indicated otherwise, 2.5 mM 4-nitrophenyl-@D-glucopyranoside and 2.5 mM salicin (benzohydroquinone-O+D-glucopyranoside) were included in order to protect the product formed from degradation by endogenous P-glucosidase activity (cf. (9)). The reaction was started by addition of 25-200 pg protein from the described purification steps. After incubation for 60 min at 28”C, the reaction was terminated by addition of 5 ~1 3 N H3P04, followed by extraction with 3 X 200 ~1 portions of ethyl acetate. The combined organic phases were analyzed by TLC in solvent system A. The product appeared at R, 0.35 (PCP; R,, 0.9). Control assays were carried out with heat-inactivated enzyme (10 min, 100°C) or by omitting UDP-glucose from the reaction mixture.

0-Malonyltransferase Assay

Procedure A. The 0-GT assay was performed with inclusion of 20 ~1 20 mM malonyl-CoA (in water). The O-(malonyl)-O-D-glucoside of PCP had R, 0.1.

Procedure B. The assay mixture (final volume, 200 ~1) contained 3- 5 pM [i4C]PCP-@-D-glucopyranoside (0.1 &i), and 2 mM malonyl-CoA in buffer 1. Usually, 2.5 mM 4-nitrophenyl-B-D-glucopyranoside and 2.5 mM salicin were included in order to protect the labeled P-D-glucoside. Even in the presence of the potential competitive inhibitors, high conversion rates of up to 90% were achieved. Incubation and product determination were as described for the 0-GT assay.

PLANT BIOCHEMISTRY OF XENOBIOTICS 343

N-Malonyltransferase Assay

[U-i4C]DCA (41 nmol in 10 ~1 methanol; 0.25 FCi) was added to 145 ~1 buffer 3 and malonyl-CoA (200 nmol in 20 ~1 water). The assay was started by addition of 25 ~1 enzyme solution, followed by incubation for 40 min at 4O’C. The reaction was terminated by addition of 5 ~1 acetic acid and 200 ~1 ethyl acetate. After thorough mixing and centrifugation, 20.~1 aliquots of the upper phase were fractionated by TLC. The radio- active product was detected at R,O.35,0.17, and 0.34 in solvent systems A, C, and D, respectively.

N-Glucosyltransferase Assay

[U-‘“C]DCA (41 nmol in 10 ~1 methanol; 0.25 &i) was added to UDP- glucose (2 pmol) in buffer 3 (165 ~1). The assay was started by addition of 25 gl enzyme solution followed by incubation for 40 min at 40°C. The reaction was terminated by addition of 200 ~1 ethyl acetate. In this case, acetic acid could not be added in view of the acid sensitivity of the product (cf. (10)). A 20.~1 aliquot of the upper phase was fractionated by TLC (solvent systems A or D) and scanning for radioactivity. The product appeared at R, 0.05 and 0.15, respectively.

Purification of 0-Glucosyltransferase

All steps were carried out at 0-4°C. At each step portions of the enzyme solution were withdrawn, frozen in liquid nitrogen, and stored at minus 70°C for further purification or analysis.

step 1: Preparation of extract. Soybean cell suspensions were cultured as previously described (9, 10). After 7 days of growth, the cells were harvested by filtration, frozen in liquid nitrogen, and ground to a powder using a mortar and pestle. The cells (600 g) were taken up with grinding in 900 ml of buffer 1 with the addition of a small amount of quartz sand and 10% (w/v) Dowex 1 X 2 ion-exchange resin (Serva) equilibrated in buffer 1. Immediately after thawing of the cell material 1 mM phenylmethylsulfonyl fluoride was added to the homogenization mixture. The cell slurry was filtered through Miracloth and the filtrate was cleared by centrifugation (52OOg, 20 min). The supernatant was used as cell free extract for further purification.

Step 2: Ammonium sulfate fractionation. Finely powdered solid ammonium sulfate was added slowly at 0°C to the stirred cell-free extract to yield a final salt concentration of 35% (w/v). The protein precipitate was removed by centrifugation (15 min, lO,OOOg), and the supernatant was adjusted to 80% (w/v) ammonium sulfate. The precipitated protein was again isolated by centrifugation and dissolved in 80 ml buffer 2. The protein solution was dialyzed overnight at 4°C against excess buffer 2.

Step 3: Ion-exchange chromatography. The dialyzed solution (83 ml) was applied to a column (5 X 11.5 cm) of DEAE-Sepharose Fast-Flow (Pharmacia) packed in buffer 2. After elution of nonabsorbed material (600 ml buffer 2) two linear gradients between 0 and 400 mM NaCl in buffer 2 (2 X 300 ml) and between 400 mM and 2 M NaCl in buffer 2 (2 X 300 ml) were applied. The O-glucosyltransferase activity as well as the N-malonyltransferase activity appeared as coinciding peaks at the end of the first gradient program. Fractions of 8.8 ml were collected. The active fractions eluting between fractions 150 and 180 (corresponding to 200-400 mM NaCl) were combined, concentrated by ammonium sulfate precipitation to 80% (w/v), redissolved in a small volume of buffer 1, and dialyzed overnight against excess buffer 1.

Step 4: Hydrophobic interaction chromatography. The retentate from step 3 (lo-30 ml) was either frozen in liquid nitrogen and stored at -20°C up to several weeks (no detectable loss of activity) or it was directly applied to a column (2.5 X 33.5 cm) of phenyl-Sepharose CL- 4B equilibrated in degassed buffer 1. The column of phenyl-Sepharose was washed with about 400 ml buffer 1. The protein that eluted in this step was detected by absorbance at 280 nm. This initial protein fraction was designated “pool 1”. Elution of the phenyl-Sepharose column was continued with a linear gradient of O-60% ethylene glycol in buffer 1

(600 ml, fraction size 5 ml; flow-rate, 0.42 ml/min)). Active fractions appeared in the initial part of the second protein peak. These fractions were combined (“pool 2”), and dialyzed against buffer 4 in order to remove excess ethylene glycol. The protein solution was then concentrated by precipitation with 75% (w/v) ammonium sulfate. After centrifugation, the protein precipitate was redissolved in buffer 4 (4 ml) and dialyzed overnight against 10% (v/v) ethylene glycol in buffer 4.

Step 5: Chromatofocusirzg. The dialyzed solution from step 4 (6 ml) was used for further purification on a chromatofocusing column (0.9 X 28 cm) packed with polybuffer-Exchanger 94 and equilibrated in degassed buffer 4 containing 10% (v/v) ethylene glycol. The instruction manual (Pharmacia, Freiburg, FRG) was followed. Fractionation was performed by elution with 200 ml of diluted polybuffer 74 in a linear pH range of 6.2-4.0. Fractions of 3 ml were collected. Fractions with enzyme activity (fractions 28 to 38; corresponding to pH 4.4 to 5.0) were immediately combined and concentrated by 75% (w/v) ammonium sulfate precipitation. The enzyme appeared to be unstable near its isoelectric point. The precipitated protein was redissolved in a small volume of buffer 1 and was dialyzed over night against buffer 1 with 1 mM phenylmethylsulfonyl fluoride, or further used without prior dialysis.

Step 6: Gel-permeation chromatography. The protein solution from step 5 (2-5 ml) was applied to a calibrated column (2.6 X 63 cm) of Sephacryl S-200 packed in buffer 1. Elution was performed with buffer 1, and fractions of 2 ml were collected. The enzyme appeared as a relatively broad peak at the approximate position of the ovalbumin standard. O-MAT (assay procedure A) showed the same elution profile. Fractions with 0-glucosyltransferase activity (fractions 80 to 110) were combined and concentrated by 75% ammonium sulfate precipitation. The redissolved protein precipitate was dialyzed against excess buffer 5.

Step 7: Fast protein liquid chromatography (FPLC). A prepacked Mono Q HR 5/5 anion-exchange column equilibrated in degassed buffer

I

POOL1

-N-GT

I I ,

POOL 2

?L&y ^ _-bz& lJ 150 260

0'

FRACTION NUMBER

FIG. 2. Hydrophobic interaction chromatography on phenyl-sepharose 4B. Step 3 enzyme was chromatographed as described under Ma- terials and Methods, applying a gradient of ethylenglycol (- - -). Absor- bance was followed at 280 nm (--; arbitrary units). N-Malonyltransferase activity (N-MAT; 0) was measured in the standard assay with 100.~1 aliquots of the fraction to be tested. One unit of enzyme activity was defined as 10% conversion into product, the peak fractions giving complete conversion. 0-Glucosyltransferase activity (O-GT; 0) was measured by the standard assay with 100.~1 aliquots of the fraction to be tested. One unit of enzyme activity was defined as 1% conversion into product. A maximum of 10% conversion was observed. 0-Malonyl transferase activity (O-MAT; X) was tested by assay procedure A. Enzyme units in this coupled assay were the same as in the 0-GT procedure. A maximum of 80-90% conversion of the P-D-glucoside to the double conjugate was observed. For further analysis and purification the fractions labeled pool 1 and pool 2 were combined and stored in aliquots at ~70°C. N-Glu- cosyltransferase activity was studied in the combined fractions by the standard procedure and was found only in pool 1.

344 SANDERMANN ET AL.

% ENZYME ACTIVITY 3 L 16 22 23 15 IO 6

14 15 16 17 18 19 20 21 S FRACTION NUMBER

FIG. 3. SDS-gel electrophoresis of 0-GT enzyme fractions obtained from FPLC (purification step 7). Enzyme activity appeared upon in- creasing the NaCl concentration in a linear gradient from 100 to 250 mM. Most of the total protein appeared at 160 mM NaCl and the fractions analyzed were located in the last part of the protein peak. The graph shows the SDS gels and the percentage of substrate converted in the standard 0-GT assay of 180~~1 aliquots of the indicated fraction numbers. The lane labeled S contained the marker proteins bovine serum albumin (67 kDa) and ovalbumin (45 kDa), followed by an impurity and by tryp sinogen (24 kDa) and fl-lactoglobulin (18.4 kDa). The proposed position of enzyme protein is marked by a dot near the lane S. The gel was silver stained. The fractions containing 0-GT activity also contained O-MAT activity but no N-GT or N-MAT activity.

5 was used in connection with the Pharmacia FPLC System. The dialyzed solution from step 6 (1.5 ml in buffer 5) was applied to the column in several consecutive loading cycles. After extensive washing with buffer 5, a linear gradient between O-500 mM NaCl in buffer 5 (40 ml) was applied. Fractions of 1 ml were collected at a flow rate of 0.5 ml/min. Active fractions (fractions 14 to 21) appeared at -160 mM NaCl. O- MAT activity (assay procedure A) had a nearly identical elution profile. These fractions were analyzed by SDSgel electrophoresis, then combined, frozen in liquid nitrogen, and stored at -70°C for further inves- tigation.

Step 8: Dye ligand chromatography. Pooled enzyme from step 7 (2.5 ml) was diluted lo-fold into buffer 2 containing 10% (v/v) ethylene glycol and applied to a column (9 X 15 cm) of Matrex Gel Red A (Amicon, Witten, Germany) packed in buffer 2 containing 10% (v/v) ethylene glycol. The column was developed with buffer 2, containing 10% (v/v) ethylene glycol, followed by a linear gradient (2 X 18 ml) toward 1.2 M

NaCl in the same buffer and then elution with 2.0 M NaCl. Fractions of 3 ml were collected at a flow rate of 0.2 ml/min. Elution with NaCl was employed since the enzyme could not be specifically eluted with 10 mM UDPG or 10 mM CoASH.

Purification of N-Malonyltransferase

The general procedures used were as described for the O-glucosyltransferase. Step 1 (preparation of extract) and step 2 (ammonium sulfate fractionation) were the same as described above. Desalting was by chromatography on a column (5 X 70 cm) of Sephadex G-25. In the above step 3 (ion-exchange chromatography) and also in gel permeation chromatography on Sephadex G-100, the N-malonyltransferase was eluted within the peak of 0-glucosyltransferase activity. In the above step 4 (hydrophobic interaction chromatography), N-malonyltransferase and most of the 0-glucosyltransferase activities were separated from each other. The active N-malonyltransferase fractions were combined, enzyme protein was precipitated with ammonium sulfate (80% (w/v)) and the resulting pellet was dissolved in 2.5 ml buffer 1. In step 5 (gel permeation chromatography), the protein solution was applied to a calibrated column

(2.6 X 91 cm) of Ultrogel AcA 54 (LKB) in buffer 3, followed by devel- opment with the same buffer. The enzyme appeared as a symmetrical peak corresponding to a molecular weight of -48 kDa. Enzyme in the pooled active fractions was precipitated by ammonium sulfate (80% (w/ v)), redissolved in 0.6 ml buffer 3, and stored in aliquots at -70°C.

Preparation of N-Glucosyltransferase

Pool 1 obtained in step 5 of 0-GT purification or step 5 enzyme of N-MAT purification showed high activity in the N-GT standard assay. The specific activity amounted to lo-20% of the specific N-MAT activity present in the same fractions.

RESULTS

Purification of 0-Glucosyltransferase and 0-Malonyltransferase

Homogenates from cultured soybean cells were found to contain an 0-glucosyltransferase active with PCP (9). About 90% of this enzyme activity was in the soluble fraction and about 10% was in the microsomal fraction which was prepared either by ultracentrifugation, or by Mg” + precipitation (25). Only the soluble activity was further studied. P-Glucosidase activity detected in the crude extract was suppressed by the addition of 2.5 mM salicin and 2.5 mM p-nitrophenyl-P-D-glucoside. Enzyme purification proceeded first by ammonium sulfate precipitation and by ion-exchange chromatography on DEAE- Sepharose. A key step in the further purification was hydrophobic interaction chromatography on phenyl- Sepharose (step 4) where the N-malonyl- and N-gluco- syltransferases activities acting on chlorinated anilines were not bound (Fig. 2). Both the 0-glucosyl- and the O- malonyltransferase activities acting on PCP (9) were

0-GT 5 I I I I

0 FR%TION N%BER

. 18 L----f

FIG. 4. Partial separation of 0-malonyl transferase (O-MAT; 0) and 0-glucosyltransferase (O-GT; 0) activities by dye-ligand chromatography. Step 7 enzyme was applied and eluted as described under Materials and Methods (step 8). The applied gradient of sodium chloride (NaCl) is indicated (---). The eluted fractions were assayed for 0-GT (30 min incubation) in the absence of protective @-D-glucosides, and for O-MAT activity (procedure B). One unit of enzyme activity corresponded to 40% conversion in the case of O-MAT and to 7% conversion in the case of 0-GT activity. In the right-hand panel the SDS gels of a standard protein mixture (lane S; bovine serum albumin, ovalbumin, trypsinogen, fl-lactoglobulin) and of the pooled 0-GT peak fractions are shown. The proposed position of the enzyme is marked by a dot.


strongly bound and could only be eluted in the presence a polypeptide band of molecular weight - 47 kDa. This of 20-40s (v/v) ethylene glycol. Two pools were formed band was present in the final purification steps as a weak for further purification, as indicated in Fig. 2. Pool 1 con- band (Figs. 4 and 5), and could be more clearly detected tained a significant portion of 0-GT activity but only after FPLC (Fig. 3). In Fig. 3 and during the column chro- little O-MAT activity. Early 0-GT elution was not due matographic runs of purification steps 5-8 (gels not to column overloading as shown by rechromatography, shown), the 47-kDa band showed a maximum coincident which only led to elution in the position of pool 1. The with 0-GT activity. In purification steps 5-8, where a 0-malonyltransferase further copurified with the bound number of other polypeptides were also present, the en- portion of 0-glucosyltransferase activity during the sub- zyme band could nevertheless be detected since it reacted sequent chromatofocussing and gel permeation steps. strongly in the silver stain procedure. No assignment of FPLC led to further enrichment of 0-GT as illustrated O-MAT activity to a defined polypeptide could be made. in Fig. 3. However, O-MAT activity was only partially In an attempt at further purification, step 8 enzyme in removed. Separation of the two enzymes was achieved in buffer 2 containing 0.5 M NaCl was applied to concanav- the final purification step, chromatography on Matrex alin A-Sepharose (Pharmacia). However, 0-GT and O- Gel Red A, Fig. 4. Mat were eluted together in the unbound protein fraction.

The entire purification sequence is summarized in Ta- ble I. SDS-polyacrylamide gel patterns obtained for the various purification steps are shown in Fig. 5. The final enzyme preparation displayed three major protein bands upon polyacrylamide gel electrophoresis under nondenaturing conditions. Only one of these bands had 0-GT activity when gel slices were tested in the standard assay (Fig. 6). However, even at this stage, the two prominent polypeptides copurifying throughout the entire purification scheme (cf. Fig. 5) were still present. Since these polypeptides of individual molecular weight - 22 and - 24 kDa were not removed by gel permeation chromatography, they appeared to be present as dimers under nondenaturing conditions. 0-GT activity was assigned to

Purification of N-Malonyltransferase and N-Glucosyltransferase

These enzymes copurified with 0-GT and O-MAT during ammonium sulfate fractionation and ion-exchange chromatography. N-GT and N-MAT were, however, well separated from 0-GT and O-MAT activity by hydrophobic interaction chromatography (Fig. 2). A considerable purification was achieved by gel permeation chromatography on Ultrogel AcA 54 (Fig. 7). The peak of N-MAT activity was enriched in a -4%kDa polypeptide band. N- GT was not studied in this experiment. The purification sequence leading to -70-fold-purified N-MAT is summarized in Table I.

TABLE I

Purification Schemes for 0Glucosyltransferase and N-Malonyltransferase Activities

Purification step Protein Specific activity

(mg) (d=t/kg)

O-Glucosyltransferase

Enrichment (fold)

Yield ( % )

Step 1 Crude extract Step 2 Ammonium sulfate fractionation Step 3 Ion-exchange chromatography Step 4 Hydrophobic interaction chromatography Step 5 Chromatofocusing Step 6 Gel-permeation chromatography Step I FPLC Step 8 Dye-ligand chromatography

Step 1 Crude extract Step 2 Ammonium sulfate fractionation Step 3 Ion-exchange chromatography Step 4 Gel-permeation on Sephadex G-100 Step 5 Hydrophobic interaction chromatography Step 6 Gel-permeation on Ultrogel AcA 54

2530 0.96 1 100 863 1.1 1.2 39 215 4.4 4.8 39

19 15 16 12 4.3 33 35 6 1.8 34 36 2.5 0.04 545 577 0.9 0.01 950 1005 0.04

N-Malonyltransferase

5590 32 1 100 2730 39 1.2 60 1180 86 2.6 57

110 780 24 48 17 1360 42 13

8 2380 74 10

Note. These data refer to the work-up procedures and standard assays given under Materials and Methods. The protective substances 4- nitrophenyl-$-D-glucopyranoside and salicin (2.5 mM each) were included in the 0-GT assay. The specific activity values given were calculated from the percentage conversion rates on TLC plates.


S12345670 S

I

FIG. 5. SDS-gel electrophoresis for O-glucosyltransferase purification. The following protein standards were applied to the lanes S (top to bottom): bovine serum albumin (67 kDa), ovalbumin (45 kDa), trypsinogen (24 kDa), P-lactoglobulin (18.4 kDa). The enzyme samples came from the active fractions of the following steps of 0-GT purification: 1, soluble extract; 2, ammonium sulfate fractionation; 3, ion-exchange chromatography; 4, hydrophobic interaction chromatography; 5, chromatofocusing; 6, gel permeation chromatography; 7, FPLC; 8, dye-ligand chromatography. The proposed position of 0-glucosyltransferase at 47 kDa is labeled by a dot near the standard lanes. A corresponding band appeared in lanes 6 and 7 and weakly in lane 8. Staining was with Coomassie brilliant blue.

Molecular Weight Determination

As previously reported (9) 0-glucosyltransferase activity appeared at an apparent molecular weight of 47 kDa on a calibrated gel permeation column of Sephacryl S- 200. The same result was also obtained on calibrated columns of Sephadex G-100 as well as Ultrogel AcA 54 (LKB) and Superose 12 (Pharmacia). SDS-polyacrylamide gel electrophoresis of step 7 and step 8 enzyme revealed two prominent polypeptide bands with apparent molecular weight values of -24 and 22 kDa and a minor band of - 47 kDa. The latter band consistently amounted to about lo-15% of step 7 or step 8 protein (cf. Figs. l- 3). 0-GT activity was assigned to the -47-kDa polypeptide band (see above) so that the enzyme appeared to be monomeric. The same conclusion was drawn for N-MAT. In this case, gel permeation chromatography on Sephadex G-100 and Ultrogel AcA-54 led to a native molecular weight estimate of 48 f 3 kDa. A corresponding polypeptide band appeared under denaturing conditions upon SDS-gel electrophoresis (Fig. 7). Gel permeation chromatography on Sephacryl S-200 and Ultrogel AcA 54 led to native molecular weight values of 48-49 kDa for O- MAT (cf (9)) and 42-43 kDa for N-GT.

Isoelectric Points

Upon chromatofocusing (step 5 of the purification scheme) 0-GT appeared as a sharp peak corresponding to a pH value of 4.7-4.9. Isoelectric focusing using the LKB-Ampholine gel system led to an isoelectric point of PI - 4.8 (Fig. 8). The same result was obtained with LKB- Immobiline dry plates (not shown). In the case of N-MAT, a pl estimate of -6.1 was obtained (Fig. 8).

Catalytic Properties of 0-GT

The enzyme had a pH optimum between pH 6.8 and 7.5 as determined by use of 0.2 M Tris-HCl buffers adjusted to pH values between 3.5 and 10. The reaction rate in the standard assay increased with incubation temperature up to 45°C followed by a rapid decline at temperatures above 50°C. Complete loss of activity occurred after heating for 10 min at 60°C. The organic solvents methanol and ethylene glycol monomethyl ether could be included in the assay mixture up to 10% (v/v) with -10% loss of activity. The proteinase inhibitor phenylmethylsulfonyl fluoride (1 mM) could be included without loss of activity. Activity was, however, completely inhibited by 0.5 mM p- chloromercuribenzoate.

Studies on substrate specificity are summarized in Ta- ble II. The two sets of experiments indicated that high enzyme activity existed with regard to chlorophenols, 2,4,5-trichlorophenol, and 2,4-dichlorophenol being the best substrates. A number of additional compounds were utilized as substrates of low efficiency, the relative reaction rate with natural compounds was below 1%. However, these reactions did take place as further shown by use of the labeled aglycones (Table IIB). No systematic studies to determine V values for each of the tested substrates were performed. In separate experiments using step 5 enzyme and the conditions of Table IIA, [‘4C]bis(4-chlorophenyl)acetic acid was glucosylated as well as pentachlorophenol. Bis(4chlorophenyl)-acetic acid is a plant metabolite of DDT, and has previously been isolated from cultured soybean cells as the fl-D-glucopyranosylester (26).

0-GT S

FIG. 6. Nondenaturing gel electrophoresis of step 7 enzyme. The sample was applied in sample buffer of pH 6.8 containing 10% (v/v) glycerol to a stacking and separation gel prepared in a glass tube according to a published procedure (7, 21). After the electrophoretic run at 1.5 mA per tube, parallel gels were either stained with Comassie brilliant blue as shown or alternatively stored at -70°C and later on sliced. The gel slices were tested for O-glucosyltransferase activity (upper part), or used for SDS-polyacrylamide gel electrophoresis. For the enzyme assay, the gel slices were treated for 2 h at 25°C with 170 11 buffer 1 (containing 10% (v/v) ethyleneglycol) followed by addition of 20 ~1 200 mM UDP glucose and 10 ~1 1 mM [U’“C]PCP (1 PCi). After incubation for 1 h at 28°C in a shaking water bath, 5 gl 3 N H,PO, was added, and the product was determined as in the standard assay. Two SDS gel lanes are shown. In lane S the standard proteins (from top to bottom; bovine serum albumin (67 kDa), ovalbumin (45 kDa), and trypsinogen (24 kDa) were applied. Lane 0-GT corresponds to the region of the native gel containing 0-GT activity. In the native gel available for SDSgel electrophoresis the three native protein bands were not well separated and the three bands partially overlapped.


2 s .-. .

67

LS

2L

FIG. 7. SDS gel documenting N-MAT purification. In lane 1, pool 1 of step 4 was analyzed. The lanes S show the same protein standards (bovine serum albumin, ovalbumin, trypsinogen, and p-lactoglobulin). The proposed positions of the 0-GT band are marked by dots. In gel lane 2, the most highly purified pool of N-MAT activity (step 5) was analyzed. The right-hand panel came from a separate experiment.

Preliminary kinetic constants were determined with step 4 and step 5 enzyme preparations: K, for PCP, 30 PM (at constant 0.1 mM UDP-glucose); & for UDP-glu- case 77 PM (at constant 7 PM PCP).

Catalytic Properties of O-MAT

There was an unusual copurification of 0-GT and O- MAT through eight different purification steps. The O- MAT activity converted 90% of newly generated [U- “C]-PCP-fi-~-glucoside to the (0-malonyl)0-glucoside conjugate (assay procedure A). This conversion took place even in the presence of 5 mM protective nonradioactive @-D-glucosides as illustrated here by the data of Fig. 2. No transfer of the malonyl group to PCP occurred.

Catalytic Properties of N-MAT

The enzyme had a pH optimum between pH 6.3 and 7.2 as determined by the use of 0.1 M potassium phosphate buffers with adjusted pH values between 5 and 11. The reaction rate in the standard assay increased with incubation temperature up to 4O”C, followed by rapid loss of activity at higher temperatures. Linearity of the standard assay with regard to time existed up to -70 min incubation time and with regard to protein concentrations up to -0.2 mg protein per assay. A KM value of 0.1 mM was obtained for DCA (at constant 1 mM malonyl-CoA).

Enzyme activity was completely inhibited by 0.1 mM p-chloromercuribenzoate, but activity was regained upon 30 min incubation (0°C) with 2 mM dithiothreitol. Studies on substrate specificity are summarized in Table III. The two sets of experiments indicated that high enzyme ac-

tivity existed with regard to chlorinated anilines, 3,4-dichloroaniline and 4-chloroaniline being the best substrates. Very low or no reaction occurred with a number of natural substrates and herbicides. The following CoA- esters were not able to substitute for malonyl-CoA: acetyl-, propionyl-, crotonyl-, n-hexanoyl-, benzoyl-, and phenyl- acetyl-CoA. Only succinyl-CoA reacted with DCA. The product was identified as the N-succinyl conjugate of DCA by cochromatography (TLC) with the authentic reference compound (27) in solvent systems A (Rf, 0.72) and C (R,, 0.31). However, product formation was independent of enzyme protein. The nonenzymatic reaction was -five- fold higher at pH 5 than at pH 6.5. No reaction occurred at pH > 7.5.

Catalytic Properties of N-GT

This enzyme has been studied with regard to substrate specificity. As is shown in Table IV, a number of chlorinated anilines were utilized as substrates, but the natural compounds and various herbicides tested were not used. In contrast to the N-MAT reaction, aniline and l-amino- 4-methyl-coumarin did not serve as substrates.

DISCUSSION

Use of the two xenobiotic standard substrates PCP and DCA had led to the isolation and partial purification of

FIG. 8. Isoelectric focusing. The commercial Ampholine PAG-plate system (Pharmacia) was used according to the manufacturer’s instruction manual. The following marker proteins were studied separately: a, glucose oxidase, pZ, 4.25; band c, phycocyanin bands, p14.75 and 4.85; d, soybean trypsin inhibitor, pl 4.55; e, b-lactoglobulin (Sigma), pI 5.13; f and g, (Y- and @-lactoglobulin (LKB), pZ5.25 and 5.35; h, azurin, p15.65. These proteins were detected by silver staining. In a first experiment, an O- GT protein solution (step 7) was applied near the cathode via a small piece of filter paper. For determination of enzyme activity (- - -) the gel lane was sliced into 0.5 X 0.5.cm segments which were eluted with 100 ~1 buffer 1. These solutions were used for the standard assay for 0-GT, including the use of the protective substances. A maximal conversion rate of 2.5% was obtained at pZ - 4.8. N-MAT was studied in a separate experiment with -7.5 fig pool 1 enzyme from step 5. In this case, the gel slices were eluted with 200 ~1 buffer 3, followed by the standard N- MAT assay. A conversion rate of 3.8% occurred at pl - 6.1 (---). For determination of pH (&) gel lanes were sliced into 0.5 X O&cm segments and were eluted with 400 ~1 water, followed by pH determination by means of a microelectrode. These measurements led to the pH curve shown which was further defined by positions a-h of the marker proteins used. SDS-gel electrophoresis of the pZ 4.8 and 6.1 fractions indicated the presence of the same polypeptides that were present in the samples used for isoelectric focusing (see Figs. 5, 7).

348 SANDERMANN ET AL

TABLE II

Substrate Specificity of Soybean 0-Glucosyltransferase

Substrate Relative conversion

A. Comparison of 14C-labeled phenols

2,4-Dichlorophenol 100 PCP 20 2,4$Trichlorophenol 18 4-Nitrophenol 6

B. General screening program using UDP-[‘“Clglucose

2,4$Trichlorophenol 100 2,3,4-Trichlorophenol 77 2,3,5-Trichlorophenol 28 2,4$Trichlorophenol 25 4-Nitrophenol 23 2,4-Dichlorophenol 23 Vanillin 4 Vanillic acid 3 2-Nitrophenol 2 4-Hydroxy-3,5 dichlorobenzoic acid 2 Salicylic acid 2 4-Hydroxy-2,5-dichlorophenoxyacetic Acid 1 Ioxynil 1 4-Hydroxybenzoic acid 1

Note. (A) Step 7 and step 8 enzymes were tested with similar results. Y-labeled phenol (10 ~1; 2 mM; -2 pCi; in ethylene glycol monomethyl ether), 165 ~1 buffer 1, 5 ~1 28 mM 4-nitrophenyl-fl-D-glucoside, 5 ~1 28 mM salicin, 10 ~1200 mM UDP-glucose, and 5 ~1 enzyme (0.3 pg protein) were mixed for incubation (30 min, 28°C). The reaction was stopped by addition of 5 ~1 3 N H3P04. followed by extraction with 3 X 200 ~1 ethylacetate and TLC in solvent systems A, B, or D. The conversion rate (25.5%) of the best substrate (2.4-dichlorophenol) was set at 100%. Reaction rates of ~3% were found for [4-‘4C]cholesterol, [5-methyl- ‘Hltocopherol, 2-nitro[*4C]phenol, [2,3-‘?]maleic hydrazide, and 2,6- di-t-butyl-[“Clphenol. (B) Substrate solution in ethylene glycol monomethyl ether (200 nmol in 10 Al), 65 ~1 buffer 1,20 ~1 UDP-[‘4C]glucose (-0.5 &i, 1.7 nmol) and 5 ~1 step 8 enzyme (0.25 pg protein) were mixed for incubation (30 min, 28°C). The reaction was stopped by addition of 5 ~13 N H,PO, and 200 ~1 ethylacetate. Aliquots of the organic phase were counted, as well as analyzed for product by TLC in solvent systems A, B, C, or D, or by PC in solvent system F. The conversion rate of the best substrate (2,4,5-trichlorophenol, 35%) was set 100%. Reaction rates of ~1% were under these special conditions (very low concentration of UPD-glucose) obtained with PCP, D(-)-quinic acid, daidzein, 2’,4’,4-trihydroxychalcone, prunasin, apigenin, 4-methylum- belliferone, genistein, naringenin, quercetin, D-tyrosine, 2,4,6-trihy- droxybenzoic acid, benzoic acid, vanillyl alcohol, coniferyl alcohol, ferulic acid, 4.coumaric acid, caffeic acid, sinapic acid. Use of 14C-labeled daidzein, naringenin, genistein, coniferyl alcohol, and kgmpferol, as well as [3H]dihydroxypterocarpan with 10 mM UDP-glucose and step 4 enzyme has shown that glucosylation was in all cases possible at low rates (as analyzed by TLC on cellulose with 15% aqueous acetic acid as solvent system; data not shown).

four transferase activities from cultured soybean cells. Native molecular weight values of 45-50 kDa and the monomer composition determined in two cases (0-GT, N-MAT) as well as isoelectric points of 4.8 to 6.1 are in agreement with data reported for 0-GT, N-GT, O-MAT,

and N-MAT enzyme preparations purified from other plant sources (13, 15, 16, 28). The maize bronze gene en- coding a flavonol 0-glucosyl transferase has been se- quenced and corresponded to a protein subunit molecular weight of 48.8 kDa and ~16.0 (29). Soybean 0-GT activities for flavonols (30) and coniferyl alcohol (31) have previously been isolated. However, due to insufficient reported data on substrate specificity and protein properties, a comparison with the present soybean 0-GT for chlorinated phenols is not possible.

TABLE III

Substrate Specificity of Soybean N-Malonyltransferase


A. Comparison of 14C!-labeled chloroanilines

3,4-Dichloroaniline 4-Chloroaniline 3,4,5Trichloroaniline

100 a7 72

B. General screening using [“Clmalonyl-CoA

3,4-Dichloroaniline 100 4-Chloroaniline 82 4-Bromoaniline 75 7-Amino-4-methyl coumarin 70 3-Chloroaniline 67 Aniline 65 2-Chloroaniline 61 2,4-Dichloroaniline 53 2,5-Dichloroaniline 53 2,3,4-Trichloroaniline 50 3,5-Dichloroaniline 48 4.Nitroaniline 43 3,4,5-Trichloroaniline 30 2,3,4$Tetrachloroaniline 29 2,4,5-Trichloroaniline 22 4-Aminophenol 20

Note. (A) [‘4C]Chloroaniline in methanol (10 ~1; -0.5 &i, 41 nmol), 140 +I buffer 3, 20 ~1 malonyl-CoA (200 nmol in water), and 25 ~1 enzyme solution (1 part pool 1 plus 9 parts buffer 3; 2 pg protein) were mixed for incubation (40 min, 40°C). The reaction was stopped by addition of 5 ~1 acetic acid and 200 ~1 ethylacetate. After TLC and scanning the percentage conversion rate of the best substrate (3,4-dichloroaniline, 15.7%) was set at 100%. (B) Substrate solution in methanol (40 nmol in 4 pl), 146 ~1 buffer 3, 25 111 [‘4C]malonyl-CoA (202 nmol, -0.1 &i.) and 25 ~1 step 6 enzyme (28 pg protein) were mixed for incubation (40 min, 40°C). The reaction was stopped by addition of 5 ~1 acetic acid and 200 ~1 ethylacetate. Amounts of product were determined by counting an aliquot of the organic phase, and by scanning the following samples: TLC of the organic phase in solvent systems A and D; TLC and PC of the aqueous phase in solvent systems A, D, and F, respectively. The conversion rate of the best substrate (3,4-dichloroaniline; 25% of total 14C) was set at 100%. Reaction rates of ~3% were obtained with: 2,6- dichloroaniline, 2,4,6-trichloroaniline, 2,3,5,6-tetrachloroaniline, pentachloroaniline, l-aminobutane, 2-aminobutane, chloramben, picloram, metribuzin, asulam, chloridazon, brompyrazon, dichloran, metamitron, 4.aminophenyl-o-D-glucopyranoside, nicotinic acid, D- and L-trypto- phane, D- and L-phenylalanine, 1-aminocyclopropyl carboxylic acid, S- benzyl-L-cysteine, indole, indole-3-acetic acid, tryptamine.


TABLE IV

Substrate Specificity of Soybean N-Glucosyltransferase


2,4,5-Trichloroaniline 100 3Chloroaniline 99 3,4-Dichloroaniline 62 2$Dichloroaniline 40 3,4,5Trichloroaniline 32 2,3,4-Trichloroaniline 29 2,3Dichloroaniline 24 2,4-Dichloroaniline 24 4.Nitroaniline 24 3,5Dichloroaniline 23

Note. General screening using UDP-[‘“C]glucose. Substrate solution in methanol (20 nmol in 2 (.d), 66 ~1 buffer 3, 12 ~1 UDP-[i4C]glucose (21 nmol, 0.25 FCi), and 10 ~1 step 5 enzyme (22 fig protein) were mixed for incubation (40 min. 4O’C). The reaction was stopped by addition of 100 ~1 ethylacetate and was followed by counting of aliquots of the organic and aqueous phases. The amounts of product were further determined by radioscanning after TLC or PC of both phases in solvent systems A, C, D, E, or F. The conversion rate of the best substrate (2,4,5-dichloroaniline; 3% of total 14C) was set at 100%. Reaction rates of <5% were obtained with: aniline, 2,6dichloroaniline, 2,4&trichloroaniline, 2,3,5,6- tetrachloroaniline, pentachloroaniline, 4-aminophenol, l-aminobutane, Zaminobutane, 7-amino-4-methyl-coumarin, metribuzin, chloramben, amitrol, asulam, chloridazon, brompyrazon, dichloran, picloram, metamitron, nicotinic acid, 1-aminocyclopropylcarboxylic acid, tryptamine, indole. indole-3-acetic acid.

The present extensive copurification of the 0-GT and O-MAT enzymes appears remarkable and has not previously been reported. Both enzymes may work in concert and facilitate the vacuolar deposition of (O-malonyl)-P- D-glucosides (13, 32). The present O-MAT activity has not been characterized with regard to substrate specificity. However, previously purified O-MAT enzyme preparations have been shown to act on the xenobiotic 4-nitrophenyl-a-D-glucoside (32,33) and on the P-D-glucoside of 4-hydroxy-2,5-dichlorophenoxyacetic acid (32). The (O- malonyl)-0-glucoside conjugate resulting from the latter transferase reaction has been detected as a 2,4-D metabolite of cultured soybean cells by a novel DEAE Sephacel ion-exchange procedure (11).

The present data on substrate specificity of 0-GT (Ta- ble II), N-MAT (Table III), and N-GT (Table IV) reveal a marked preference for chlorinated phenols or anilines. These data are of preliminary nature since no complete kinetics (V&J were determined for each of the substrates tested. It appeared nevertheless remarkable that two of the transferases (N-MAT; N-GT) did not accept sterically hindered aryl-substrates with 2,6-substituents, whereas 0-GT and O-MAT utilized these substrates. The conclusion of narrow substrate specificity is in agreement with literature studies on transferases (13, 28-33). However, in many of the reported studies, as well as in the present Table II, substrates other than the preferred ones were utilized at low rates.

The observation of narrow substrate specificity may be of practical interest. A selective action of herbicides is required in agriculture and is often due to metabolic de- toxification in the crop plant. The genes for the N-glucosyltransferase for metribuzin (28) and for the present 0-glucosyltransferase for PCP may thus be regarded as potential resistance genes (34).

The present isoenzymes with specificity for chlorinated phenols and anilines can well explain the formation of the reported soluble in uiuo metabolites of PCP and chlorinated anilines ((9, 10); Fig. 1). Specific plant isoenzymes have also been shown to be involved in the formation of xenobiotic glutathione S-conjugates (4-6) and in the es- terase cleavage of a plasticizer chemical (7). It is at present unknown why plants should possess isoenzymes with specificity for xenobiotics, but these enzymes may have an important role in the sink function of the global plant biomass.

ACKNOWLEDGMENTS

The excellent technical assistance of C. Reiss and E. Mattes and advice and experimental help by M. Arjmand and R. Winkler are grate- fully acknowledged. This work has been supported by the Deutsche Forschungsgemeinschaft (Sa 180/15-5) and in part by the Bundesmin- isterium fur Forschung and Technologie (PBE 038657) and the Fonds der Chemischen Industrie.

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plant biochemistry of xenobiotics: isolation and properties of soybean o- and n-glucosyl and o- and...

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