THE JOURNAL OF BIOLOGICAL CHEMISTRY 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 41, Issue of October 12, pp. 38179 38184, 2001 Printed in U.S.A.

Morphine Metabolism in the Opium Poppy and Its Possible Physiological Function
BIOCHEMICAL CHARACTERIZATION OF THE MORPHINE METABOLITE, BISMORPHINE*
Received for publication, July 26, 2001, and in revised form, August 8, 2001 Published, JBC Papers in Press, August 9, 2001, DOI 10.1074/jbc.M107105200

Satoshi Morimoto, Kazunari Suemori, Jun Moriwaki, Futoshi Taura, Hiroyuki Tanaka, Mariko Aso, Masakazu Tanaka, Hiroshi Suemune, Yasuyuki Shimohigashi, and Yukihiro Shoyama
From the Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582 and the Faculty and Graduate School of Sciences, Kyushu University, Fukuoka, 812-8581, Japan

We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in crosslinking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.

In response to mechanical damage, the immature capsules of the opium poppy (Papaver somniferum) and related species (Papaver setigerum, etc.) immediately secrete opium consisting of various secondary constituents such as morphine, codeine, papaverine, and noscapine. Among these, morphine has attracted a great deal of attention as one of the most medicinally important analgesics and narcotics. Therefore, numerous studies on morphine have been carried out since its first isolation in 1804 by Sertu rner (1), but why the opium poppy produces this compound remains unknown. Morphine is structurally classified into alkaloid, based on the presence of nitrogen in its molecule. Many higher plants including the opium poppy synthesize a variety of alkaloids, and like urea and uric acid in animals, these alkaloids have often been suggested to be produced as nitrogenous waste products with little physiological importance for host plants. However, this hypothesis lacks precise experimental evidence
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. Tel.: 81-92-642-6581; Fax: 81-92-642-6545; E-mail: morimoto@shoyaku.phar.kyushu-u.ac.jp.
This paper is available on line at http://www.jbc.org

(2). In addition, the significant physiological roles of some alkaloids in host plants have been demonstrated by characterizing the biological properties of the alkaloids and their metabolites; steroid alkaloids such as tomatine and solanidine are involved in protecting plants against herbivores and microbial pathogens (3), whereas the pyridine alkaloids, trigonelline (Nmethylnicotinic acid) and N-arabinosylnicotinic acid act as precursors of the vitamin nicotinic acid (4, 5). Therefore, it is not reasonable to regard all alkaloids including morphine as waste products, although in contrast to these steroidal and pyridine alkaloids, little information is available concerning the physiological function of morphine in opium poppy. Important information on the physiological roles of several plant secondary constituents as well as the above pyridine alkaloids can be obtained by investigating the properties of their metabolites. For example, the main steroid saponins of oat (Avena sativa), avenacosides A and B, have been shown to be metabolized by endogenous -glucosidase into 26-desglucoavenacosides A and B, respectively, which function as antibacterial substances against pathogens (6, 7). Furthermore, we recently investigated the metabolic pathway of flavone glucuronide in the skullcap plant (Scutellaria baicalensis Georgi), and the metabolite (baicalein) of baicalein 7-O--D-glucuronide has been shown to play an important role in detoxification of the large amount of H2O2 produced by the oxidative burst (8 11). These results indicated that precise understanding of morphine metabolism may provide the useful evidence from which its physiological importance in the opium poppy is inferred. Recent studies have almost completely elucidated the biosynthetic mechanism of morphine (12), but metabolism of morphine in the opium poppy is largely unknown. Therefore, to determine the physiological importance of morphine, we investigated its metabolism in the opium poppy. Our results indicated that in response to mechanical damage, morphine immediately undergoes oxidation by bismorphine-forming peroxidase (BFP)1 and is metabolized into the dimer of morphine, bismorphine. Biochemical characterization of bismorphine demonstrated that the two amino groups of this alkaloid ionically bind to the carboxyl groups of the galacturonic acid residues of cell wall polysaccharides pectins, resulting in cross-linking of pectins to each other. Furthermore, we confirmed that binding of bismorphine to pectins significantly contributes to their resistance to hydrolysis by pectinase. We report here the metabolism of morphine and its novel physiological role in the opium poppy.

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1 The abbreviations used are: BFP, bismorphine-forming peroxidase; HPLC, high performance liquid chromatography.

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EXPERIMENTAL PROCEDURES

Plant MaterialsOpium poppies (P. somniferum L.) were grown in the herbal garden of the Graduate School of Pharmaceutical Sciences of Kyushu University. Opium poppies at 10 15 days after flowering were used in this study. Wounding of the capsules was carried out as follows. The immature capsules (fresh weight, 1520 g) were scratched using a blade (15 scratches/capsule; length of scratch, 2.0 cm; interval of each scratch, 2.0 mm). The opium poppies of which the capsules had been scratched were incubated for 24 h at 25 C in a greenhouse. The scratched regions were cut off the capsules and used as the wounded capsules. We confirmed that reproducible results were obtained by these procedures. Isolation and Structural Characterization of BismorphineCrude cell walls prepared from the wounded capsules (200 g) were sonicated in 20 mM HCl (100 ml) for 10 min. Insoluble materials were removed by centrifugation at 20,000 g for 5 min, and the HCl extracts were neutralized with 0.1 M NaOH. After concentration under vacuum, the residue was washed with 1% (v/v) ammonia water (50 ml) and then applied to high performance liquid chromatography (HPLC) to afford bismorphine (2.5 mg). The structure of bismorphine was determined by obtaining its 1H and 13C NMR (Varian) spectra. NMR data were assigned as follows: 1H NMR (diemethylsulfoxide-d6, 500 MHz) ppm; 1.68 (2H,H-15,15), 1.99 (2H,H-15,15), 2.09 (2H,H-9,9), 2.28 (2H,H-16,16), 2.38 (6H,N-Me), 2.48 (2H,H-16,16), 2.56 (2H,H-13,13), 2.88 (2H,H9,9), 3.28 (2H,H-14,14), 4.10 (2H,H-6,6), 4.71 (1H,H-5,5), 5.26 (2H,H7,7), 5.57 (2H,H-8,8), and 6.30 (2H,H-2,2); 13C NMR (dimethyl sulfoxide-d6, 100 MHz) ppm; 20.0 (C-9,9), 35.3 (C-15,15), 40.6 (C-13,13), 42.8 (C-12,12, Me), 46.0 (C-16,16), 58.1 (C-14,14), 66.3 (C-6,6), 91.7 (C-5,5), 120.9 (C-2,2), 124.9 (C-10,10), 128.1 (C-11,11), 128.3 (C-8,8), 129.5 (C-1,1), 133.4 (C-7,7), 135.6 (C-3,3), and 147.1 (C-4,4). Preparation and Fractionation of Crude Cell WallsThe immature capsules (100 g) from which seeds were removed were homogenized with distilled water (2,000 ml) and filtered using filter papers. The residue was consecutively washed with distilled water (2,000 ml), 0.1% (w/v) Tween 20 (2,000 ml), EtOH (2,000 ml), acetone (2,000 ml), and hexane (2,000 ml). After drying under vacuum, the residue was used as the crude cell wall fraction. Fractionation of the crude cell walls was carried out by a modification of the method of Srisuma et al. (13). The crude cell wall fraction (10 g) was heated twice in 0.5% (w/v) ammonium oxalate (500 ml) at 85 C to remove pectins and then washed with water (500 ml). After drying under vacuum, the residue was incubated twice in 1 M NaOH (500 ml) at room temperature for 18 h and washed with water (2,000 ml) to afford the lignocellulose fraction. This fraction was incubated in 72% (v/v) H2SO4 at 4 C for 36 h to give the lignin fraction. HPLC Analysis of Bismorphine Bound to Cell WallsThe crude cell wall fraction (50 200 mg) of opium poppies was homogenized with 20 mM HCl (20 80 ml), and the homogenate was centrifuged at 20,000 g for 5 min. The amount of bismorphine in the supernatant was measured using an HPLC system equipped with a 0.46 15-cm column of Cosmosil 5C18 AR-II (Nacalai, Kyoto). Bismorphine was eluted with 60% (for quantitative analysis) or 50% (v/v) aqueous acetonitrile (for identification and isolation of bismorphine) containing 5 mM sodium di-2-ethylhexyl sulfosuccinate at a flow rate of 1 ml/min (14). The pH of the mobile phase was adjusted to 3.3 using acetic acid. The eluate was monitored by absorption at 280 nm. The amount of bismorphine was calculated from the standard curve obtained with the authentic sample. In Vitro Binding of BismorphineAssay mixtures consisting of 35 M bismorphine, crude cell wall fraction (5 mg), and 10 mM Tris-HCl (pH 7.5, 500 l) were incubated at 30 C for 1 h. The cell walls were removed by centrifugation at 20,000 g for 5 min, and the amount of bismorphine in the supernatant was quantified by HPLC. The amount of bismorphine bound to the cell walls was calculated by subtracting the amount of bismorphine in the assay mixture incubated without the cell walls from that in the supernatant. We confirmed that 668 g of bismorphine bound to 1 g of the crude cell wall fraction under these conditions and that bound bismorphine was completely solubilized with 20 mM HCl. Binding of Pectins to Uronic Acid-conjugated Sepharose 6BUronic acid-conjugated Sepharose 6B was prepared as follows. Epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech; dry weight, 3 g) preswollen with water was suspended in 0.2 N NaOH (20 ml) solution containing galacturonic acid or glucuronic acid (each 400 mg). After incubation at room temperature for 24 h, the gel was washed with 1 M NaCl (100 ml), and then water (100 ml) was used as uronic acid-conjugated Sepharose 6B. A 60 M bismorphine solution (300 ml) was loaded onto the column (1.0 5.0 cm) containing uronic acid-conjugated Sepharose 6B. After application of 0.2% (w/v) polysaccharide solution (5 ml) to the

same column, the gel was washed with water (20 ml). The bound polysaccharides were eluted with 20 mM HCl (20 ml), and the amounts of polysaccharides in the HCl eluate were quantified by phenol/sulfuric acid analysis. Pectinase Treatment of Crude Cell WallThe crude cell wall fraction (50 mg) was incubated at 30 C for 1 h in 50 ml of 10 mM citrate buffer (pH 5.5) containing bismorphine (30 g). We confirmed that all bismorphine was bound to the cell walls. Pectinase treatment of the crude cell sample was carried out by a modification of the method of Cervone et al. (15). Aspergillus pectinase (Sigma; 1 unit) was added to these samples and incubated at 30 C for 5 min. Ammonium oxalate (100 mg) was added to the enzymatic reaction mixture and then heated at 85 C for 1 h. After centrifugation at 20,000 g for 5 min, the reducing groups in the supernatant were quantified using dinitrosalicylic acid reagent. To determine the amounts of pectin fragments released from the cell walls, the above enzymatic reaction mixture was centrifuged at 20,000 g for 5 min, and the pectin fragments in the supernatant were quantified by phenol/sulfuric acid analysis. Extraction and Purification of BFPAll procedures were carried out at 4 C, unless otherwise indicated. The immature capsules of opium poppies (30 g) were homogenized in 100 mM phosphate buffer (pH 7.0, 100 ml) containing 3 mM mercaptoethanol and then filtered through Nylon filters. The filtrate was centrifuged at 100,000 g for 15 min, and the supernatant was fractionated by the addition of ammonium sulfate. Proteins precipitating at 65% saturation were collected by centrifugation at 20,000 g for 30 min and then dialyzed overnight against 10 mM phosphate buffer (3000 ml, pH 7.0). The dialyzed sample (crude enzyme solution) was applied to a DEAE-cellulose column (1.5 10 cm) equilibrated with 10 mM phosphate buffer (pH 7.0). The column was washed with three column volumes of the same buffer, and the bound proteins were eluted with a 600-ml linear gradient of NaCl (0 0.4 m) at a flow rate of 1 ml/min. Fractions containing BFP were collected, concentrated, and dialyzed against 10 mM sodium phosphate buffer (pH 7.0). The dialysate was applied to a hydroxylapatite column (1.0 10.0 cm). The column was washed with the same buffer (100 ml) and then with a 200-ml gradient of 10 200 mM phosphate buffer. Potent BFP activity was detected in fractions eluted with 10 mM phosphate buffer at a flow rate of 1 ml/min. The most active fractions were directly loaded onto a column containing morphine-conjugated Sepharose 6B (1.0 10.0 cm), which was prepared using 50 mg of morphine and epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech; dry weight, 3 g) according to the manufacturers protocol. After washing with 30 ml of 10 mM phosphate buffer (pH 7.0), a 100-ml linear gradient of 0 0.4 M NaCl was passed through the column at a flow rate of 0.1 ml/min. The fractions containing BFP activity were pooled, concentrated, and used for the kinetic studies. Assay for BFPThe assay mixtures consisted of 5 mM morphine, 5 mM H2O2, 50 mM Tris-HCl (pH 9.0) and enzyme solution (100 l) in a final volume of 500 l. The samples were incubated at 30 C for 10 min, and the reaction was terminated with 500 l of HPLC mobile phase. The amount of bismorphine liberated was quantified by HPLC. Large Scale Preparation of BismorphineA solution (100 ml) containing 143 mg of morphine, 50 mM Tris-HCl, and 5 mM H2O2 was incubated at 30 C for 12 h with crude enzyme solution prepared from 30 g of the immature capsules. The precipitates containing bismorphine were collected by centrifugation at 20,000 g for 10 min and washed twice with distilled water (50 ml) and then with EtOH (50 ml). The washed samples were dissolved in dimethyl sulfoxide (5 ml), and insoluble materials were removed by centrifugation at 20,000 g for 10 min. Water (30 ml) was added to the supernatant, and the resulting precipitates were collected by similar centrifugation to afford bismorphine (62 mg).
RESULTS

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Identification of Morphine Metabolites in the Opium PoppyTo determine the metabolic pathway of morphine, we first attempted to identify morphine metabolites in the opium poppy by HPLC analysis and by feeding with 3H-labeled morphine. However, no morphine metabolites were detected in the intact immature capsules of opium poppies using these methods. In contrast, we found that an unknown alkaloid accumulated at the site of damage in the wounded capsules (Fig. 1A). Furthermore, incorporation of 3H-labeled morphine into this unknown compound was observed by feeding experiments (data not shown), confirming that this was a metabolite of morphine. This metabolite was purified from the damaged capsules of

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FIG. 1. Identification of the metabolite of morphine. A, HPLC analysis of the opium poppy capsules. The crude cell walls prepared from the intact capsules (left panel) or from the wounded capsules (right panel) were homogenized with 20 mM HCl. The insoluble materials were removed by centrifugation at 20,000 g for 5 min, and the supernatant was analyzed by HPLC. B, structure of bismorphine.

FIG. 2. Amount of bismorphine after wounding. Opium poppies of which the capsules were wounded were incubated at 30 C for different times. The crude cell walls were prepared from the wounded capsules, homogenized in 20 mM HCl, and centrifuged at 20,000 g for 5 min. The amount of bismorphine in the supernatant was estimated as described under Experimental Procedures. The data are the means of five replicate assays.

opium poppies by preparative HPLC, and its mass spectrum and NMR spectra were obtained. The fast atom bombardment mass spectrum showed an [MH] ion peak at m/z 569, indicating the dimeric nature of the metabolite. Furthermore, the 1H NMR and 13 C NMR spectra, which were almost identical to those of morphine except for the aromatic signals, indicated that this dimeric compound possessed a symmetrical structure. Based on analyses of the two-dimensional NMR spectra, the metabolite was finally determined to be dimeric morphine (called bismorphine in this study), in which two morphine units are oxidatively coupled to each other through a biphenyl bond (Fig. 1B). Bismorphine is a new alkaloid with a dimeric structure. Bismorphine Level after WoundingChanges in the amount of bismorphine after wounding of the immature capsules are assessed by HPLC. As shown in Fig. 2, bismorphine was not detected in the intact capsules, and its production was induced immediately after wounding. The amount of bismorphine increased rapidly until 2 h after wounding, and thereafter its production rate became relatively slow. The capsules at 24 h after wounding produced 600 g/g cell walls of bismorphine (Fig. 2). Furthermore, the accumulation (450 g/g cell walls) of bismorphine was also confirmed in the dead, brown tissue of the immature capsules infected by pathogens. Taken together with the absence of bismorphine in the intact capsules, these results suggested that production of bismorphine is induced rapidly by various forms of stress. Localization of BismorphineThe localization of bismorphine accumulating in the wounded capsules was deduced from its solubility in various solvents. Solvents such as water, ethanol, and acetone were less effective for bismorphine extraction from the damaged capsules, whereas bismorphine was readily solubilized using aqueous solvents containing HCl, CaCl2, NaCl, and EDTA (Fig. 3A), which are often used for extraction of components ionically associated with cell walls (16, 17). Furthermore, more than 90% of the bismorphine produced by the wounded capsules was recovered in the crude cell wall fraction (Fig. 3B). These results indicated that most of the bismorphine induced by wounding was ionically bound to the cell walls. As shown in Fig. 3A, NaCl or EDTA accelerated the extraction of bismorphine in a concentration-dependent manner, although bismorphine exhibited low solubility in 100 mM HCl or CaCl2 as compared with respective 20 mM solutions. Acidic conditions and divalent ions significantly change the physical properties of cell wall polysaccharide pectins, resulting in the solidification of pectins (18, 19). We hypothesized that such changes in the pectin moiety caused the inhibition of bismorphine extraction.

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FIG. 3. Extraction of bismorphine. A, effects of solvents on bismorphine extraction. Bismorphine was extracted from the crude cell walls of the wounded capsules using various solvents and quantified by HPLC. The pH of EDTA solution was adjusted to 7.0 using NaOH. B, amounts of bismorphine in the wounded capsules (CP) and the crude cell walls (CW). Bismorphine was extracted from the wounded capsules (dry weight, 1.2 g) or cell walls prepared from the same amount of wounded capsules and quantified by HPLC. The data are the means of five replicate assays.

We also investigated to which cell wall components bismorphine binds. The cell walls from the wounded capsules were fractionated into three fractions (pectins, hemicellulose, and lignocellulose), and the bismorphine in each fraction was quantified. However, it was impossible to precisely quantify bismorphine, because during fractionation most bismorphine was solubilized from the cell wall. Therefore, the localization of bismorphine was determined by in vitro binding assay. Bismorphine was incubated with cell wall samples fractionated according to Srisumas procedure (13), and the amount of bismorphine bound to each fraction was then analyzed. As shown in Fig. 4A, bismorphine exhibited apparently lower affinity for the cell wall fraction when pectins were removed, as compared with the crude cell walls. Thus, it became evident that bismorphine is predominantly bound to pectins. In the cell walls from which pectins and hemicellulose had been removed, a further decrease was observed in the amount of bound bismorphine, suggesting that a small amount of bismorphine is also bound to the hemicellulose moiety. Little bismorphine was bound to the lignin fraction. Pectins, which are structurally classified into three types (polygalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II), possess high galacturonic acid residue contents, whereas xylans belonging to hemicelluloses have low glucuronic acid residue contents (18, 19). Because morphinan alkaloids readily form alkaloid salts with organic acids (2), we assumed that bismorphine is ionically bound to the carboxyl groups of these uronic acid residues. This was unequivocally confirmed by the observation that bismorphine did not bind to the cell walls where carboxyl groups were extensively esterified by diazomethane treatment (Fig. 4B).

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FIG. 4. In vitro binding of bismorphine to various cell wall fractions. Bismorphine was incubated with ammonium oxalatetreated cell walls (AOCW), lignocellulose fraction (LGC), and lignin fraction (LG), each prepared from 1 g of the crude cell walls (A). CH2N2-treated cell walls were obtained by incubation of the crude cell walls (1 g) at 4 C for 4 days in CH2N2 ether solution (20 ml) (B). The amount of bismorphine bound to these samples was compared with that bound to crude cell walls (1 g, CW). The relative amount of 100% was 668 g.

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Cross-linking Ability of Bismorphine for Various CarbohydratesConsidering the dibasic nature of bismorphine, it is likely that the two amino groups simultaneously form ionic bonds with carboxyl groups of pectins, leading to cross-linking of pectins to each other through bismorphine bridges (Fig. 5A). To confirm this hypothesis, we developed a chromatographic method using artificial polysaccharides as a matrix, prepared by coupling of galacturonic acid with Sepharose 6B through ether linkages (Fig. 5B). Bismorphine was readily absorbed to galacturonic acid-conjugated Sepharose 6B, whereas HCl solution or NaCl solution effectively solubilized bound bismorphine, confirming that bismorphine is ionically bound to the galacturonic acid residues of the matrix. If bismorphine can form bismorphine bridges between this matrix and polysaccharides, the polysaccharides should bind to the matrix as shown in Fig. 5B. Pectins were loaded onto the galacturonic acidconjugated Sepharose 6B column pretreated with bismorphine, and the substances absorbed to the column were then eluted with HCl solution. Quantitative analysis using phenol/sulfuric acid reagent confirmed that pectins bound to the bismorphinetreated matrix (Fig. 6A). Thus, it was evident that bismorphine possesses the ability to cross-link pectins to each other through bismorphine bridges (Fig. 5A). Small amounts of pectins bound to the matrix without any pretreatment (control), and we assumed that this may have been due to interaction between pectins and few charged groups in Sepharose. A similar experiment was carried out using the same matrix treated with the monobasic alkaloid morphine instead of bismorphine, but the amount of pectins bound to the matrix was almost identical to that in the control sample (Fig. 6A), indicating that morphine lacks the ability to form cross-linkages. Furthermore, glucuronic acid-conjugated Sepharose 6B was also prepared, and pectins were applied to this matrix pretreated with bismorphine. Binding of pectins to the matrix was observed, although its amount was lower than the values obtained using galacturonic acid-conjugated Sepharose 6B (Fig. 6B). These results were consistent with the above finding that bismorphine was predominantly bound to the pectin fraction rather than the hemicellulose fraction with low glucuronic acid residue contents. On the other hand, like starch, polysaccharides containing no uronic acid residues were not cross-linked to uronic acid-conjugated Sepharose 6B, even if each matrix was

FIG. 5. Possible model for interaction between bismorphine and polysaccharides. A, cross-linking of pectins (black) through bismorphine bridges (red). B, interaction between pectins (black) and galacturonic acid-conjugated Sepharose 6B (blue) pretreated with bismorphine (red).

FIG. 6. Amount of pectin bound to uronic acid-conjugated Sepharose 6B. Pectins were loaded onto galacturonic acid-conjugated (A) or glucuronic acid-conjugated Sepharose 6B (B) pretreated with bismorphine (12 mol), morphine (11 mol), or Ca2 (12 mol). Control experiments were carried out using the same matrix without any pretreatment. Pectins bound to the matrix were eluted with 20 mM HCl and quantified by phenol/sulfuric acid analysis. The data are the means of five replicate assays.

treated with bismorphine, indicating that cross-linking of polysaccharides through bismorphine bridges is significantly dependent on the contents of uronic acid. Similarly to bismorphine, divalent ions such as Ca2 are also known to bind to carboxyl groups of galacturonic acid residues in pectins and cross-link these polysaccharides to each other. When the ability of Ca2 to cross-link pectins was compared with that of bismorphine using both uronic acid-conjugated gels, we found that the amount of pectins cross-linked by bismorphine was much higher than that by Ca2 (Fig. 6). Effects of Bismorphine on Hydrolysis of Pectins by PectinasesPreviously, two groups reported that xylans in several

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FIG. 7. Properties of BFP. A, BFP activity after wounding. The wounded capsules were incubated at 30 C for different times, and the peroxidase activity was measured. The data are the means of five replicate assays. B, SDS-polyacrylamide gel electrophoresis analysis of bismorphine. The samples were resolved by electrophoresis on a 12.5% acrylamide gel, and the proteins were stained with Coomassie Brilliant Blue. Lane 1, molecular standards with the indicated molecular mass; lane 2, BFP. C, substrate specificity of BFP. Morphinan alkaloids (each 5 mM) were incubated with BFP (0.1 g) under standard assay conditions. The reaction mixture was analyzed by HPLC.

tated by 65% saturation was applied to a DE-cellulose (DE-52) column and then to a hydroxylapatite column. Finally this peroxidase was purified to homogeneity by affinity column chromatography over morphine-conjugated Sepharose 6B. SDS-polyacrylamide gel electrophoresis of purified BFP displayed a monomeric molecular mass of about 33 kDa (Fig. 7B), whereas the pI for this peroxidase was estimated to be 5.0 by isoelectric focusing. The enzyme activity of BFP was maximal at pH 9.0, with half-maximal activities at pH values around 7.5. and 10.0. Therefore, standard assay conditions included Tris-HCl buffer (pH 9.0) containing 5 mM H2O2. Under these conditions, BFP catalyzed the dimerization of morphine, whereas the other oligomers and polymers of morphine were not detected in the assay solution. We also tested the substrate specificity of BFP using several morphinan alkaloids. In contrast to morphine, its biosynthetic precursors thebaine and codeine were not substrates of this peroxidase (Fig. 7C). In addition, BFP did not catalyze oxidation of various phenolics (guaiacol, catechol, pyrogallol, ferulic acid, and caffeic acid), which are often used as substrates of phenol peroxidase (22 24). Thus, we found that BFP displayed restricted substrate specificity.
DISCUSSION

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monocots are cross-linked through diferuloylester bridges and assumed that such cross-linking results in resistance to enzymatic digestion (20, 21). Bismorphine is structurally different from diferulic acid, but both compounds have a biphenyl bond. Therefore, we hypothesized that cross-linking of pectins through bismorphine bridges may lead to resistance to digestion by pectin-hydrolyzing enzymes. To confirm this hypothesis, the effects of bismorphine on pectinase reaction were examined using the crude cell wall fraction prepared from the intact capsules. The crude cell wall fraction treated with bismorphine was incubated in the presence of pectinase, and the reducing groups, which were produced by enzymatic hydrolysis, were quantified. The amount (23 nmol) of the reducing groups from bismorphine-treated cell walls was lower than that (69 nmol) from the untreated cell walls. Furthermore, we confirmed that bismorphine treatment also reduced the amounts (185 g) of pectin fragments released by the enzymatic reaction from the cell walls, as compared with that (335 g) from the untreated cell walls. Because these results were obtained at a level of bismorphine (600 g/g cell walls) that the wounded capsules can produce, we concluded that bismorphine accumulating in the wounded capsules contributes to protection of pectins from hydrolysis by pectinase. Identification of Enzymes Involved in Bismorphine FormationBecause production of bismorphine was regarded as a novel defense response of the opium poppy, we attempted to determine its biosynthetic mechanism by identifying and characterizing the enzymes involved in this reaction. Morphine was incubated under various conditions in the presence of crude enzyme extracts prepared from the immature capsules of opium poppies, and reaction products were then analyzed by HPLC. Bismorphine was produced only in the presence of H2O2, indicating that bismorphine formation is catalyzed by peroxidase. This peroxidase (BFP) was also found in the leaves of opium poppies (0.7 microkatal/g leaves), but its activity was lower than that in the immature capsules (1.3 microkatal/g capsules). In contrast, no activity was detected in the roots of this plant. We tested whether the BFP activity is induced by wounding of immature capsules, but the enzyme activity did not increase by wounding the immature capsules (Fig. 7A). Purification and Characterization of BFPPurification of BFP was carried out by a four-step procedure. Crude enzyme extracts prepared from the immature capsules were fractionated by ammonium sulfate saturation. The fraction precipi-

Despite numerous studies on morphine, nothing is known about its roles in the opium poppy. In the present study, we identified the novel metabolic pathway by which morphine is oxidized into the dibasic alkaloid bismorphine in the opium poppy and established the physiological importance of this metabolism. Various alkaloids have been isolated from opium poppies, but the morphine dimer has not been identified previously. Because several morphine derivatives have been shown to have strong affinity for opioid receptors and to display potent analgesic effects (25), we also investigated the binding ability of bismorphine to opioid receptors. However, its affinity for receptor was markedly lower than that of morphine.2 The physiological importance of morphine metabolism in the opium poppy was confirmed by investigating the properties of bismorphine. Interestingly, bismorphine was bound readily to cell wall polysaccharides containing uronic acid residues such as pectins and hemicellulose. In addition, our newly developed method using artificial polysaccharides demonstrated that these polysaccharides are cross-linked through bismorphine bridges and that bismorphine more effectively cross-links pectins than other polysaccharides including hemicellulose. Taken together with the observation that bismorphine accumulated in the site of damage in the capsules, these results suggested that bismorphine may hold together pectin fragments produced by wounding. On the other hand, morphine, the precursor of bismorphine, was also ionically bound to the uronic acid residues of these artificial polysaccharides, whereas this alkaloid did not cross-link pectins. Because the morphine molecule possesses only one amino group, it is reasonable that this alkaloid lacks cross-linking ability. The divalent ions such as Ca2 are known to cross-link pectins to each other, resulting in formation of gels (18, 19). Based on the observation that treatment of plant tissues with Ca2 or chelating agents stiffens or softens them, respectively, it has been proposed that cross-linking of pectins through Ca2 contributes to strengthening of the plant wall and adhesion of plant cells (26, 27). Surprisingly, our experiments using uronic acid-conjugated Sepharose 6B indicated that the cross-linking ability of bismorphine for uronic acid-containing polysaccharides is much higher than that of Ca2. In addition, we confirmed that pectins form gels in the presence of bismorphine
2

S. Morimoto, Y. Shoyama, and Y. Shimohigashi, unpublished data.

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Morphine Metabolism in the Opium Poppy

Because stress treatment of plant cells causes observed rapid production of large amounts of reactive oxygen species, a reaction known as the oxidative burst (30 32), H2O2 induced during the oxidative burst may be used for bismorphine formation. In conclusion, the metabolism of morphine to bismorphine is regarded as a defense system of the opium poppy. Higher plants can immediately initiate production of various defenserelated secondary constituents to protect themselves from microbial phytopathogens. Most act as antimicrobial substances, and several constituents contribute to strengthening of the lignin moiety in cell walls. Bismorphine is the first alkaloid involved in the repair and strengthening of pectins to be identified to date. Several Papaver species produce morphinan alkaloids other than morphine. Among these, Papaver pseudoorientale and Papaver lasiothrix produce the biosynthetic precursor of morphine, salutaridine, as a major alkaloid. In both plants, Sariyar et al. (33) identified salutadimerine, in which two morphine units are oxidatively coupled to each other through a biphenylether bond. Because cross-linking of pectins through bismorphine bridges is based on the ionic interaction between the two amino groups of bismorphine and the uronic acid residues of polysaccharides, salutadimerine may display similar properties for pectins. In addition, the occurrence of many dibasic alkaloids in the plant kingdom suggested that similar defense systems may be found in other plants.
AcknowledgmentsWe thank Yoshitsugu Tanaka and Kyoko Soeda for NMR measurements of morphine and bismorphine.
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(data not shown). These results suggested that bismorphine may have physiological functions similar to those of Ca2. It is of interest that the opium poppy synthesizes bismorphine with Ca2-like function by dimerization of the monobasic alkaloid morphine. Cross-linking of polysaccharides through other secondary constituents has been demonstrated in monocots. For example, diferulic acid in which two ferulic acid units are oxidatively coupled to each other through a biphenyl bond has been shown to be bound to the cell wall polysaccharides xylans of Triticum aestivum and Lolium multiflorum via ester bonds, involving both of its carboxyl groups (20, 21). Such cross-linking is believed to have significant effects on the ability to resist enzymatic digestion of xylans, but to date no experimental evidence for this notion has been reported. In the present study, we established that binding of bismorphine to pectins resulted in resistance against their degradation by pectinases. It is noteworthy that the protective effect of bismorphine for pectins was found at a bismorphine level that can be produced by the wounded capsules. From these results, we concluded that production of bismorphine is a defense response. Pectins are polysaccharides that play important roles in cell adhesion, cell wall stiffening, and assembly of cell wall components, and degradation of pectins is well known to cause serious damage in plants (26, 27). Therefore, the repair and stiffening system for pectins is quite important. The formation of bismorphine is catalyzed by endogenous anionic peroxidase, BFP. This enzyme activity was also found in the leaves of the opium poppy, although at somewhat lower levels than that in the capsules. This distribution showed a good correlation with the morphine content; greater amounts of morphine are found in the immature capsules than in the leaves. Because bismorphine was also detected in wounded leaves of the opium poppy (data not shown), it is likely that the same defense system also exists in the leaves of the opium poppy. We found that the properties of BFP are different from those of other plant peroxidases. Plant phenol peroxidases usually catalyze various substrates including both natural and synthetic substrates; for example, peroxidases in tobacco and mung bean have the ability to oxidize several phenolics (22, 23). In contrast, BFP catalyzed oxidation only of morphine, and none of the other phenolic compounds tested were substrates for this enzyme. Thus, BFP is quite a unique peroxidase. Although bismorphine has a biphenyl bond that is often found in lignans and lignins, judged from the inability to oxidize ferulic acid and caffeic acid, it is unlikely that BFP catalyzes biosynthesis of lignin, one of the cell wall components. Bismorphine is not detected in the intact plant, and its production is initiated within 1 h by stress. This response is faster than transcription-dependent defense reactions such as phytoalexin accumulation (28), which are usually observed more than 2 h after stress treatment. Hence, bismorphine production is one of the earliest defense reactions. Albersheim et al. (29) reported that in many cases, the first stage of the infectious process of phytopathogens is the production of cell wall-degrading enzymes such as pectinases. Therefore, rapid production of bismorphine involved in the stiffening of pectins may play an important role in protecting the opium poppy against infection by pathogens at the early stages. In the present study, we confirmed the pre-existence of large amounts of BFP as well as morphine in the immature capsules of the opium poppy, and such constitutive expression was considered to enable the observed rapid responses. In addition to both constituents, H2O2 is also essential for bismorphine formation.

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