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.

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 (2). In addition, the significant physiological roles of some al-
the opium poppy (Papaver somniferum L.). In response kaloids in host plants have been demonstrated by characteriz-
to stress, morphine is quickly metabolized to bismor- ing the biological properties of the alkaloids and their metab-
phine consisting of two morphine units, followed by ac- olites; steroid alkaloids such as tomatine and solanidine are
cumulation in the cell wall. This bismorphine binds pre- involved in protecting plants against herbivores and microbial
dominantly to pectins, which possess high galacturonic pathogens (3), whereas the pyridine alkaloids, trigonelline (N-
acid residue contents, through ionical bonds. Our newly methylnicotinic acid) and N-arabinosylnicotinic acid act as pre-
developed method using artificial polysaccharides dem- cursors of the vitamin nicotinic acid (4, 5). Therefore, it is not
onstrated that bismorphine bridges are formed between reasonable to regard all alkaloids including morphine as waste
the two amino groups of bismorphine and the carboxyl
products, although in contrast to these steroidal and pyridine
groups of galacturonic acid residues, resulting in cross-
alkaloids, little information is available concerning the physi-
linking of galacturonic acid-containing polysaccharides
ological function of morphine in opium poppy.
to each other. The ability of bismorphine to cross-link
pectins is much higher than that of Ca2ⴙ, which also acts Important information on the physiological roles of several
as a cross-linker of these polysaccharides. Furthermore, plant secondary constituents as well as the above pyridine
we confirmed that cross-linking of pectins through alkaloids can be obtained by investigating the properties of
bismorphine bridges leads to resistance against hydrol- their metabolites. For example, the main steroid saponins of
ysis by pectinases. These results indicated that produc- oat (Avena sativa), avenacosides A and B, have been shown to
tion of bismorphine is a defense response of the opium be metabolized by endogenous ␤-glucosidase into 26-desglu-
poppy. Bismorphine formation is catalyzed by anionic coavenacosides A and B, respectively, which function as anti-
peroxidase that pre-exists in the capsules and leaves of bacterial substances against pathogens (6, 7). Furthermore, we
opium poppies. The constitutive presence of morphine, recently investigated the metabolic pathway of flavone glucu-
together with bismorphine-forming peroxidase, enables ronide in the skullcap plant (Scutellaria baicalensis Georgi),
the opium poppy to rapidly induce the defense system. 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
In response to mechanical damage, the immature capsules of (8 –11). These results indicated that precise understanding of
the opium poppy (Papaver somniferum) and related species morphine metabolism may provide the useful evidence from
(Papaver setigerum, etc.) immediately secrete opium consisting which its physiological importance in the opium poppy is in-
of various secondary constituents such as morphine, codeine, ferred. Recent studies have almost completely elucidated the
papaverine, and noscapine. Among these, morphine has at- biosynthetic mechanism of morphine (12), but metabolism of
tracted a great deal of attention as one of the most medicinally morphine in the opium poppy is largely unknown.
important analgesics and narcotics. Therefore, numerous stud- Therefore, to determine the physiological importance of mor-
ies on morphine have been carried out since its first isolation in phine, we investigated its metabolism in the opium poppy. Our
1804 by Sertürner (1), but why the opium poppy produces this results indicated that in response to mechanical damage, mor-
compound remains unknown. phine immediately undergoes oxidation by bismorphine-forming
Morphine is structurally classified into alkaloid, based on peroxidase (BFP)1 and is metabolized into the dimer of morphine,
the presence of nitrogen in its molecule. Many higher plants bismorphine. Biochemical characterization of bismorphine dem-
including the opium poppy synthesize a variety of alkaloids, onstrated that the two amino groups of this alkaloid ionically
and like urea and uric acid in animals, these alkaloids have bind to the carboxyl groups of the galacturonic acid residues of
often been suggested to be produced as nitrogenous waste cell wall polysaccharides pectins, resulting in cross-linking
products with little physiological importance for host plants. of pectins to each other. Furthermore, we confirmed that binding
However, this hypothesis lacks precise experimental evidence of bismorphine to pectins significantly contributes to their resist-
ance to hydrolysis by pectinase. We report here the metabolism of
* The costs of publication of this article were defrayed in part by the morphine and its novel physiological role in the opium poppy.
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.
1
§ To whom correspondence should be addressed. Tel.: 81-92-642-6581; The abbreviations used are: BFP, bismorphine-forming peroxidase;
Fax: 81-92-642-6545; E-mail: [email protected]. HPLC, high performance liquid chromatography.

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

This is an Open Access article under the CC BY license.
38180 Morphine Metabolism in the Opium Poppy
EXPERIMENTAL PROCEDURES same column, the gel was washed with water (20 ml). The bound
Plant Materials—Opium poppies (P. somniferum L.) were grown in polysaccharides were eluted with 20 mM HCl (20 ml), and the amounts
the herbal garden of the Graduate School of Pharmaceutical Sciences of of polysaccharides in the HCl eluate were quantified by phenol/sulfuric
Kyushu University. Opium poppies at 10 –15 days after flowering were acid analysis.
used in this study. Wounding of the capsules was carried out as follows. Pectinase Treatment of Crude Cell Wall—The crude cell wall fraction
The immature capsules (fresh weight, 15–20 g) were scratched using a (50 mg) was incubated at 30 °C for 1 h in 50 ml of 10 mM citrate buffer
blade (15 scratches/capsule; length of scratch, 2.0 cm; interval of each (pH 5.5) containing bismorphine (30 ␮g). We confirmed that all bismor-
scratch, 2.0 mm). The opium poppies of which the capsules had been phine was bound to the cell walls. Pectinase treatment of the crude cell
scratched were incubated for 24 h at 25 °C in a greenhouse. The 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
scratched regions were cut off the capsules and used as the wounded
and incubated at 30 °C for 5 min. Ammonium oxalate (100 mg) was
capsules. We confirmed that reproducible results were obtained by
added to the enzymatic reaction mixture and then heated at 85 °C for
these procedures.
1 h. After centrifugation at 20,000 ⫻ g for 5 min, the reducing groups in
Isolation and Structural Characterization of Bismorphine—Crude
the supernatant were quantified using dinitrosalicylic acid reagent. To
cell walls prepared from the wounded capsules (200 g) were sonicated in
determine the amounts of pectin fragments released from the cell walls,
20 mM HCl (100 ml) for 10 min. Insoluble materials were removed by
the above enzymatic reaction mixture was centrifuged at 20,000 ⫻ g for
centrifugation at 20,000 ⫻ g for 5 min, and the HCl extracts were
5 min, and the pectin fragments in the supernatant were quantified by
neutralized with 0.1 M NaOH. After concentration under vacuum, the
phenol/sulfuric acid analysis.
residue was washed with 1% (v/v) ammonia water (50 ml) and then
Extraction and Purification of BFP—All procedures were carried out
applied to high performance liquid chromatography (HPLC) to afford
at 4 °C, unless otherwise indicated. The immature capsules of opium
bismorphine (2.5 mg). The structure of bismorphine was determined by
poppies (30 g) were homogenized in 100 mM phosphate buffer (pH 7.0,
obtaining its 1H and 13C NMR (Varian) spectra. NMR data were as-
100 ml) containing 3 mM mercaptoethanol and then filtered through
signed as follows: 1H NMR (diemethylsulfoxide-d6, 500 MHz) ppm; 1.68
Nylon filters. The filtrate was centrifuged at 100,000 ⫻ g for 15 min,
(2H,H-15,15⬘), 1.99 (2H,H-15,15⬘), 2.09 (2H,H-9,9⬘), 2.28 (2H,H-16,16⬘),
and the supernatant was fractionated by the addition of ammonium
2.38 (6H,N-Me), 2.48 (2H,H-16,16⬘), 2.56 (2H,H-13,13⬘), 2.88 (2H,H-
sulfate. Proteins precipitating at 65% saturation were collected by
9,9⬘), 3.28 (2H,H-14,14⬘), 4.10 (2H,H-6,6⬘), 4.71 (1H,H-5,5⬘), 5.26 (2H,H-
centrifugation at 20,000 ⫻ g for 30 min and then dialyzed overnight
7,7⬘), 5.57 (2H,H-8,8), and 6.30 (2H,H-2,2⬘); 13C NMR (dimethyl sulfox- against 10 mM phosphate buffer (3000 ml, pH 7.0). The dialyzed sample
ide-d6, 100 MHz) ppm; 20.0 (C-9,9⬘), 35.3 (C-15,15⬘), 40.6 (C-13,13⬘), (crude enzyme solution) was applied to a DEAE-cellulose column (1.5 ⫻
42.8 (C-12,12⬘, Me), 46.0 (C-16,16⬘), 58.1 (C-14,14⬘), 66.3 (C-6,6⬘), 91.7 10 cm) equilibrated with 10 mM phosphate buffer (pH 7.0). The column
(C-5,5⬘), 120.9 (C-2,2⬘), 124.9 (C-10,10⬘), 128.1 (C-11,11⬘), 128.3 (C-8,8⬘), was washed with three column volumes of the same buffer, and the
129.5 (C-1,1⬘), 133.4 (C-7,7⬘), 135.6 (C-3,3⬘), and 147.1 (C-4,4⬘). bound proteins were eluted with a 600-ml linear gradient of NaCl
Preparation and Fractionation of Crude Cell Walls—The immature (0 – 0.4 m) at a flow rate of 1 ml/min. Fractions containing BFP were
capsules (100 g) from which seeds were removed were homogenized collected, concentrated, and dialyzed against 10 mM sodium phosphate
with distilled water (2,000 ml) and filtered using filter papers. The buffer (pH 7.0). The dialysate was applied to a hydroxylapatite column
residue was consecutively washed with distilled water (2,000 ml), 0.1% (1.0 ⫻ 10.0 cm). The column was washed with the same buffer (100 ml)
(w/v) Tween 20 (2,000 ml), EtOH (2,000 ml), acetone (2,000 ml), and and then with a 200-ml gradient of 10 –200 mM phosphate buffer. Potent
hexane (2,000 ml). After drying under vacuum, the residue was used as BFP activity was detected in fractions eluted with 10 mM phosphate
the crude cell wall fraction. Fractionation of the crude cell walls was buffer at a flow rate of 1 ml/min. The most active fractions were directly
carried out by a modification of the method of Srisuma et al. (13). The loaded onto a column containing morphine-conjugated Sepharose 6B
crude cell wall fraction (10 g) was heated twice in 0.5% (w/v) ammonium (1.0 ⫻ 10.0 cm), which was prepared using 50 mg of morphine and
oxalate (500 ml) at 85 °C to remove pectins and then washed with water epoxy-activated Sepharose 6B (Amersham Pharmacia Biotech; dry
(500 ml). After drying under vacuum, the residue was incubated twice weight, 3 g) according to the manufacturer’s protocol. After washing
in 1 M NaOH (500 ml) at room temperature for 18 h and washed with with 30 ml of 10 mM phosphate buffer (pH 7.0), a 100-ml linear gradient
water (2,000 ml) to afford the lignocellulose fraction. This fraction was of 0 – 0.4 M NaCl was passed through the column at a flow rate of 0.1
incubated in 72% (v/v) H2SO4 at 4 °C for 36 h to give the lignin fraction. ml/min. The fractions containing BFP activity were pooled, concen-
HPLC Analysis of Bismorphine Bound to Cell Walls—The crude cell trated, and used for the kinetic studies.
wall fraction (50 –200 mg) of opium poppies was homogenized with 20 Assay for BFP—The assay mixtures consisted of 5 mM morphine, 5
mM HCl (20 – 80 ml), and the homogenate was centrifuged at 20,000 ⫻ mM H2O2, 50 mM Tris-HCl (pH 9.0) and enzyme solution (100 ␮l) in a
g for 5 min. The amount of bismorphine in the supernatant was meas- final volume of 500 ␮l. The samples were incubated at 30 °C for 10 min,
ured using an HPLC system equipped with a 0.46 ⫻ 15-cm column of and the reaction was terminated with 500 ␮l of HPLC mobile phase.
Cosmosil 5C18 AR-II (Nacalai, Kyoto). Bismorphine was eluted with The amount of bismorphine liberated was quantified by HPLC.
60% (for quantitative analysis) or 50% (v/v) aqueous acetonitrile (for Large Scale Preparation of Bismorphine—A solution (100 ml) con-
identification and isolation of bismorphine) containing 5 mM sodium taining 143 mg of morphine, 50 mM Tris-HCl, and 5 mM H2O2 was
di-2-ethylhexyl sulfosuccinate at a flow rate of 1 ml/min (14). The pH of incubated at 30 °C for 12 h with crude enzyme solution prepared from
the mobile phase was adjusted to 3.3 using acetic acid. The eluate was 30 g of the immature capsules. The precipitates containing bismorphine
monitored by absorption at 280 nm. The amount of bismorphine was were collected by centrifugation at 20,000 ⫻ g for 10 min and washed
calculated from the standard curve obtained with the authentic sample. twice with distilled water (50 ml) and then with EtOH (50 ml). The
In Vitro Binding of Bismorphine—Assay mixtures consisting of 35 washed samples were dissolved in dimethyl sulfoxide (5 ml), and insol-
␮M bismorphine, crude cell wall fraction (5 mg), and 10 mM Tris-HCl uble materials were removed by centrifugation at 20,000 ⫻ g for 10 min.
(pH 7.5, 500 ␮l) were incubated at 30 °C for 1 h. The cell walls were Water (30 ml) was added to the supernatant, and the resulting precip-
removed by centrifugation at 20,000 ⫻ g for 5 min, and the amount of itates were collected by similar centrifugation to afford bismorphine
bismorphine in the supernatant was quantified by HPLC. The amount (62 mg).
of bismorphine bound to the cell walls was calculated by subtracting the
amount of bismorphine in the assay mixture incubated without the cell RESULTS
walls from that in the supernatant. We confirmed that 668 ␮g of Identification of Morphine Metabolites in the Opium Pop-
bismorphine bound to 1 g of the crude cell wall fraction under these
py—To determine the metabolic pathway of morphine, we first
conditions and that bound bismorphine was completely solubilized with
20 mM HCl. attempted to identify morphine metabolites in the opium poppy
Binding of Pectins to Uronic Acid-conjugated Sepharose 6B—Uronic by HPLC analysis and by feeding with 3H-labeled morphine.
acid-conjugated Sepharose 6B was prepared as follows. Epoxy-activated However, no morphine metabolites were detected in the intact
Sepharose 6B (Amersham Pharmacia Biotech; dry weight, 3 g) preswol- immature capsules of opium poppies using these methods. In
len with water was suspended in 0.2 N NaOH (20 ml) solution contain- contrast, we found that an unknown alkaloid accumulated at
ing galacturonic acid or glucuronic acid (each 400 mg). After incubation the site of damage in the wounded capsules (Fig. 1A). Further-
at room temperature for 24 h, the gel was washed with 1 M NaCl (100
more, incorporation of 3H-labeled morphine into this unknown
ml), and then water (100 ml) was used as uronic acid-conjugated Sepha-
rose 6B. A 60 ␮M bismorphine solution (300 ml) was loaded onto the compound was observed by feeding experiments (data not
column (1.0 ⫻ 5.0 cm) containing uronic acid-conjugated Sepharose 6B. shown), confirming that this was a metabolite of morphine.
After application of 0.2% (w/v) polysaccharide solution (5 ml) to the This metabolite was purified from the damaged capsules of
 Morphine Metabolism in the Opium Poppy 38181

FIG. 2. Amount of bismorphine after wounding. Opium poppies

of which the capsules were wounded were incubated at 30 °C for differ-
ent times. The crude cell walls were prepared from the wounded cap-
FIG. 1. Identification of the metabolite of morphine. A, HPLC
sules, homogenized in 20 mM HCl, and centrifuged at 20,000 ⫻ g for 5
analysis of the opium poppy capsules. The crude cell walls prepared
min. The amount of bismorphine in the supernatant was estimated as
from the intact capsules (left panel) or from the wounded capsules (right
described under “Experimental Procedures.” The data are the means of
panel) were homogenized with 20 mM HCl. The insoluble materials
five replicate assays.
were removed by centrifugation at 20,000 ⫻ g for 5 min, and the
supernatant was analyzed by HPLC. B, structure of bismorphine.

opium poppies by preparative HPLC, and its mass spectrum and

NMR spectra were obtained. The fast atom bombardment mass
spectrum showed an [M⫹H]⫹ 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 mor-
phine 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
FIG. 3. Extraction of bismorphine. A, effects of solvents on bismor-
study), in which two morphine units are oxidatively coupled to
phine extraction. Bismorphine was extracted from the crude cell walls
each other through a biphenyl bond (Fig. 1B). Bismorphine is a of the wounded capsules using various solvents and quantified by
new alkaloid with a dimeric structure. HPLC. The pH of EDTA solution was adjusted to 7.0 using NaOH. B,
Bismorphine Level after Wounding—Changes in the amount amounts of bismorphine in the wounded capsules (CP) and the crude
of bismorphine after wounding of the immature capsules are cell walls (CW). Bismorphine was extracted from the wounded capsules
(dry weight, 1.2 g) or cell walls prepared from the same amount of
assessed by HPLC. As shown in Fig. 2, bismorphine was not wounded capsules and quantified by HPLC. The data are the means of
detected in the intact capsules, and its production was induced five replicate assays.
immediately after wounding. The amount of bismorphine in-
creased rapidly until 2 h after wounding, and thereafter its We also investigated to which cell wall components bismor-
production rate became relatively slow. The capsules at 24 h phine binds. The cell walls from the wounded capsules were
after wounding produced ⬃600 ␮g/g cell walls of bismorphine fractionated into three fractions (pectins, hemicellulose, and
(Fig. 2). Furthermore, the accumulation (450 ␮g/g cell walls) of lignocellulose), and the bismorphine in each fraction was
bismorphine was also confirmed in the dead, brown tissue of quantified. However, it was impossible to precisely quantify
the immature capsules infected by pathogens. Taken together bismorphine, because during fractionation most bismorphine
with the absence of bismorphine in the intact capsules, these was solubilized from the cell wall. Therefore, the localization
results suggested that production of bismorphine is induced of bismorphine was determined by in vitro binding assay.
rapidly by various forms of stress. Bismorphine was incubated with cell wall samples fractionated
Localization of Bismorphine—The localization of bismor- according to Srisuma’s procedure (13), and the amount of
phine accumulating in the wounded capsules was deduced from bismorphine bound to each fraction was then analyzed. As
its solubility in various solvents. Solvents such as water, eth- shown in Fig. 4A, bismorphine exhibited apparently lower af-
anol, and acetone were less effective for bismorphine extraction finity for the cell wall fraction when pectins were removed, as
from the damaged capsules, whereas bismorphine was readily compared with the crude cell walls. Thus, it became evident
solubilized using aqueous solvents containing HCl, CaCl2, that bismorphine is predominantly bound to pectins. In the cell
NaCl, and EDTA (Fig. 3A), which are often used for extraction walls from which pectins and hemicellulose had been removed,
of components ionically associated with cell walls (16, 17). a further decrease was observed in the amount of bound
Furthermore, more than 90% of the bismorphine produced by bismorphine, suggesting that a small amount of bismorphine is
the wounded capsules was recovered in the crude cell wall also bound to the hemicellulose moiety. Little bismorphine was
fraction (Fig. 3B). These results indicated that most of the bound to the lignin fraction. Pectins, which are structurally
bismorphine induced by wounding was ionically bound to classified into three types (polygalacturonan, rhamnogalactu-
the cell walls. As shown in Fig. 3A, NaCl or EDTA accelerated ronan I, and rhamnogalacturonan II), possess high galactu-
the extraction of bismorphine in a concentration-dependent ronic acid residue contents, whereas xylans belonging to hemi-
manner, although bismorphine exhibited low solubility in 100 celluloses have low glucuronic acid residue contents (18, 19).
mM HCl or CaCl2 as compared with respective 20 mM solutions. Because morphinan alkaloids readily form alkaloid salts with
Acidic conditions and divalent ions significantly change the organic acids (2), we assumed that bismorphine is ionically
physical properties of cell wall polysaccharide pectins, result- bound to the carboxyl groups of these uronic acid residues. This
ing in the solidification of pectins (18, 19). We hypothesized was unequivocally confirmed by the observation that bismor-
that such changes in the pectin moiety caused the inhibition of phine did not bind to the cell walls where carboxyl groups were
bismorphine extraction. extensively esterified by diazomethane treatment (Fig. 4B).
38182 Morphine Metabolism in the Opium Poppy

FIG. 4. In vitro binding of bismorphine to various cell wall

fractions. Bismorphine was incubated with ammonium oxalate-
treated 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.

Cross-linking Ability of Bismorphine for Various Carbohy-

drates—Considering 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).
FIG. 5. Possible model for interaction between bismorphine
To confirm this hypothesis, we developed a chromatographic and polysaccharides. A, cross-linking of pectins (black) through
method using artificial polysaccharides as a matrix, prepared bismorphine bridges (red). B, interaction between pectins (black) and
by coupling of galacturonic acid with Sepharose 6B through galacturonic acid-conjugated Sepharose 6B (blue) pretreated with
ether linkages (Fig. 5B). Bismorphine was readily absorbed to bismorphine (red).
galacturonic acid-conjugated Sepharose 6B, whereas HCl solu-
tion or NaCl solution effectively solubilized bound bismor-
phine, confirming that bismorphine is ionically bound to the
galacturonic acid residues of the matrix. If bismorphine can
form bismorphine bridges between this matrix and polysaccha-
rides, the polysaccharides should bind to the matrix as shown
in 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 eluted
with HCl solution. Quantitative analysis using phenol/sulfuric
acid reagent confirmed that pectins bound to the bismorphine-
treated 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 FIG. 6. Amount of pectin bound to uronic acid-conjugated
to the matrix without any pretreatment (control), and we as- Sepharose 6B. Pectins were loaded onto galacturonic acid-conjugated
sumed that this may have been due to interaction between (A) or glucuronic acid-conjugated Sepharose 6B (B) pretreated with
pectins and few charged groups in Sepharose. A similar exper- bismorphine (12 ␮mol), morphine (11 ␮mol), or Ca2⫹ (12 ␮mol). Control
experiments were carried out using the same matrix without any pre-
iment was carried out using the same matrix treated with the
treatment. Pectins bound to the matrix were eluted with 20 mM HCl
monobasic alkaloid morphine instead of bismorphine, but the and quantified by phenol/sulfuric acid analysis. The data are the means
amount of pectins bound to the matrix was almost identical to of five replicate assays.
that in the control sample (Fig. 6A), indicating that morphine
lacks the ability to form cross-linkages. treated with bismorphine, indicating that cross-linking of
Furthermore, glucuronic acid-conjugated Sepharose 6B was polysaccharides through bismorphine bridges is significantly
also prepared, and pectins were applied to this matrix pre- dependent on the contents of uronic acid.
treated with bismorphine. Binding of pectins to the matrix was Similarly to bismorphine, divalent ions such as Ca2⫹ are also
observed, although its amount was lower than the values ob- known to bind to carboxyl groups of galacturonic acid residues
tained using galacturonic acid-conjugated Sepharose 6B (Fig. in pectins and cross-link these polysaccharides to each other.
6B). These results were consistent with the above finding that When the ability of Ca2⫹ to cross-link pectins was compared
bismorphine was predominantly bound to the pectin fraction with that of bismorphine using both uronic acid-conjugated
rather than the hemicellulose fraction with low glucuronic acid gels, we found that the amount of pectins cross-linked by
residue contents. On the other hand, like starch, polysacchar- bismorphine was much higher than that by Ca2⫹ (Fig. 6).
ides containing no uronic acid residues were not cross-linked to Effects of Bismorphine on Hydrolysis of Pectins by Pecti-
uronic acid-conjugated Sepharose 6B, even if each matrix was nases—Previously, two groups reported that xylans in several
 Morphine Metabolism in the Opium Poppy 38183
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 dis-
played 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
FIG. 7. Properties of BFP. A, BFP activity after wounding. The not detected in the assay solution. We also tested the substrate
wounded capsules were incubated at 30 °C for different times, and the specificity of BFP using several morphinan alkaloids. In con-
peroxidase activity was measured. The data are the means of five trast to morphine, its biosynthetic precursors thebaine and
replicate assays. B, SDS-polyacrylamide gel electrophoresis analysis of
bismorphine. The samples were resolved by electrophoresis on a 12.5%
codeine were not substrates of this peroxidase (Fig. 7C). In
acrylamide gel, and the proteins were stained with Coomassie Brilliant addition, BFP did not catalyze oxidation of various phenolics
Blue. Lane 1, molecular standards with the indicated molecular mass; (guaiacol, catechol, pyrogallol, ferulic acid, and caffeic acid),
lane 2, BFP. C, substrate specificity of BFP. Morphinan alkaloids (each which are often used as substrates of phenol peroxidase (22–
5 mM) were incubated with BFP (0.1 ␮g) under standard assay condi-
24). Thus, we found that BFP displayed restricted substrate
tions. The reaction mixture was analyzed by HPLC.
specificity.
DISCUSSION
monocots are cross-linked through diferuloylester bridges and
assumed that such cross-linking results in resistance to enzy- Despite numerous studies on morphine, nothing is known
matic digestion (20, 21). Bismorphine is structurally different about its roles in the opium poppy. In the present study, we
from diferulic acid, but both compounds have a biphenyl bond. identified the novel metabolic pathway by which morphine is
Therefore, we hypothesized that cross-linking of pectins oxidized into the dibasic alkaloid bismorphine in the opium
through bismorphine bridges may lead to resistance to diges- poppy and established the physiological importance of this
tion by pectin-hydrolyzing enzymes. To confirm this hypothe- metabolism. Various alkaloids have been isolated from opium
sis, the effects of bismorphine on pectinase reaction were ex- poppies, but the morphine dimer has not been identified pre-
amined using the crude cell wall fraction prepared from the viously. Because several morphine derivatives have been
intact capsules. The crude cell wall fraction treated with shown to have strong affinity for opioid receptors and to display
bismorphine was incubated in the presence of pectinase, and potent analgesic effects (25), we also investigated the binding
the reducing groups, which were produced by enzymatic hy- ability of bismorphine to opioid receptors. However, its affinity
drolysis, were quantified. The amount (23 nmol) of the reducing for receptor ␮ was markedly lower than that of morphine.2
groups from bismorphine-treated cell walls was lower than The physiological importance of morphine metabolism in the
that (69 nmol) from the untreated cell walls. Furthermore, we opium poppy was confirmed by investigating the properties of
confirmed that bismorphine treatment also reduced the bismorphine. Interestingly, bismorphine was bound readily to
amounts (185 ␮g) of pectin fragments released by the enzy- cell wall polysaccharides containing uronic acid residues such
matic reaction from the cell walls, as compared with that (335 as pectins and hemicellulose. In addition, our newly developed
␮g) from the untreated cell walls. Because these results were method using artificial polysaccharides demonstrated that
obtained at a level of bismorphine (600 ␮g/g cell walls) that the these polysaccharides are cross-linked through bismorphine
wounded capsules can produce, we concluded that bismorphine bridges and that bismorphine more effectively cross-links pec-
accumulating in the wounded capsules contributes to protec- tins than other polysaccharides including hemicellulose. Taken
tion of pectins from hydrolysis by pectinase. together with the observation that bismorphine accumulated in
Identification of Enzymes Involved in Bismorphine Forma- the site of damage in the capsules, these results suggested that
tion—Because production of bismorphine was regarded as a bismorphine may hold together pectin fragments produced by
novel defense response of the opium poppy, we attempted to wounding. On the other hand, morphine, the precursor of
determine its biosynthetic mechanism by identifying and char- bismorphine, was also ionically bound to the uronic acid resi-
acterizing the enzymes involved in this reaction. Morphine was dues of these artificial polysaccharides, whereas this alkaloid
incubated under various conditions in the presence of crude did not cross-link pectins. Because the morphine molecule pos-
enzyme extracts prepared from the immature capsules of sesses only one amino group, it is reasonable that this alkaloid
opium poppies, and reaction products were then analyzed by lacks cross-linking ability.
HPLC. Bismorphine was produced only in the presence of The divalent ions such as Ca2⫹ are known to cross-link
H2O2, indicating that bismorphine formation is catalyzed by pectins to each other, resulting in formation of gels (18, 19).
peroxidase. This peroxidase (BFP) was also found in the leaves Based on the observation that treatment of plant tissues with
of opium poppies (0.7 microkatal/g leaves), but its activity was Ca2⫹ or chelating agents stiffens or softens them, respectively,
lower than that in the immature capsules (1.3 microkatal/g it has been proposed that cross-linking of pectins through Ca2⫹
capsules). In contrast, no activity was detected in the roots of contributes to strengthening of the plant wall and adhesion of
this plant. We tested whether the BFP activity is induced by plant cells (26, 27). Surprisingly, our experiments using uronic
wounding of immature capsules, but the enzyme activity did acid-conjugated Sepharose 6B indicated that the cross-linking
not increase by wounding the immature capsules (Fig. 7A). ability of bismorphine for uronic acid-containing polysacchar-
Purification and Characterization of BFP—Purification of ides is much higher than that of Ca2⫹. In addition, we con-
BFP was carried out by a four-step procedure. Crude enzyme firmed that pectins form gels in the presence of bismorphine
extracts prepared from the immature capsules were fraction-
2
ated by ammonium sulfate saturation. The fraction precipi- S. Morimoto, Y. Shoyama, and Y. Shimohigashi, unpublished data.
38184 Morphine Metabolism in the Opium Poppy
(data not shown). These results suggested that bismorphine Because stress treatment of plant cells causes observed rapid
may have physiological functions similar to those of Ca2⫹. It is production of large amounts of reactive oxygen species, a reac-
of interest that the opium poppy synthesizes bismorphine with tion known as the oxidative burst (30 –32), H2O2 induced dur-
Ca2⫹-like function by dimerization of the monobasic alkaloid ing the oxidative burst may be used for bismorphine formation.
morphine. In conclusion, the metabolism of morphine to bismorphine is
Cross-linking of polysaccharides through other secondary regarded as a defense system of the opium poppy. Higher
constituents has been demonstrated in monocots. For example, plants can immediately initiate production of various defense-
diferulic acid in which two ferulic acid units are oxidatively related secondary constituents to protect themselves from mi-
coupled to each other through a biphenyl bond has been shown crobial phytopathogens. Most act as antimicrobial substances,
to be bound to the cell wall polysaccharides xylans of Triticum and several constituents contribute to strengthening of the
aestivum and Lolium multiflorum via ester bonds, involving lignin moiety in cell walls. Bismorphine is the first alkaloid
both of its carboxyl groups (20, 21). Such cross-linking is be- involved in the repair and strengthening of pectins to be iden-
lieved to have significant effects on the ability to resist enzy- tified to date. Several Papaver species produce morphinan al-
matic digestion of xylans, but to date no experimental evidence kaloids other than morphine. Among these, Papaver pseudo-
for this notion has been reported. In the present study, we orientale and Papaver lasiothrix produce the biosynthetic
established that binding of bismorphine to pectins resulted in precursor of morphine, salutaridine, as a major alkaloid. In
resistance against their degradation by pectinases. It is note- both plants, Sariyar et al. (33) identified salutadimerine, in
worthy that the protective effect of bismorphine for pectins was which two morphine units are oxidatively coupled to each other
found at a bismorphine level that can be produced by the through a biphenylether bond. Because cross-linking of pectins
wounded capsules. From these results, we concluded that pro- through bismorphine bridges is based on the ionic interaction
duction of bismorphine is a defense response. Pectins are poly- between the two amino groups of bismorphine and the uronic
saccharides that play important roles in cell adhesion, cell wall acid residues of polysaccharides, salutadimerine may display
stiffening, and assembly of cell wall components, and degrada- similar properties for pectins. In addition, the occurrence of
tion of pectins is well known to cause serious damage in plants many dibasic alkaloids in the plant kingdom suggested that
(26, 27). Therefore, the repair and stiffening system for pectins similar defense systems may be found in other plants.
is quite important.
The formation of bismorphine is catalyzed by endogenous Acknowledgments—We thank Yoshitsugu Tanaka and Kyoko Soeda
for NMR measurements of morphine and bismorphine.
anionic peroxidase, BFP. This enzyme activity was also found
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