Morphine Metabolism in the Opium Poppy and Its PossiblePhysiological FunctionBIOCHEMICAL CHARACTERIZATION OF THE MORPHINE METABOLITE, BISMORPHINE*

Received for publication, July 26, 2001, and in revised form, August 8, 2001Published, 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 andGraduate School of Sciences, Kyushu University, Fukuoka, 812-8581, Japan

We identified a novel metabolic system of morphine inthe opium poppy (Papaver somniferum L.). In responseto stress, morphine is quickly metabolized to bismor-phine consisting of two morphine units, followed by ac-cumulation in the cell wall. This bismorphine binds pre-dominantly to pectins, which possess high galacturonicacid residue contents, through ionical bonds. Our newlydeveloped method using artificial polysaccharides dem-onstrated that bismorphine bridges are formed betweenthe two amino groups of bismorphine and the carboxylgroups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharidesto each other. The ability of bismorphine to cross-linkpectins is much higher than that of Ca2�, which also actsas a cross-linker of these polysaccharides. Furthermore,we confirmed that cross-linking of pectins throughbismorphine bridges leads to resistance against hydrol-ysis by pectinases. These results indicated that produc-tion of bismorphine is a defense response of the opiumpoppy. Bismorphine formation is catalyzed by anionicperoxidase that pre-exists in the capsules and leaves ofopium poppies. The constitutive presence of morphine,together with bismorphine-forming peroxidase, enablesthe opium poppy to rapidly induce the defense system.

In response to mechanical damage, the immature capsules ofthe opium poppy (Papaver somniferum) and related species(Papaver setigerum, etc.) immediately secrete opium consistingof various secondary constituents such as morphine, codeine,papaverine, and noscapine. Among these, morphine has at-tracted a great deal of attention as one of the most medicinallyimportant analgesics and narcotics. Therefore, numerous stud-ies on morphine have been carried out since its first isolation in1804 by Serturner (1), but why the opium poppy produces thiscompound remains unknown.

Morphine is structurally classified into alkaloid, based onthe presence of nitrogen in its molecule. Many higher plantsincluding the opium poppy synthesize a variety of alkaloids,and like urea and uric acid in animals, these alkaloids haveoften been suggested to be produced as nitrogenous wasteproducts with little physiological importance for host plants.However, this hypothesis lacks precise experimental evidence

(2). In addition, the significant physiological roles of some al-kaloids in host plants have been demonstrated by characteriz-ing the biological properties of the alkaloids and their metab-olites; steroid alkaloids such as tomatine and solanidine areinvolved in protecting plants against herbivores and microbialpathogens (3), whereas the pyridine alkaloids, trigonelline (N-methylnicotinic acid) and N-arabinosylnicotinic acid act as pre-cursors of the vitamin nicotinic acid (4, 5). Therefore, it is notreasonable to regard all alkaloids including morphine as wasteproducts, although in contrast to these steroidal and pyridinealkaloids, little information is available concerning the physi-ological function of morphine in opium poppy.

Important information on the physiological roles of severalplant secondary constituents as well as the above pyridinealkaloids can be obtained by investigating the properties oftheir metabolites. For example, the main steroid saponins ofoat (Avena sativa), avenacosides A and B, have been shown tobe metabolized by endogenous �-glucosidase into 26-desglu-coavenacosides A and B, respectively, which function as anti-bacterial substances against pathogens (6, 7). Furthermore, werecently investigated the metabolic pathway of flavone glucu-ronide in the skullcap plant (Scutellaria baicalensis Georgi),and the metabolite (baicalein) of baicalein 7-O-�-D-glucuronidehas been shown to play an important role in detoxification ofthe large amount of H2O2 produced by the oxidative burst(8–11). These results indicated that precise understanding ofmorphine metabolism may provide the useful evidence fromwhich its physiological importance in the opium poppy is in-ferred. Recent studies have almost completely elucidated thebiosynthetic mechanism of morphine (12), but metabolism ofmorphine in the opium poppy is largely unknown.

Therefore, to determine the physiological importance of mor-phine, we investigated its metabolism in the opium poppy. Ourresults indicated that in response to mechanical damage, mor-phine immediately undergoes oxidation by bismorphine-formingperoxidase (BFP)1 and is metabolized into the dimer of morphine,bismorphine. Biochemical characterization of bismorphine dem-onstrated that the two amino groups of this alkaloid ionicallybind to the carboxyl groups of the galacturonic acid residues ofcell wall polysaccharides pectins, resulting in cross-linkingof pectins to each other. Furthermore, we confirmed that bindingof bismorphine to pectins significantly contributes to their resist-ance to hydrolysis by pectinase. We report here the metabolism ofmorphine and its novel physiological role in the opium poppy.* 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 toindicate 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.

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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 41, Issue of October 12, pp. 38179–38184, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 38179

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from

EXPERIMENTAL PROCEDURES

Plant Materials—Opium poppies (P. somniferum L.) were grown inthe herbal garden of the Graduate School of Pharmaceutical Sciences ofKyushu University. Opium poppies at 10–15 days after flowering wereused in this study. Wounding of the capsules was carried out as follows.The immature capsules (fresh weight, 15–20 g) were scratched using ablade (15 scratches/capsule; length of scratch, 2.0 cm; interval of eachscratch, 2.0 mm). The opium poppies of which the capsules had beenscratched were incubated for 24 h at 25 °C in a greenhouse. Thescratched regions were cut off the capsules and used as the woundedcapsules. We confirmed that reproducible results were obtained bythese procedures.

Isolation and Structural Characterization of Bismorphine—Crudecell walls prepared from the wounded capsules (200 g) were sonicated in20 mM HCl (100 ml) for 10 min. Insoluble materials were removed bycentrifugation at 20,000 � g for 5 min, and the HCl extracts wereneutralized with 0.1 M NaOH. After concentration under vacuum, theresidue was washed with 1% (v/v) ammonia water (50 ml) and thenapplied to high performance liquid chromatography (HPLC) to affordbismorphine (2.5 mg). The structure of bismorphine was determined byobtaining its 1H and 13C NMR (Varian) spectra. NMR data were as-signed 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,H-9,9�), 3.28 (2H,H-14,14�), 4.10 (2H,H-6,6�), 4.71 (1H,H-5,5�), 5.26 (2H,H-7,7�), 5.57 (2H,H-8,8), and 6.30 (2H,H-2,2�); 13C NMR (dimethyl sulfox-ide-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 Walls—The immaturecapsules (100 g) from which seeds were removed were homogenizedwith distilled water (2,000 ml) and filtered using filter papers. Theresidue 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), andhexane (2,000 ml). After drying under vacuum, the residue was used asthe crude cell wall fraction. Fractionation of the crude cell walls wascarried out by a modification of the method of Srisuma et al. (13). Thecrude cell wall fraction (10 g) was heated twice in 0.5% (w/v) ammoniumoxalate (500 ml) at 85 °C to remove pectins and then washed with water(500 ml). After drying under vacuum, the residue was incubated twicein 1 M NaOH (500 ml) at room temperature for 18 h and washed withwater (2,000 ml) to afford the lignocellulose fraction. This fraction wasincubated in 72% (v/v) H2SO4 at 4 °C for 36 h to give the lignin fraction.

HPLC Analysis of Bismorphine Bound to Cell Walls—The crude cellwall fraction (50–200 mg) of opium poppies was homogenized with 20mM HCl (20–80 ml), and the homogenate was centrifuged at 20,000 �g for 5 min. The amount of bismorphine in the supernatant was meas-ured using an HPLC system equipped with a 0.46 � 15-cm column ofCosmosil 5C18 AR-II (Nacalai, Kyoto). Bismorphine was eluted with60% (for quantitative analysis) or 50% (v/v) aqueous acetonitrile (foridentification and isolation of bismorphine) containing 5 mM sodiumdi-2-ethylhexyl sulfosuccinate at a flow rate of 1 ml/min (14). The pH ofthe mobile phase was adjusted to 3.3 using acetic acid. The eluate wasmonitored by absorption at 280 nm. The amount of bismorphine wascalculated from the standard curve obtained with the authentic sample.

In Vitro Binding of Bismorphine—Assay 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 wereremoved by centrifugation at 20,000 � g for 5 min, and the amount ofbismorphine in the supernatant was quantified by HPLC. The amountof bismorphine bound to the cell walls was calculated by subtracting theamount of bismorphine in the assay mixture incubated without the cellwalls from that in the supernatant. We confirmed that 668 �g ofbismorphine bound to 1 g of the crude cell wall fraction under theseconditions and that bound bismorphine was completely solubilized with20 mM HCl.

Binding of Pectins to Uronic Acid-conjugated Sepharose 6B—Uronicacid-conjugated Sepharose 6B was prepared as follows. Epoxy-activatedSepharose 6B (Amersham Pharmacia Biotech; dry weight, 3 g) preswol-len with water was suspended in 0.2 N NaOH (20 ml) solution contain-ing galacturonic acid or glucuronic acid (each 400 mg). After incubationat room temperature for 24 h, the gel was washed with 1 M NaCl (100ml), and then water (100 ml) was used as uronic acid-conjugated Sepha-rose 6B. A 60 �M bismorphine solution (300 ml) was loaded onto thecolumn (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 boundpolysaccharides were eluted with 20 mM HCl (20 ml), and the amountsof polysaccharides in the HCl eluate were quantified by phenol/sulfuricacid analysis.

Pectinase Treatment of Crude Cell Wall—The 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 bismor-phine was bound to the cell walls. Pectinase treatment of the crude cellsample was carried out by a modification of the method of Cervone et al.(15). Aspergillus pectinase (Sigma; 1 unit) was added to these samplesand incubated at 30 °C for 5 min. Ammonium oxalate (100 mg) wasadded to the enzymatic reaction mixture and then heated at 85 °C for1 h. After centrifugation at 20,000 � g for 5 min, the reducing groups inthe supernatant were quantified using dinitrosalicylic acid reagent. Todetermine the amounts of pectin fragments released from the cell walls,the above enzymatic reaction mixture was centrifuged at 20,000 � g for5 min, and the pectin fragments in the supernatant were quantified byphenol/sulfuric acid analysis.

Extraction and Purification of BFP—All procedures were carried outat 4 °C, unless otherwise indicated. The immature capsules of opiumpoppies (30 g) were homogenized in 100 mM phosphate buffer (pH 7.0,100 ml) containing 3 mM mercaptoethanol and then filtered throughNylon filters. The filtrate was centrifuged at 100,000 � g for 15 min,and the supernatant was fractionated by the addition of ammoniumsulfate. Proteins precipitating at 65% saturation were collected bycentrifugation at 20,000 � g for 30 min and then dialyzed overnightagainst 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 columnwas washed with three column volumes of the same buffer, and thebound 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 werecollected, concentrated, and dialyzed against 10 mM sodium phosphatebuffer (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. PotentBFP activity was detected in fractions eluted with 10 mM phosphatebuffer at a flow rate of 1 ml/min. The most active fractions were directlyloaded onto a column containing morphine-conjugated Sepharose 6B(1.0 � 10.0 cm), which was prepared using 50 mg of morphine andepoxy-activated Sepharose 6B (Amersham Pharmacia Biotech; dryweight, 3 g) according to the manufacturer’s protocol. After washingwith 30 ml of 10 mM phosphate buffer (pH 7.0), a 100-ml linear gradientof 0–0.4 M NaCl was passed through the column at a flow rate of 0.1ml/min. The fractions containing BFP activity were pooled, concen-trated, and used for the kinetic studies.

Assay for BFP—The assay mixtures consisted of 5 mM morphine, 5mM H2O2, 50 mM Tris-HCl (pH 9.0) and enzyme solution (100 �l) in afinal 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 Bismorphine—A solution (100 ml) con-taining 143 mg of morphine, 50 mM Tris-HCl, and 5 mM H2O2 wasincubated at 30 °C for 12 h with crude enzyme solution prepared from30 g of the immature capsules. The precipitates containing bismorphinewere collected by centrifugation at 20,000 � g for 10 min and washedtwice with distilled water (50 ml) and then with EtOH (50 ml). Thewashed samples were dissolved in dimethyl sulfoxide (5 ml), and insol-uble materials were removed by centrifugation at 20,000 � g for 10 min.Water (30 ml) was added to the supernatant, and the resulting precip-itates were collected by similar centrifugation to afford bismorphine(62 mg).

RESULTS

Identification of Morphine Metabolites in the Opium Pop-py—To determine the metabolic pathway of morphine, we firstattempted to identify morphine metabolites in the opium poppyby HPLC analysis and by feeding with 3H-labeled morphine.However, no morphine metabolites were detected in the intactimmature capsules of opium poppies using these methods. Incontrast, we found that an unknown alkaloid accumulated atthe site of damage in the wounded capsules (Fig. 1A). Further-more, incorporation of 3H-labeled morphine into this unknowncompound was observed by feeding experiments (data notshown), confirming that this was a metabolite of morphine.

This metabolite was purified from the damaged capsules of

Morphine Metabolism in the Opium Poppy38180

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from

opium poppies by preparative HPLC, and its mass spectrum andNMR spectra were obtained. The fast atom bombardment massspectrum showed an [M�H]� ion peak at m/z 569, indicating thedimeric nature of the metabolite. Furthermore, the 1H NMR and13C NMR spectra, which were almost identical to those of mor-phine except for the aromatic signals, indicated that this dimericcompound possessed a symmetrical structure. Based on analysesof the two-dimensional NMR spectra, the metabolite was finallydetermined to be dimeric morphine (called bismorphine in thisstudy), in which two morphine units are oxidatively coupled toeach other through a biphenyl bond (Fig. 1B). Bismorphine is anew alkaloid with a dimeric structure.

Bismorphine Level after Wounding—Changes in the amountof bismorphine after wounding of the immature capsules areassessed by HPLC. As shown in Fig. 2, bismorphine was notdetected in the intact capsules, and its production was inducedimmediately after wounding. The amount of bismorphine in-creased rapidly until 2 h after wounding, and thereafter itsproduction rate became relatively slow. The capsules at 24 hafter wounding produced �600 �g/g cell walls of bismorphine(Fig. 2). Furthermore, the accumulation (450 �g/g cell walls) ofbismorphine was also confirmed in the dead, brown tissue ofthe immature capsules infected by pathogens. Taken togetherwith the absence of bismorphine in the intact capsules, theseresults suggested that production of bismorphine is inducedrapidly by various forms of stress.

Localization of Bismorphine—The localization of bismor-phine accumulating in the wounded capsules was deduced fromits solubility in various solvents. Solvents such as water, eth-anol, and acetone were less effective for bismorphine extractionfrom the damaged capsules, whereas bismorphine was readilysolubilized using aqueous solvents containing HCl, CaCl2,NaCl, and EDTA (Fig. 3A), which are often used for extractionof components ionically associated with cell walls (16, 17).Furthermore, more than 90% of the bismorphine produced bythe wounded capsules was recovered in the crude cell wallfraction (Fig. 3B). These results indicated that most of thebismorphine induced by wounding was ionically bound tothe cell walls. As shown in Fig. 3A, NaCl or EDTA acceleratedthe extraction of bismorphine in a concentration-dependentmanner, although bismorphine exhibited low solubility in 100mM HCl or CaCl2 as compared with respective 20 mM solutions.Acidic conditions and divalent ions significantly change thephysical properties of cell wall polysaccharide pectins, result-ing in the solidification of pectins (18, 19). We hypothesizedthat such changes in the pectin moiety caused the inhibition ofbismorphine extraction.

We also investigated to which cell wall components bismor-phine binds. The cell walls from the wounded capsules werefractionated into three fractions (pectins, hemicellulose, andlignocellulose), and the bismorphine in each fraction wasquantified. However, it was impossible to precisely quantifybismorphine, because during fractionation most bismorphinewas solubilized from the cell wall. Therefore, the localizationof bismorphine was determined by in vitro binding assay.Bismorphine was incubated with cell wall samples fractionatedaccording to Srisuma’s procedure (13), and the amount ofbismorphine bound to each fraction was then analyzed. Asshown in Fig. 4A, bismorphine exhibited apparently lower af-finity for the cell wall fraction when pectins were removed, ascompared with the crude cell walls. Thus, it became evidentthat bismorphine is predominantly bound to pectins. In the cellwalls from which pectins and hemicellulose had been removed,a further decrease was observed in the amount of boundbismorphine, suggesting that a small amount of bismorphine isalso bound to the hemicellulose moiety. Little bismorphine wasbound to the lignin fraction. Pectins, which are structurallyclassified into three types (polygalacturonan, rhamnogalactu-ronan I, and rhamnogalacturonan II), possess high galactu-ronic acid residue contents, whereas xylans belonging to hemi-celluloses have low glucuronic acid residue contents (18, 19).Because morphinan alkaloids readily form alkaloid salts withorganic acids (2), we assumed that bismorphine is ionicallybound to the carboxyl groups of these uronic acid residues. Thiswas unequivocally confirmed by the observation that bismor-phine did not bind to the cell walls where carboxyl groups wereextensively esterified by diazomethane treatment (Fig. 4B).

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

FIG. 2. Amount of bismorphine after wounding. Opium poppiesof which the capsules were wounded were incubated at 30 °C for differ-ent times. The crude cell walls were prepared from the wounded cap-sules, homogenized in 20 mM HCl, and centrifuged at 20,000 � g for 5min. The amount of bismorphine in the supernatant was estimated asdescribed under “Experimental Procedures.” The data are the means offive replicate assays.

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

Morphine Metabolism in the Opium Poppy 38181

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from

Cross-linking Ability of Bismorphine for Various Carbohy-drates—Considering the dibasic nature of bismorphine, it islikely that the two amino groups simultaneously form ionicbonds with carboxyl groups of pectins, leading to cross-linkingof pectins to each other through bismorphine bridges (Fig. 5A).To confirm this hypothesis, we developed a chromatographicmethod using artificial polysaccharides as a matrix, preparedby coupling of galacturonic acid with Sepharose 6B throughether linkages (Fig. 5B). Bismorphine was readily absorbed togalacturonic acid-conjugated Sepharose 6B, whereas HCl solu-tion or NaCl solution effectively solubilized bound bismor-phine, confirming that bismorphine is ionically bound to thegalacturonic acid residues of the matrix. If bismorphine canform bismorphine bridges between this matrix and polysaccha-rides, the polysaccharides should bind to the matrix as shownin Fig. 5B. Pectins were loaded onto the galacturonic acid-conjugated Sepharose 6B column pretreated with bismorphine,and the substances absorbed to the column were then elutedwith HCl solution. Quantitative analysis using phenol/sulfuricacid reagent confirmed that pectins bound to the bismorphine-treated matrix (Fig. 6A). Thus, it was evident that bismorphinepossesses the ability to cross-link pectins to each other throughbismorphine bridges (Fig. 5A). Small amounts of pectins boundto the matrix without any pretreatment (control), and we as-sumed that this may have been due to interaction betweenpectins and few charged groups in Sepharose. A similar exper-iment was carried out using the same matrix treated with themonobasic alkaloid morphine instead of bismorphine, but theamount of pectins bound to the matrix was almost identical tothat in the control sample (Fig. 6A), indicating that morphinelacks the ability to form cross-linkages.

Furthermore, glucuronic acid-conjugated Sepharose 6B wasalso prepared, and pectins were applied to this matrix pre-treated with bismorphine. Binding of pectins to the matrix wasobserved, although its amount was lower than the values ob-tained using galacturonic acid-conjugated Sepharose 6B (Fig.6B). These results were consistent with the above finding thatbismorphine was predominantly bound to the pectin fractionrather than the hemicellulose fraction with low glucuronic acidresidue contents. On the other hand, like starch, polysacchar-ides containing no uronic acid residues were not cross-linked touronic acid-conjugated Sepharose 6B, even if each matrix was

treated with bismorphine, indicating that cross-linking ofpolysaccharides through bismorphine bridges is significantlydependent on the contents of uronic acid.

Similarly to bismorphine, divalent ions such as Ca2� are alsoknown to bind to carboxyl groups of galacturonic acid residuesin pectins and cross-link these polysaccharides to each other.When the ability of Ca2� to cross-link pectins was comparedwith that of bismorphine using both uronic acid-conjugatedgels, we found that the amount of pectins cross-linked bybismorphine was much higher than that by Ca2� (Fig. 6).

Effects of Bismorphine on Hydrolysis of Pectins by Pecti-nases—Previously, two groups reported that xylans in several

FIG. 4. In vitro binding of bismorphine to various cell wallfractions. Bismorphine was incubated with ammonium oxalate-treated cell walls (AOCW), lignocellulose fraction (LGC), and ligninfraction (LG), each prepared from 1 g of the crude cell walls (A).CH2N2-treated cell walls were obtained by incubation of the crude cellwalls (1 g) at 4 °C for 4 days in CH2N2 ether solution (20 ml) (B). Theamount of bismorphine bound to these samples was compared with thatbound to crude cell walls (1 g, CW). The relative amount of 100% was668 �g.

FIG. 5. Possible model for interaction between bismorphineand polysaccharides. A, cross-linking of pectins (black) throughbismorphine bridges (red). B, interaction between pectins (black) andgalacturonic acid-conjugated Sepharose 6B (blue) pretreated withbismorphine (red).

FIG. 6. Amount of pectin bound to uronic acid-conjugatedSepharose 6B. Pectins were loaded onto galacturonic acid-conjugated(A) or glucuronic acid-conjugated Sepharose 6B (B) pretreated withbismorphine (12 �mol), morphine (11 �mol), or Ca2� (12 �mol). Controlexperiments were carried out using the same matrix without any pre-treatment. Pectins bound to the matrix were eluted with 20 mM HCland quantified by phenol/sulfuric acid analysis. The data are the meansof five replicate assays.

Morphine Metabolism in the Opium Poppy38182

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from

monocots are cross-linked through diferuloylester bridges andassumed that such cross-linking results in resistance to enzy-matic digestion (20, 21). Bismorphine is structurally differentfrom diferulic acid, but both compounds have a biphenyl bond.Therefore, we hypothesized that cross-linking of pectinsthrough bismorphine bridges may lead to resistance to diges-tion by pectin-hydrolyzing enzymes. To confirm this hypothe-sis, the effects of bismorphine on pectinase reaction were ex-amined using the crude cell wall fraction prepared from theintact capsules. The crude cell wall fraction treated withbismorphine was incubated in the presence of pectinase, andthe reducing groups, which were produced by enzymatic hy-drolysis, were quantified. The amount (23 nmol) of the reducinggroups from bismorphine-treated cell walls was lower thanthat (69 nmol) from the untreated cell walls. Furthermore, weconfirmed that bismorphine treatment also reduced theamounts (185 �g) of pectin fragments released by the enzy-matic reaction from the cell walls, as compared with that (335�g) from the untreated cell walls. Because these results wereobtained at a level of bismorphine (600 �g/g cell walls) that thewounded capsules can produce, we concluded that bismorphineaccumulating in the wounded capsules contributes to protec-tion of pectins from hydrolysis by pectinase.

Identification of Enzymes Involved in Bismorphine Forma-tion—Because production of bismorphine was regarded as anovel defense response of the opium poppy, we attempted todetermine its biosynthetic mechanism by identifying and char-acterizing the enzymes involved in this reaction. Morphine wasincubated under various conditions in the presence of crudeenzyme extracts prepared from the immature capsules ofopium poppies, and reaction products were then analyzed byHPLC. Bismorphine was produced only in the presence ofH2O2, indicating that bismorphine formation is catalyzed byperoxidase. This peroxidase (BFP) was also found in the leavesof opium poppies (0.7 microkatal/g leaves), but its activity waslower than that in the immature capsules (1.3 microkatal/gcapsules). In contrast, no activity was detected in the roots ofthis plant. We tested whether the BFP activity is induced bywounding of immature capsules, but the enzyme activity didnot increase by wounding the immature capsules (Fig. 7A).

Purification and Characterization of BFP—Purification ofBFP was carried out by a four-step procedure. Crude enzymeextracts prepared from the immature capsules were fraction-ated by ammonium sulfate saturation. The fraction precipi-

tated by 65% saturation was applied to a DE-cellulose (DE-52)column and then to a hydroxylapatite column. Finally thisperoxidase was purified to homogeneity by affinity columnchromatography over morphine-conjugated Sepharose 6B.

SDS-polyacrylamide gel electrophoresis of purified BFP dis-played a monomeric molecular mass of about 33 kDa (Fig. 7B),whereas the pI for this peroxidase was estimated to be 5.0 byisoelectric focusing. The enzyme activity of BFP was maximalat pH 9.0, with half-maximal activities at pH values around7.5. and 10.0. Therefore, standard assay conditions includedTris-HCl buffer (pH 9.0) containing 5 mM H2O2. Under theseconditions, BFP catalyzed the dimerization of morphine,whereas the other oligomers and polymers of morphine werenot detected in the assay solution. We also tested the substratespecificity of BFP using several morphinan alkaloids. In con-trast to morphine, its biosynthetic precursors thebaine andcodeine were not substrates of this peroxidase (Fig. 7C). Inaddition, 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 substratespecificity.

DISCUSSION

Despite numerous studies on morphine, nothing is knownabout its roles in the opium poppy. In the present study, weidentified the novel metabolic pathway by which morphine isoxidized into the dibasic alkaloid bismorphine in the opiumpoppy and established the physiological importance of thismetabolism. Various alkaloids have been isolated from opiumpoppies, but the morphine dimer has not been identified pre-viously. Because several morphine derivatives have beenshown to have strong affinity for opioid receptors and to displaypotent analgesic effects (25), we also investigated the bindingability of bismorphine to opioid receptors. However, its affinityfor receptor � was markedly lower than that of morphine.2

The physiological importance of morphine metabolism in theopium poppy was confirmed by investigating the properties ofbismorphine. Interestingly, bismorphine was bound readily tocell wall polysaccharides containing uronic acid residues suchas pectins and hemicellulose. In addition, our newly developedmethod using artificial polysaccharides demonstrated thatthese polysaccharides are cross-linked through bismorphinebridges and that bismorphine more effectively cross-links pec-tins than other polysaccharides including hemicellulose. Takentogether with the observation that bismorphine accumulated inthe site of damage in the capsules, these results suggested thatbismorphine may hold together pectin fragments produced bywounding. On the other hand, morphine, the precursor ofbismorphine, was also ionically bound to the uronic acid resi-dues of these artificial polysaccharides, whereas this alkaloiddid not cross-link pectins. Because the morphine molecule pos-sesses only one amino group, it is reasonable that this alkaloidlacks cross-linking ability.

The divalent ions such as Ca2� are known to cross-linkpectins to each other, resulting in formation of gels (18, 19).Based on the observation that treatment of plant tissues withCa2� 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 ofplant cells (26, 27). Surprisingly, our experiments using uronicacid-conjugated Sepharose 6B indicated that the cross-linkingability of bismorphine for uronic acid-containing polysacchar-ides is much higher than that of Ca2�. In addition, we con-firmed that pectins form gels in the presence of bismorphine

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

FIG. 7. Properties of BFP. A, BFP activity after wounding. Thewounded capsules were incubated at 30 °C for different times, and theperoxidase activity was measured. The data are the means of fivereplicate assays. B, SDS-polyacrylamide gel electrophoresis analysis ofbismorphine. The samples were resolved by electrophoresis on a 12.5%acrylamide gel, and the proteins were stained with Coomassie BrilliantBlue. Lane 1, molecular standards with the indicated molecular mass;lane 2, BFP. C, substrate specificity of BFP. Morphinan alkaloids (each5 mM) were incubated with BFP (0.1 �g) under standard assay condi-tions. The reaction mixture was analyzed by HPLC.

Morphine Metabolism in the Opium Poppy 38183

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from

(data not shown). These results suggested that bismorphinemay have physiological functions similar to those of Ca2�. It isof interest that the opium poppy synthesizes bismorphine withCa2�-like function by dimerization of the monobasic alkaloidmorphine.

Cross-linking of polysaccharides through other secondaryconstituents has been demonstrated in monocots. For example,diferulic acid in which two ferulic acid units are oxidativelycoupled to each other through a biphenyl bond has been shownto be bound to the cell wall polysaccharides xylans of Triticumaestivum and Lolium multiflorum via ester bonds, involvingboth of its carboxyl groups (20, 21). Such cross-linking is be-lieved to have significant effects on the ability to resist enzy-matic digestion of xylans, but to date no experimental evidencefor this notion has been reported. In the present study, weestablished that binding of bismorphine to pectins resulted inresistance against their degradation by pectinases. It is note-worthy that the protective effect of bismorphine for pectins wasfound at a bismorphine level that can be produced by thewounded capsules. From these results, we concluded that pro-duction of bismorphine is a defense response. Pectins are poly-saccharides that play important roles in cell adhesion, cell wallstiffening, and assembly of cell wall components, and degrada-tion of pectins is well known to cause serious damage in plants(26, 27). Therefore, the repair and stiffening system for pectinsis quite important.

The formation of bismorphine is catalyzed by endogenousanionic peroxidase, BFP. This enzyme activity was also foundin the leaves of the opium poppy, although at somewhat lowerlevels than that in the capsules. This distribution showed agood correlation with the morphine content; greater amounts ofmorphine are found in the immature capsules than in theleaves. Because bismorphine was also detected in woundedleaves of the opium poppy (data not shown), it is likely that thesame defense system also exists in the leaves of the opiumpoppy.

We found that the properties of BFP are different from thoseof other plant peroxidases. Plant phenol peroxidases usuallycatalyze various substrates including both natural and syn-thetic substrates; for example, peroxidases in tobacco andmung bean have the ability to oxidize several phenolics (22,23). In contrast, BFP catalyzed oxidation only of morphine, andnone of the other phenolic compounds tested were substratesfor this enzyme. Thus, BFP is quite a unique peroxidase. Al-though bismorphine has a biphenyl bond that is often found inlignans and lignins, judged from the inability to oxidize ferulicacid and caffeic acid, it is unlikely that BFP catalyzes biosyn-thesis of lignin, one of the cell wall components.

Bismorphine is not detected in the intact plant, and itsproduction is initiated within 1 h by stress. This response isfaster than transcription-dependent defense reactions such asphytoalexin accumulation (28), which are usually observedmore than 2 h after stress treatment. Hence, bismorphineproduction is one of the earliest defense reactions. Albersheimet al. (29) reported that in many cases, the first stage of theinfectious process of phytopathogens is the production of cellwall-degrading enzymes such as pectinases. Therefore, rapidproduction of bismorphine involved in the stiffening of pectinsmay play an important role in protecting the opium poppyagainst infection by pathogens at the early stages. In the pres-ent study, we confirmed the pre-existence of large amounts ofBFP as well as morphine in the immature capsules of theopium poppy, and such constitutive expression was consideredto enable the observed rapid responses. In addition to bothconstituents, H2O2 is also essential for bismorphine formation.

Because stress treatment of plant cells causes observed rapidproduction of large amounts of reactive oxygen species, a reac-tion known as the oxidative burst (30–32), H2O2 induced dur-ing the oxidative burst may be used for bismorphine formation.

In conclusion, the metabolism of morphine to bismorphine isregarded as a defense system of the opium poppy. Higherplants can immediately initiate production of various defense-related secondary constituents to protect themselves from mi-crobial phytopathogens. Most act as antimicrobial substances,and several constituents contribute to strengthening of thelignin moiety in cell walls. Bismorphine is the first alkaloidinvolved in the repair and strengthening of pectins to be iden-tified to date. Several Papaver species produce morphinan al-kaloids other than morphine. Among these, Papaver pseudo-orientale and Papaver lasiothrix produce the biosyntheticprecursor of morphine, salutaridine, as a major alkaloid. Inboth plants, Sariyar et al. (33) identified salutadimerine, inwhich two morphine units are oxidatively coupled to each otherthrough a biphenylether bond. Because cross-linking of pectinsthrough bismorphine bridges is based on the ionic interactionbetween the two amino groups of bismorphine and the uronicacid residues of polysaccharides, salutadimerine may displaysimilar properties for pectins. In addition, the occurrence ofmany dibasic alkaloids in the plant kingdom suggested thatsimilar defense systems may be found in other plants.

Acknowledgments—We thank Yoshitsugu Tanaka and Kyoko Soedafor NMR measurements of morphine and bismorphine.

REFERENCES

1. Serturner, F. W. A. F. (1806) J. Pharm. Arzte Apotheker Chem. 14, 47–932. Robinson, T. (1981) Molecular Biology, Biochemistry and Biophysics: The

Biochemistry of Alkaloids, 2nd Ed., Springer-Verlag, Berlin, Germany3. Robinson, T. (1979) in Habivores: Their Interaction with Secondary Plant

Metabolism (Rosenthal, G., and Janzen, D. H., eds) pp. 413–448, AcademicPress, New York

4. Joshi, J. G., and Handler, P. (1962) J. Biol. Chem. 237, 3185–31885. Neuhann, H., Leienbach, K.-W., and Barz, W. (1979) Phytochemistry 18, 61–646. Tschesche, R., and Lauren, P. (1971) Chemische Berichtung 104, 3549–35557. Tschesche, R., and Wiemann, W. (1977) Chemische Berichtung 110,

2416–24238. Morimoto, S., Harioka, T., and Shoyama, Y. (1995) Planta 195, 535–5409. Morimoto, S., Tateishi, N., Matsuda, T., Tanaka, H., Taura, F., Furuya, N.,

Matsuyama, N., and Shoyama, Y. (1998) J. Biol. Chem. 273, 12606–1261110. Morimoto, S., Tateishi, N., Inuyama, M., Taura, F., Tanaka, H., and Shoyama,

Y. (1999) J. Biol. Chem. 274, 26192–2619811. Sasaki, K., Taura, F., Shoyama, S., and Morimoto, S. (2000) J. Biol. Chem. 274,

27466–2747212. Kutchan, T. M. (1998) in The Alkaloid (Cordell, G., ed) Vol. 50, pp. 257–316,

Academic Press, New York13. Srisuma, N., Ruengsakulrach, S., Uebersax, M. A., Bennink, M. R., and

Hammerschmidt, R. (1991) J. Agric. Food. Chem. 39, 855–85814. Kubiak, E. J., and Munson, J. W. (1980) J. Pharmacol. Sci. 69, 152–15615. Cervone, F., Scala, A., Foresti, M., Cacacr, M. G., and Noviello, C. (1977)

Biochim. Biopys. Acta 482, 379–38516. Ridge, I., and Osborne, D. J. (1971) Nat. New Biol. 229, 205–20817. Bradley, J. D., Kjellbom, P., and Lamb, C. J. (1992) Cell 70, 21–3018. McNeil, M., Darvill, A. G., Fry, S. C., and Albersheim, P. (1984) Annu. Rev.

Biochem. 53, 625–66319. Varner, J. E., and Lin, L. S. (1989) Cell 56, 231–23920. Markwalder, H. U., and Neukom, H. (1976) Phytochemistry 15, 836–83721. Hartley, R. D., and Jones, E. C. (1976) Phytochemistry 15, 1157–116022. Kim, S. S., Wender, S. H., and Smith, E. C. (1980) Phytochemistry 19, 165–16823. Chabanet, A., Catesson, A. M., and Goldberg, R. (1993) Phytochemistry 33,

759–76324. Vitali, A., Botta, B., Monache, G. D., Zappitelli, S., Ricciardi, P., Melino, S.,

Petruzzelli, R., and Giardina, B. (1998) Biochem. J. 331, 513–51925. Oguri, K., Yamada-Mori, I., Shigezane, J., Hirano, H., and Yoshimura, H.

(1984) Eur. J. Pharmacol. 102, 229–23526. Roberts, K. (1990) Curr. Opin. Cell Biol. 2, 920–92827. Levy, S., and Staehelin, L. A. (1992) Curr. Opin. Cell Biol. 4, 856–86228. Dixon, R. A., and Harrison, M. J. (1990) Adv. Genet. 28, 165–23429. Albersheim, P., Jones, T. M., and English, P. D. (1969) Annu. Rev. Pytopathol.

7, 171–19430. Doke, N. (1983) Physiol. Plant. Pathol. 23, 345–35731. Sutherland, M. W. (1991) Physiol. Mol. Plant Pathol. 39, 79–9332. Baker, C. J., and Orlandi, E. W. (1995) Annu. Rev. Phytopathol. 33, 299–32133. Sariyar, G., Freyer, A. J., Guinaudeau, H., and Shamma, M. (1990) J. Nat.

Prod. 53, 1383–1386

Morphine Metabolism in the Opium Poppy38184

by guest, on October 12, 2012

ww

w.jbc.org

Dow

nloaded from