Purification and molecular analysis of a monoamine oxidase isolated from narcissus tazetta

Article abstract of DOI:10.1271/bbb.130291

Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS- 520 HQ column, was estimated to be 135 kDa. SDS- PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The Km value for n-propylamine was 5.9 × 10^-5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.

Full text of DOI:10.1271/bbb.130291

Biosci. Biotechnol. Biochem., 77 (8), 1728–1733, 2013
Purification and Molecular Analysis of a Monoamine Oxidase Isolated
from Narcissus tazetta
y
1;
Zhifeng CUI,1 Yongming ZHANG,2 Hironori INOUE,1 Syun YOGO,3 and Eiji HIRASAWA
1Department of Bio-Geosciences, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
2College of Life Science and Technology, Inner Mongolia Normal University,
Hohhot 010022, Inner Mongolia, China
3Kansaikako Co., Ltd., 9-9 Hiroshiba, Suita, Osaka 564-0052, Japan
Received April 8, 2013; Accepted May 27, 2013; Online Publication, August 7, 2013
[doi:10.1271/bbb.130291]
Semicarbazide-sensitive amine oxidase activity was
detected in Narcissus tazetta. The enzyme was purified to
homogeneity by the criterion of native polyacrylamide
gel electrophoresis (PAGE) with DEAE-Sephacel,
hydroxyapatite, and phenyl-Sepharose columns. The
molecular mass of the enzyme, determined using a GS-
520 HQ column, was estimated to be 135 kDa. SDS–
PAGE yielded two bands of, 75 kDa and 65 kDa. The
enzyme, which had catalytic activity for some aliphatic
and aromatic monoamines, belongs to a class of mono-
amine oxidases (MAOs). The Km value for n-propyl-
amine was 5:9 Â 10À5 M. A substrate analog, 2-bromoe-
thylamine, inhibited enzyme activity. Redox-cycling
staining detected a quinone in the MAO protein. By
inductively coupled plasma mass analysis, it was deter-
mined that there were 2.44 moles of copper atoms per
mole of the enzyme. Protein sequence analysis revealed
that there was no identity between two N-terminal
residues of the 75 kDa and 65 kDa proteins of narcissus
MAO.
the secondary amine of polyamines.2,3) Plant DAO is one
of the best known enzymes involved in amine catabo-
lism. Spermidine and spermine are specific substrates
for PAO. Additionally, MAO activity has been reported
in several plants.4–7) In the plant species reported,
Tsushida and Takeo used tea leaves to purify an AO that
catalyzes monoamines including some alkylamines,
ethanolamine, and benzylamine, although this last was
not classified as an MAO.7) The cofactor in the tea-
derived AO was not determined. Recently we reported a
semicarbazide-insensitive MAO from Avena sativa.8)
This FAD-containing enzyme had high substrate speci-
ficity for benzylamine and 2-phenethylamine, which are
oxidized to benzaldehyde and 2-phenylactaldehyde.
Many aromatic and flavor compounds containing fatty
acid derivatives, benzenoids, and isoprenoids are found
in many parts of the plant.9) The flavor volatiles 2-
phenylacetaldehyde and 2-phenylethanol are presuma-
bly produced in vivo from 2-phenethylamine by an AO,
dehydrogenase, or transaminase in tomatoes, although
the plant genes responsible for the synthesis of the
benzenoids have not yet been identified.10) AO activity
has yet to be detected in the fragrant flowers of plant
species examined in our laboratory, but it has been
reported that AO activity was detected in the flowers of
Narcissus tazetta (Y. Suzuki, personal communication).
In this study, we purified an AO from Narcissus tazetta
and examined its properties to determine whether it is
involved in the flower-scent-producing pathway.
Key words: 2-bromoethylamine; copper; monoamine
oxidase; Narcissus tazetta; quinoprotein
Amine oxidases (AOs) catalyze the oxidation of
amines, monoamines, diamines, and/or polyamines to
their corresponding aldehydes, hydrogen peroxide and
ammonia. The superfamily of AOs isolated from
mammals, higher plants, fungi, and bacteria are formed
by a group of enzymes involved in the cellular and
extracellular metabolism of amines. AOs have been
divided into two categories depending on the cofactor,
FAD or a copper-containing quinone. Enzymes contain-
ing FAD (EC.1.4.3.3) are further subdivided into mono-
amine oxidases (MAOs) and polyamine oxidases
(PAOs), depending on substrate specificity. In the
second category, AOs (EC 1.4.3.6) are characterized
by the presence of topaquinone (TPQ) or lysyl tyrosyl-
quinone,1) cupric Cu-containing quinone cofactors. The
copper AOs include diamine oxidase (DAO) and semi-
carbazide-sensitive, mammalian MAO. In plants, there
are two well-established types of AOs: copper-contain-
ing DAOs that act at the primary amine of many amine
compounds, and flavin-containing PAOs that function at
Materials and Methods
Plant materials. Flowering shoots of narcissus (Narcissus tazetta L.
var. chinensis) were perchased from Kansaikako (Osaka, Japan). The
shoots were used for enzyme purification.
Enzyme activity. MAO activity at the purification steps and in the
other experiments was assayed by spectrophotometric detection of the
H2O2 produced in a reaction mixture by the modification of the
quinoneimine dye method.11) The standard assay mixture contained
0.67 mM propylamine, 36 mM K-Pi buffer (pH 7.0), 0.33 mM N-ethyl-
N-(2-hydroxy-3-sulfopropyl)-3-methylamine (Dojin Laboratories,
Kumamoto, Japan), 0.33 mM 4-aminoantipyrine, and 3.3 purpurogallin
units of horseradish-peroxidase (Wako Pure Chemical, Osaka, Japan)
and enzyme solution in a total volume of 3 mL. The reaction was
initiated by the addition of the amine. The mixture was incubated at
y
To whom correspondence should be addressed. Fax: +81-6-6605-2522; E-mail: hirasawa@sci.osaka-cu.ac.jp
Abbreviations: AO, amine oxidase; DAO, diamine oxidase; ICP-MS, inductive coupled plasma-mass; MAO, monoamine oxidase; NTB, nitroblue
tetrazolium; PAGE, polyacrylamide gel electrophoresis; PAO, polyamine oxidase; TPQ, topaquinone

Monoamine Oxidase from Narcissus tazetta
1729
37 ꢀC, and enzyme activity was determined by the linear increase in the
absorbance at 555 nm for 5 min from initiation of the reaction.
The optimum pH of the assay mixture was determined at 37 ꢀC
using acetate-NaOH buffer (pH 4.0, 4.5, 5.0, and 5.5), phosphate-KOH
buffer (pH 6.0, 6.5, 7.0, and 7.5), and Tris–HCl buffer (pH 8.0, 8.5,
and 9.0). Enzyme activity was expressed in katal (kat, mol sꢁ1).
the molecular mass of the narcissus MAO. The column was calibrated
by the following protein standards: ovalbumin, ꢀ-glucosidase (yeast),
lactate dehydrogenase (yeast), glucose oxidase (Aspergillus nigar), and
ferritin type I (horse spleen). The purified enzyme and protein
standards were applied to a column equilibrated with 0.1 M K-Pi buffer
(pH 7.0) containing 0.3 M NaCl and then eluted with the same buffer.
Enzyme purification. All procedures were carried out at 4 ꢀC. Shoots
of the narcissus plant (100 g) were ground in a Waring blender (MX-
151S, Panasonic, Osaka) with 5 volumes of 100 mM K-Pi buffer
(pH 7.0). The homogenate was filtered through four layers of gauze,
and the filtrate was centrifuged at 10;000 ꢂ g for 25 min. The
supernatant (500 mL) was supplemented with solid (NH4)2SO4 to
65% saturation and incubated for 30 min. The precipitate obtained by
centrifugation of the supernatant at 10;000 ꢂ g for 25 min was
dissolved in 75 mL of 20 mM K-Pi buffer (pH 7.0), yielding buffer
A, and centrifuged again at 10;000 ꢂ g for 25 min. The supernatant
was applied to a Sephadex G-25 column (3:5 ꢂ 35 cm) equilibrated
with buffer A, and then eluted with the same buffer to remove
(NH4)2SO4. The eluted protein fraction (70 mL) was heated at 60 ꢀC
for 5 min in plastic tubes using a metal-block heater. The heated
solution was centrifuged at 12;000 ꢂ g for 10 min, and the supernatant
was applied to a DEAE-Sephacel (GE Healthcare, Little Chalfont, UK)
column (2:5 ꢂ 25 cm) equilibrated with buffer A and eluted with the
same buffer. The active fractions were collected and applied to a
hydroxyapatite (Nacalai Tesque, Kyoto, Japan) column (2:0 ꢂ 15 cm)
equilibrated with buffer A and washed with the same buffer. The bound
enzyme was eluted with 100 mL of a linear gradient of buffer A to
0.8 M K-Pi buffer (pH 7.0). The active fractions were pooled (20 mL)
and concentrated to 4 mL using an Ultracel-100K centrifugal filter
(Millipore Billerica, MA). The concentrated fraction was applied to a
phenyl-Sepharose (GE Healthcare) column (1:5 ꢂ 5 cm) equilibrated
with 0.8 M (NH4)2SO4 in buffer A and then washed with 0.4 M
(NH4)2SO4 in buffer A. The bound enzyme was eluted stepwise with
100 mM and then with 50 mM (NH4)2SO4 in buffer A. The active
fractions eluted were through buffer A Ultracel-100K centrifugal filter.
The flowthrough fraction from the Ultracel-100K filter was applied to
an Amicon Ultracel-30K centrifugal filter. The concentrated fraction
was washed with buffer A on the Ultracel-30K filter by centrifugation.
The purified MAO preparation was stored at ꢁ20 ꢀC in 25% (v/v)
glycerol in buffer A.
Substrate specificity and inhibitor efficacy. Monoamines, diamines,
and polyamines were used to determine the substrate specificity of the
enzyme using a standard assay mixture containing 0.66 mM of the
substrate. The inhibitors of human MAOs, pargyline and clorgyline,
and those of Cu-AOs, o-phenanthroline, 2,20-bipyridyl, 2-bromoethyl-
amine, and semicarbazide, were employed using the standard assay to
assess their effects on the catalytic function of the narcissus MAO. The
enzyme was pre-incubated with 1.0 or 0.1 mM of each inhibitor for
5 min before activity was measured. The Km values of some mono-
amines were determined from double reciprocal plots.
Cofactor identification. To determine copper and manganese
content by atomic absorption analysis, an X5 inductive coupled
plasma-mass spectrometer (ICP-MS, Bruker, Billerica, MA) was used.
The spectrophotometric nitroblue tetrazolium (NBT)/glycinate test
and quinone staining on PVDF membrane after SDS–PAGE were
conducted as described by Paz et al.15)
Protein sequence analysis. Amino acid sequencing of the narcissus
MAO was carried out by the Edman degradation method by the Protein
Sequence System (ABI Procise 491 HT, Praha, Czech) using an
Immobilon 0.45 mm PVDF membrane (Millipore, Bedford, MA). The
protein sample was separated by SDS–PAGE, and the protein bands
were transferred to the membrane by a semi-dry electroblotting
method. Two areas, corresponding to 65 kDa and 75 kDa, were excised
from the membrane and analyzed individually by the Protein Sequence
System.
Results and Discussion
Purification of the narcissus MAO
AO activity was detected in all organs, specifically the
leaves, stems, and flowers in the shoots (data not
shown). The enzyme was purified from whole shoots
by (NH4)2SO4 fractionation, heat treatment, DEAE-
Sephacel, hydroxyapatite, and phenyl-Sepharose column
chromatography. Table 1 summarizes the steps involved
in the purification procedures for the enzyme and the
purification results when the process begins with
approximately 100 g fresh weight of shoots. Minimal
enzymatic activity was detected in the supernatant after
the first centrifugation after the purification procedures.
We postulated that small molecular material in the
supernatant would disturb the enzyme assay system. In
fact, when the supernatant was dialyzed against 20 mM
K-Pi buffer (pH 7.0), activity was detectable. The
activity of the crude extract displayed in Table 1
represents the use of the dialyzed enzyme solution in
the assay method. The enzymatic activity eluted from
the G-25 column was up to 10 min at 60 ꢀC, and
approximately 67% of the protein within the desalted
fraction was denatured by the heat treatment. The
ensuing purification steps were accomplished using
DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose
columns. As Table 1 shows, 500 mL of homogenate,
which was purified to 20% yield from the crude ground
extract, contained 37.8 nkat of total MAO activity. The
specific activity of the purified MAO was
81.85 nkat mgꢁ1 protein, and a purification factor of
1,544-fold was obtained. The purified enzyme was
applied to a gel filtration column (GS-520 HQ), and this
The protein concentration of the pooled fraction from each
purification step was estimated by the micro-assay method of
Bradford12) with bovine serum albumin as the standard.
Polyacrylamide gel electrophoresis. Electrophoresis was performed
under native and denatured conditions in an electrophoresis unit.
Native polyacrylamide gel electrophoresis (PAGE) was carried out a
7.5% (w/v) gel without SDS by the method described by Davis.13)
Propylamine-active staining on the native PAGE was used with the
assay solution described above in ‘‘Materials and Methods’’ SDS–
PAGE was done using a 15% (w/v) gel by the method described by
Laemmli.14) Full-Range Rainbow Molecular Weight Markers (GE
Healthcare) were used as protein standards.
Determination of propionaldehyde. HPLC was performed to
measure the propionaldehyde produced in the reacted mixture. The
mixture contained 0.67 mM propylamine, 33 mM K-Pi buffer (pH 7.0),
and the purified MAO solution in a total volume of 3 mL. An aliquot
(100 mL) of the reaction mixture was added to 1.0 mL of 0.25 mM 4-
(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-
H; Tokyo Chemical Industry, Tokyo) in 70% acetonitrile and 0.05%
trifluoroacetic acid (TFA). After 2 h, the DBD-H solution was
centrifuged at 10;000 ꢂ g for 10 min, and the supernatant was injected
into an HPLC sampler. Reversephase HPLC on a system composed of
a pump (LC-10AD), a spectrofluorometer (RF-535, excitation 450 nm
and emission 565 nm, Shimadzu Co., Kyoto, Japan), and a column of
COSMOSIL C18-PAQ (4.6 mm i.d. 9.25 cm; Nakarai Tesque, Kyoto,
Japan) was used. The mobile phase was 70% acetonitrile and 0.05%
TFA at a flow rate of 1 mL minꢁ1. The column temperature was
maintained at 30 ꢀC.
Determination of molecular mass using a GS-520 column. GS-520
HQ column (Shodex Asahipak, 7:8 ꢂ 300 mm) was used to determine

1730
Z. CUI et al.
Table 1. Summary of the Purification of Monoamine Oxidase from Narcissus Shoots
Total
volume
(mL)
Total
protein
(mg)
Total
activity
(nkat)
Specific
activity
(nkat mgꢁ1 protein)
Purification
(fold)
Yield
(%)
Purification step
Crude extract
(NH4)2SO4 precipitate
Heat treatment
500
70
70
60
20
4
718
728
37.80
25.87
23.52
19.15
11.08
7.53
0.053
0.036
0.097
0.632
20.52
81.85
1
0.68
1.83
11.9
387
100
68
62
51
29
20
242
DEAE-Sephacel
Hydroxyapatite
Phenyl-Sepaharose
30.3
0.54
0.092
1544
A
B
Fig. 2. Determination of Molecular Mass of Narcissus Monoamine
Oxidase.
Gel filtration (GS-520 HQ, HPLC) column; 1, ovalbumin
(45 kDa); 2, ꢀ-glucosidase from yeast (52 kDa); 3, lactate dehydro-
genase from yeast (140 kDa); 4, glucose oxidase from Aspergillus
nigar (186 kDa); 5, ferritin type I from horse spleen (440 kDa).
narcissus monoamine oxidase (135 kDa). For other details, see
‘‘Materials and Methods.’’
Fig. 1. Polyacrylamide Gel Electrophoresis of Narcissus Monoamine
Oxidase.
A, SDS-free gel of narcissus monoamine oxidase. L, Coomassie-
stained SDS-free gel; R, N-propylamine-positive spot on SDS-free
gel. B, SDS-gel of narcissus monoamine oxidase. L, Marker; R,
Coomassie-stained SDS-gel. For other details, see ‘‘Materials and
Methods.’’
during purification. However, despite the addition of
protease inhibitors (0.5 mM phenylmethylsulfonyl fluo-
ride and 1 mM EDTA) to the buffers used in enzyme
purification, SDS–PAGE of MAO purified by the
method with inhibitors gave the same results as Fig. 1B
(data not shown).
resulted in a single peak as observed in the column-
elution profile (data not shown). The enzyme was
subjected to native PAGE analysis. The gel was divided
into two portions after electrophoresis. One portion was
stained with Coomassie Brilliant Blue G-250, and the
other was stained at 37 ꢀC in enzyme-active staining (see
‘‘Materials and Methods’’). A single band migrated to
the same site in both areas of the gel (Fig. 1A),
indicating that the enzyme was purified to homogeneity
and that the active-stained band is an MAO protein.
Determination of other enzymatic parameters
When the enzyme solution was pre-incubated for
10 min at temperatures ranging from 50 to 80 ꢀC to
determine heat stability, the purified enzyme retained its
activity at temperatures of 70 ꢀC, but lost activity
quickly after pre-incubation at temperatures of 75 ꢀC
(Fig. 3). These results indicate that purified MAO is
stable at up to 70 ꢀC, which differs from the heat
stability of the MAO in desalted solution after
(NH4)2SO4 fractionation. The difference in heat stability
as between MAO in the desalted fraction and purified
MAO might have been due to the proteases in the
fraction. For comparison, tea AO is stable at up to
60 ꢀC,7) not 70 ꢀC as for the narcissus MAO. Addition-
ally, the optimum pH of the narcissus MAO was 7.0
(Fig. 4), similar to that of other Cu-AOs.3)
Determination of molecular mass
SDS–PAGE of the phenyl-Sepharose column-purified
enzyme revealed two protein bands (molecular masses
of 75 kDa and 65 kDa) (Fig. 1B). Many studies indicate
that Cu-DAOs in plants are typically homodimers.16)
Tea AO is also a homodimer, with a molecular mass of
80 kDa per subunit.7) The product of GS-520 HQ
column chromatography had an apparent molecular
mass of 135 kDa (Fig. 2). The two bands visualized on
SDS–PAGE, which represented narcissus MAO, indi-
cated that the narcissus-derived enzyme is comprised of
two different subunits, indicating a heterodimer. There is
another possibility that a 75 kDa-homodimer of MAO
was partially cleaved by an endogenous protease(s)
Substrate specificity and inhibitor efficacy
The substrate specificity of the enzyme is summarized
in Table 2. The enzyme catalyzed the oxidation of
aliphatic and aromatic short-chain monoamines. N-

Monoamine Oxidase from Narcissus tazetta
1731
A
Fig. 3. Thermal Inactivation Curve for Narcissus Monoamine Oxi-
dase.
Purified monoamine oxidase solution ( ) or desalted solution
after ammonium sulfate precipitation ( ) was incubated at various
temperatures for 10 min and activity of each was measured. See
‘‘Materials and Methods.’’
B
Fig. 4. Effects of pH on Narcissus Monoamine Oxidase Activity.
Table 2. Substrate Specificities of Narcissus MAO
C
Substrate
Relative activity (%)
Km (M)
ꢁ (sꢁ1
)
n-Propylamine
n-Butylamine
Ethylamine
Benzylamine
n-Pentylamine
Ethanolamine
Methylamine
Phenethylamine
Hexylamine
Tryptamine
Serotonin
100
98
84
74
56
50
34
9
5:9 ꢂ 10ꢁ5
1:1 ꢂ 10ꢁ4
1:3 ꢂ 10ꢁ4
4:1 ꢂ 10ꢁ4
5:0 ꢂ 10ꢁ4
2:0 ꢂ 10ꢁ3
1:1 ꢂ 10ꢁ4
1:4 ꢂ 10ꢁ3
53.4
Fig. 5. Detection of Propionaldehyde as a DBD-H Derivative by
HPLC.
A, authentic propionaldehyde solution; B, reaction mixture with
n-propylamine as substrate; C, before treatment, 2-bromoethylamine
(0.1 mM) was added to the mixture. Arrows indicate peaks of
propionaldehyde-DBD-H. For details, see ‘‘Materials and Methods.’’
7
0
Table 3. Effects of Inhibitors and Manganese Chloride on Narcissus
MAO Activity
0
Dopamine
Tyramine
0
Inhibitor
Concentrations (mM)
Inhibition (%)
0
Histamine
Putrescine
0
Semicarbazide
0.01
0.001
0.01
0.001
0.0005
1
100
51
100
71
42
50
4
0
Cadaverine
Diaminodecane
Spermidine
Spermine
0
2-Bromoethylamine
0
0
0
2,20-Bipyridyl
o-Phenanthroline
Pargyline
1
1
2
Propylamine was found to be the best substrate for the
amines. Other short-aliphatic monoamines, ethanola-
mine and benzylamine, were also good substrates for the
enzyme. By HPLC, propionaldehyde was detected
stoichiometrically, and the mixture produced hydrogen
peroxide when propylamine was incubated with the
enzyme (Fig. 5). We concluded from these results that
the enzyme from Narcissus tazetta was an MAO. The Km
values for n-propylamine and benzylamine were 5:9 ꢂ
10ꢁ5 M and 4:1 ꢂ 10ꢁ4 M respectively. The enzyme did
not catalyze the oxidation of hydroxylated benzylamines,
Clorgyline
MnCl2
1
1
1
90
122
105
0.1
0.05
diamines, or polyamines. It was not inhibited by 1 mM
pargyline or clorgyline, both of which are potent
selective inhibitors of MAOs in mammals (Table 3).
Semicarbazide, an inhibitor of enzymes having a quinone
cofactor, inhibited the narcissus-derived enzyme. The

1732
Z. CUI et al.
75 kDa subunit
65 kDa subunit
1
1
NPNPDHESETLLP
ALPPRRAASVVQF
13
13
Current study
Current study
Barley predicted AO 132 ALPPRRAVAVVRF
144 Matumoto et al. 2011
Fig. 6. Comparison of Amino Acid Sequences of Two Subunits of Narcissus Monoamine Oxidase with the Homologous Sequence of a Predicted
Barley Amine Oxidase.
Two N-terminal subunits of the narcissus MAO were sequenced by the Edman degradation method. The NCBI GenBank accession number for
barley is BAJ85075. Amino acids of barley AO that are identical the 65 kDa subunit of the narcissus MAO are shaded in gray. For details, see
‘‘Materials and Methods.’’
enzyme was also inhibited by a metal-chelating reagent,
A
B
2,20-bipyridyl. In the inhibition experiments, diethyldi-
thiocarbamate and phenylhydrazine were not available
for use owing to their ability to inhibit peroxidase
activity in the enzyme assay system. These results for
substrate specificity and inhibitory effects indicate that
narcissus MAO is comparable to tea AO,7) and is similar
to semicarbazide-sensitive AOs in mammals. Although
the cofactor of the tea AO was not identified, it appears
to be a copper- and quinone-containing enzyme based
on the effects of inhibitors. The narcissus MAO was
irreversibly inhibited by 2-bromoethylamine with an
inhibition constant (Ki) of 0.65 mM. Medda et al.
indicated that 2-bromoethylamine (an irreversible inhib-
itor of lentil DAO) is a poor substrate and combines
directly with TPQ in the enzyme.17) This strong, specific
inhibitor may be useful as a probe in searching for
physiological roles of narcissus MAO, especially for
any involvement related to fragrant aldehydes in the
narcissus flower.
Fig. 7. NBT/Glycinate Staining of Quinoproteins on an SDS-
Polyacrylamide Gel Electrophoresis Electroblot.
A and B: lane 1, marker; lane 2, purified narcissus monoamine
oxidase; lane 3, bovine serum amine oxidase; lane 4, pig kidney
diamine oxidase. The electroblot was first stained with NBT/
glycinate (A) and then counterstained with Ponceau (B). For details,
see ‘‘Materials and Methods.’’
Cofactor identification
shows two bands were stained by NTB/glycinate in the
same 65 kDa and 75 kDa positions. These results
indicate that the 65 kDa and 75 kDa subunits were
quinoproteins. Based on the other copper AOs, the
quinone in the narcissus MAO may be TPQ.19) The
oxidized form of the AOs has a distinctive pink color at
about 470–490 nm due to the presence of TPQ. The
absorbance at 490 nm was 0.29 in lentil DAO solution
(6:9 ꢂ 10ꢁ5 M enzyme c)16) and 0.05 in tea MAO
solution (8:6 ꢂ 10ꢁ6 M).7) The spectrum of pink was
shifted to that of yellow at 346, 432, and 462 after the
addition of putrescine.16) No obvious spectrum at about
470–490 nm was detectable in the narcissus enzyme
solution (6:8 ꢂ 10ꢁ7 M). The amount of 0.092 mg of the
purified enzyme must have been too little for detection
of absorption spectrum. In the future, large amounts of
enzyme will be necessary to examine electric charge of
copper by electron paramagnetic resonance.
The copper and manganese contents of a purified
MAO from Narcissus tazetta were measured by an ICP-
MS. The concentrations of copper and manganese in the
purified enzyme were estimated to be 54.37 ppb (Cu)
and 2.61 ppb (Mn)/0.3419 mM of the protein. Then, the
narcissus MAO was found to be a copper-containing
protein having 2.45 atoms of copper per mole of protein.
AOs have been divided into two categories depending
on their cofactor, FAD or Cu plus quinone. The
narcissus MAO, the latter type of AO, appears to
contain two copper atoms in a narcissus MAO molecule.
In addition, the Mn content of the protein was calculated
to be 0.14 atom of manganese per mole of protein. In
general, Cu-AOs contain one copper atom per subunit.15)
Fenugreek Cu-AO contains Mn, and its contents are 0.2
moles per mole of native enzyme as determined by
atomic absorption spectrometry.18) When manganese
ions were added to the reaction mixture for MAO,
enzyme activity accelerated to 122% (Table 3), but the
catalytic importance of manganese ions is obscure.
The NBT/glycinate method was used as a specific
stain to detect quinoproteins. Narcissus MAO protein
after SDS–PAGE was electroblotted onto a PVDF
membrane. Figure 7, panel A displays staining of the
mammalian quinoprotein, the purified narcissus MAO
(lane 2), bovine serum AO (lane 3), and pig kidney AO
(lane 4). Panel B in Fig. 7 shows the electroblot seen in
panel A counterstained red for protein with Ponceau S.
The MAO consists of a heterodimer containing 75-kDa
and 65-kDa subunits (Fig. 1B). As panel B (lane 2)
Amino acid sequencing of narcissus MAO
The N-terminal amino acid sequence of the two
subunits of the narcissus MAO was determined by the
Edman degradation method. Two identified 13 residues
from each subunit of narcissus MAO were neither
identical to partial sequences of any protein reported
elsewhere nor similar to any amino acid sequence of
leguminous DAOs or gramineous PAOs. While the
N-terminal 13 residues of the 65 kDa subunit of the
MAO showed fair homology (77% identity) to a
predicted protein of AO from barley,20) the 75 kDa
subunit had no homology anywhere with the barley

Monoamine Oxidase from Narcissus tazetta
1733
AO (Fig. 6). A comparison of the quinone-containing
References
sequences with the gene-encoded sequences indicated
that the precursor of the cofactor was a specific Tyr
residue occurring in a highly conserved sequence: Asn-
Tyr(TPQ)-Asp/Glu-Tyr.21) Researchers should examine
hereafter the sequencing of the two monomers to
determine whether narcissus MAO has two different
monomers containing TPQ.
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lated concerning the biological importance of Cu-AOs:
to remove biologically active molecules such as primary
amines; to form corresponding aldehydes that in some
cases are known to affect cell differentiation, prolifer-
ation, and survival dramatically; and to produce hydro-
gen peroxide as a signal molecule rather than a harmful
side product on-site.22) In prokaryotes and fungi, AOs
allow for the use of primary amines as sole sources of
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amine as nitrogen source.23) In plants, enzymes contain-
ing DAOs are important to the synthesis of alkaloids,
cell-wall formation, and wound healing,24) but no
physiological role of plant MAOs has yet been clearly
defined. In a previous paper, a FAD-containing MAO
(EC.1.4.3.4) purified from oat seedlings was suggested
to have a catabolic role in the oxidation of phenethyl-
amine, resulting in the decarboxylation of phenylala-
nine, which results in the hydrolysis of storage proteins
in oat grain during germination.8) The narcissus MAO
has broad substrate specificity for short-chained aliphatic
and aromatic monoamines and exerts no activity on any
diamines or polyamines. MAO, a key enzyme in the rice
sl-mutant, oxidizes tryptamine, which is biosynthesized
from tryptophan by tryptophan decarboxylase.25) Trypt-
amine-induced leaf lesions were significantly suppressed
in the presence of semicarbazide, but narcissus MAO,
despite having similar inhibitory properties, did not
oxidize tryptamine (Table 2). Tea AO, similar to
narcissus AO, is thought to have a role in the catabolism
of ethylamine as a main component of the leaves,26) but
there is no report on monoamines in the narcissus
species. The MAO may also be involved in the
catabolism of short-chained aliphatic and aromatic
monoamines in the shoots. Many volatile compounds
containing some benzenoids have been isolated from a
fragrant flower of the genus Narcissus,27) but the
narcissus MAO does not appear to be involved in the
synthesis of the flavor benzenoids 2-phenylactaldehyde
and 2-phenylethanol, because it was a lower affinity
for phenethylamine (Table 2). We intend to conduct
further studies of endogenous substrates of MAOs in
plant species.
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¨
Acknowledgment
We are very grateful to Ms. Satomi Shimonaka and
Professor Harue Masuda for the metal analyses by
ICP-MS.