Purification and properties of a monoamine oxidase from

Narcissus tazetta

(スイセンにおけるモノアミン酸化酵素の精製と諸性質)

大阪市立大学 大学院理学研究科

生物地球系専攻

平成 25年度

Zhifeng Cui

崔 智峰

Purification and properties of a monoamine oxidase from

Narcissus tazetta

A Dissertation Submitted to

the Department of Biology and GeoSciences

Graduate School of Science

Osaka City University

In partial fulfilment for the Degree of

DOCTOR OF SCIENCE

by

Zhifeng Cui

Osaka 558-8585, Japan

June 2013

CONTENTS

Page No.

Chapter Ι. General introduction........................................................... 1

Chapter ΙI. Purification and molecular analysis of a monoamine oxidase

isolated from Narcissus tazetta .......................................... 17

Chapter ΙII. General discussion.............................................................. 37

Summary…………………………………………….................................. 42

Refferences…………………………………………….............................. 43

Tables……………………………………………....................................... 64

Figures……………………………………………..................................... 67

List of abbreviations………………………………………...................... 76

Acknowledgements…………………………………………................... 78

1

Chapter Ι

General introduction

Amines are low molecular weight organic bases which widely are distributed

in plants, animals and microorganisms. They are generally divided into

monoamines, diamines and polyamines, and are shown to play important roles in

cellular metabolism, so that they are needed for growth, development and their

metabolism that appear to be coordinated with the cell cycle (Alain and Pierre

2000). Amine production have been associated with the protective mechanisms of

microorganisms against an acidic environment (Arena and Manca 2001). In higher

plants and animals, biogenic amines are important nitrogenous compounds that are

involved in significant physiological functions. If these compounds are consumed

in high quantities, they can give rise to different alterations in the organisms

(Docherty and Green 2010). These amines can be formed and degraded as a result

of normal metabolic activity in organisms.

Monoamines are observed in live organs of animals, plants and fungi

(Dannenburg and Liverman 1957, Gibson and Wightman 1972, Smith 1977a). In

animals, monoamines modulate the activity of many neurons which is a

2

prerequisite for normal brain functioning (Flugge et al. 2004). Their sympathetic

neurotransmitters are the catecholamines dopamine, noradrenaline and adrenaline,

the indoleamine serotonin (5-hydroxytryptamine), and histamine.

5-Hydroxytryptamine is found in the stinging hairs of Mucuna pruriens,

Girardinia heterophylla, Urtica spp, and probably also Luportea moroides

(Bowden et al.1954, Saxena et al. 1966, Robertson and Macfarlane 1957, Regula

et al. 1972). It is considered to be one of the active principles in these stings, and is

found also in the venoms of several animals (Welsh 1970). In a recent study,

5-hydroxytryptamine is a monoamine serving as a chemical messenger in diverse

brain regions, as well as in blood and various other organs of mammal

(Sharanappa and Adrienne 2011). The catecholamines, dopamine,

norepinephrine and epinephrine, are a group of biogenic amines possessing a

substituted 3, 4-dihydroxy phenyl ring that extensively exist in the animal

kingdom, but they have also been detected in plants (Smith 1977b, Szopa et al.

2001). The role of catecholamines in plants is poorly documented, but it is clear

that they are involved in many aspects of growth and development. The amines

were proposed as precursors for various alkaloids (Guinaudeau et al. 1993, Smith

1980). They are associated with processes such as ethylene production, nitrogen

3

fixation, defense against herbivores, flowering, prevention of 3-indole acetic acid

oxidation and gibberellins signaling (Dai et al. 1993, Kuklin and Conger 1995).

Together with several neuronal and hormonal systems, monoaminergic neurons

are activated by stress in vertebrates to induce various physiological and

behavioral changes (McEwen 2000). In monoamines, dopamine and

5-hydroxytryptamine act a part in regulation of mood and cognitive functions, and

their function is associated with a range of psychopathological conditions,

particularly with mood and anxiety disorders in mammals. A lot of experimental

procedures can be employed to investigate a role of monoamine neurotransmission

in psychopathology (Blier and De 1998, Nutt et al. 1999). Ingestion of a mass of

the tryptamines may be detrimental to humans and to some animals. For instance,

there may be a connection between the incidence of myocardial fibrosis in West

Africa and the high intake of plantains (bananas, Musa) in this region, as a result of

the high level of 5-hydroxytryptamine in this fruit. In Australia, cattle fed on

pastures (Phalaris arundinacea) develop a disease known as Phalaris staggers.

This disease is probably due to the high levels of tryptamines of the pastures

(Smith 1977c). Amounts of noradrenaline and dopamine are reported in venous

and arterial plasma origins of animals (Bell 1988). Dopamine which consists of

4

about 1-5% of the total catecholamine pool within noradrenergic neurons is

involved in stress response that is of paramount importance for both clinical and

preclinical research (Ungless and Argilli 2010, Cabib and Puglisi-Allegra 2012).

Similar to animal cells in which catecholamines stimulate glycogen mobilization,

an alike role for catecholamines in the regulation of plant carbohydrate

metabolism was suggested. Transgenic plants over-expressing tyrosine

decarboxylase, which controls an important step of catecholamine synthesis were

characterized by highly increased concentrations of norepinephrine and soluble

sugars, whereas starch level was remarkably decreased. The changes indicate a

positive correlation of norepinephrine with soluble sugars and a negative

correlation with starch (Widrych et al. 2004).

Polyamines ubiquitously distribute in all cells. Despite the proved necessity

of their presence for normal cellular growth in both prokaryotic and eukaryotic

cells, their molecular functions remain unknown (Tabors 1984, Moschou et al.

2012). Depending on species, the relative intercellular concentrations of the

polyamines are different, for that they can get high concentrations, up to the

millimolar range (Miyamoto et al. 1993). In polyamines, putrescine, spermidine

and spermine are among the major polycations in cells, together with Ca2+

and

5

Mg2+

that can bind to intracellular polyanions such as nucleic acids and ATP to

modulate their function. As an important property of these specific polyamines, it

is known that they are positively charged at a physiological pH and consequently

they have a high affection to negatively charged cellular molecules. Therefore

polyamines are very soluble in water, and they exert invigorative cation-anion

interactions with macromolecules, mainly with DNA and RNA (Marczynski 1985).

Thus a feature represents their best-known direct physiological role in cellular

functions such as cell growth, division and differentiation (Heby 1981, Jänne et

al.1978). The polyamine content of cells is regulated by biosynthesis, degradation

and transport (Igarashi et al.1999). In bacteria, it has been known that the

polyamines can modulate diverse cellular processes, including transcription and

translation. However, a detailed understanding of the mechanisms remains unclear.

Miscellaneous additional functions of bacterial polyamines have also been

uncovered. The amines that are part of outer membranes of Gram-negative

bacteria are involved in the biosynthesis of siderophores, take part in acid

resistance, protect from oxygen toxicity, play a role in signaling for cellular

differentiation, and are essential for plaque biofilm formation (Takatsuka and

Kamio 2004, Brickman and Armstrong 1996, Foster 2004, Jung et al. 2003,

6

Sturgill and Rather 2004, Patel et al. 2006). Putrescine, spermidine and spermine

are formed and degraded in cells via the ODC or ADC pathway named for the

enzymes ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). In

addition, decarboxylated S-adenosylmethionine as the donor molecule, putrescine

utilization and γ-aminobutyric acid are currently known in bacteria and archaea

(Foster 2004, Bowman et al. 1973, Tait 1976, Kurihara et al. 2010). In animals,

polyamines have an important role in cell differentiation and proliferation, and

their catabolism contributes to important physiopathological processes such as

apoptosis, amine detoxification and cell signaling through both regulation of

polyamine levels and their oxidatively deaminated reaction products that are

mainly H2O2, aminoaldehydes or dialdehyde and acrolein (Averill-Bates et al.

2008, Agostinelli et al. 2009). Furthermore, polyamine synthesis is

down-regulated as cells become senescent in many tissues of adults.

Administration of spermidine markedly extends the lifespan of yeast, flies and

their worms, and human immune cells. This treatment triggers epigenetic

deacetylation of histone H3 through inhibition of histone acetyltransferase,

suppressing oxidative stress and necrosis. The altered acetylation status of

chromatin triggers autophagy, which is crucial for enhancing longevity (Eisenberg

7

et al. 2009). In animal cells, uptake of polyamines can increase during hormonal

stimulation and cell proliferation (Kakinuma et al.1988). It has been demonstrated

that polyamines can also induce programmed cell death in various animal cell

types (Wallace et al. 2003, Igarashi and Kashiwagi 2010), thus indicating a

bivalent function for these molecules, promoting both cell growth and cell death,

likely depending on their concentration and other developmental and

environmental signals (Wallace et al. 2003, Toninello et al. 2006). It has been

suggested that plants had acquired a part of the polyamine biosynthetic pathway

from an ancestral cyanobacterial precursor of the chloroplast (Illingworth et al.

2003). Consequently, it can be supposed that this is an ancient metabolic route in

plants, which is also present in all organisms (Minguet et al. 2008). Polyamines are

considered as plant growth regulating compounds; among them, cadaverine has

been correlated with root growth promotion or osmotic stress mitigation in some

plant species. (Cassan et al. 2009) In plants, polyamines have been implicated in

many physiological processes, such as organogenesis, embryogenesis, Xoral

initiation and its development, leaf senescence, fruit development and its ripening,

and abiotic and biotic plant stress responses (Galston and Kaur-Sawhney 1990,

Kumar et al. 1997, Walden et al. 1997, Malmberg et al. 1998, Bouchereau et al.

8

1999, Bagni and Tassoni 2001, Alcázar et al. 2006, Kusano et al. 2008). The

polyamines, Put, Spd, Spm, and thermospermine are major sinks of assimilated N,

due to their high intracellular concentrations. The nitrogenous groups of

polyamines along with their carbon scaffold are efficiently recycled and

re-assimilated in various biochemical reactions. A major node of the recycling

pathway is polyamine catabolism, which recycles both C and N, producing

hydrogen peroxide. Polyamines are nitrogenous molecules which play a

well-established role in most cellular processes during growth and development

under physiological or biotic/abiotic stress conditions (Moschou et al. 2012). The

complexity of polyamine metabolism and the multitude of compensatory

mechanisms that are invoked to maintain polyamine homoeostasis argue that these

amines are critical to cell survival (Wallace et al. 2003). The studies indicate that

the intracellular levels of polyamines in plants are mostly regulated by anabolic

and catabolic processes, as well as by those conjugation to hydroxycinnamic acids.

Schematic representation of these processes and their interactions with other

metabolic pathways is depicted in Fig.1 modified from Alcázar et al. (2010).

Amine oxidases (AOs) are found in the nature widely. AOs represent a class

of enzymes that are heterogeneous in terms of structure, catalytic and

9

substrate-oxidation mechanisms. To classify AOs on the basis of their sensitivity

towards specific inhibitors or with respect to their affinity towards particular

substrates could not be satisfied adequately. Therefore a first general classification

division according to the nature of the prosthetic group contained in the enzyme

molecule was operated to decide. On the basis of this criterion, AOs are divided

into two classes: FAD-containing AOs and Cu-containing AOs. However, in this

way, there is a limit to the classification. Therefore, AOs, particularly the

copper-dependent enzymes, must in some cases be subdivided in relation to their

substrate specificity. Thus, FAD-AOs are also subdivided in mitochondrial

monoamine oxidase (MAO) and cytosolic polyamine oxidase (PAO).

Amines are oxidatively deaminated by AOs in a reaction consuming O2 and H2O,

and producing the corresponding aldehyde removed amine moiety, hydrogen

peroxide and NH4+, in stoichiometric amounts. The general equation of amine

oxidation by AOs is the following equation:

(1) Cleavage at a primary amino group (terminal-oxidative reaction, catalyzed by

Cu–AOs or MAOs)

R-CH2-NH2 + H2O + O2 → R-CHO + NH3 + H2O2

10

(2) Cleavage at the secondary amino group (interconversion reaction, catalysed by

PAOs)

R1-CH2-NH-CH2-R2 + O2 + H2O → R1-CHO + R2-CH2-NH2 + H2O2

As mentioned above, one class of AOs containing FAD as prosthetic group, and

in turn, ones are sub-divided into MAOs (A and B) and PAO which catalyzes the

oxidation of polyamines at the secondary amino group and participates in the

interconversion metabolism of polyamines. PAOs oxidize spermidine,

N1-acetylspermine (and N1-acetylspermidine, but not spermine. On the other

hand, in vertebrates spermine is directly oxidized by the cytosolic enzyme

spermine oxidase, a flavoprotein characterized in the past as a human PAO (Wang

et al. 2001) and then subsequently named SMO (Vujcic et al.2002, Cervelli et al.

2003). Sperminie oxidation leads to the production of spermidine,

3-aminopropanal and hydrogen peroxide. While N1-acetylspermine and

N1-acetylspermidine are oxidized by the peroxisomal FAD-dependent enzyme

N1-acetylPAO to produce respectively spermidine and putrescine,

3-acetoaminopropanal and H2O2. These polyamine catabolic enzymes have been

extensively characterized and it is well documented that both enzymes play an

essential role in maintaining vertebrate polyamine homeostasis, which is

11

mandatory for cellular life (Wallace et al. 2003, Thomas et al. 2001, Amendola et

al.2009).

MAOs are enzymes that are tightly bound to a component of the

mitochondrial outer membranes. The enzymes are classified into two forms, the

MAO-A and MAO-B, based on their specificity to the substrates and specific

inhibitors in animals (Abell and Kwan 2001). Both of them covalently bind to one

cofactor molecule: one FAD per 63 kDa (MAO-A sub-unit) and one FAD per 57

kDa (MAO-B sub-unit). Both MAO-A and -B sequences contain the pentapeptide

‘Serine-Glycine-Glycine-Cysteine- Tyrosine’ in which the FAD cofactor is

covalently bound to the cysteine (Cys) via a thioether link to one methyl of the

isoalloxazine ring. In addition to the covalent FAD binding site (Cys-406 in

MAO-A and Cys-397 in MAO-B), one cysteine (Cys-374) plays an important role

in MAO-A catalytic activity whereas two cysteines (Cys-156 and Cys-365) are

thought to be important for MAO-B activity (Wouters 1998). In the case of MAO,

oxidation of the substrates is coupled to the reduction of FAD (Margherita 2001).

In general, MAOs oxidize the primary amino groups of arylalkyl amines and are

widely distributed in higher eukaryotes. In mammals, MAO A and B are separate

gene products and exhibit 70% amino acid sequence identity. A more clear-cut

12

distinction is in terms of sensitivity to certain acetylenic inhibitors: MAO A is

irreversibly inhibited by very low concentrations of clorgyline, but is unaffected

by low concentrations of deprenyl and pargyline. The reverse is true of MAO B.

Certain tissues contain only MAO-A (e.g., human placenta), others only MAO-B

(e.g., blood platelets), but many tissues, like brain, contain both MAO-A and

MAO-B in varying proportions. Both MAO-A and -B contain the same covalent

amino acid adduct of FAD, 8a-5-cisteinyl FAD, and the peptide sequence at the

flavin sites is also identical (Singer 1985, Kearney et al. 1971). Both plant PAO

and human MAO-B have been recently structurally characterized by X ray

crystallography (Binda et al. 1999, 2002). In this class of FAD enzymes, the flavin

moiety is central to both substrate dehydrogenation and reduction of O2 to H2O2.

The folding of the PAO amino acid chain, relative to that of MAO-B, is crucial in

defining their relative substrate binding sites and these differences account for

their respective substrate diversities.

On the other class, Cu-AOs are only active on primary amino groups and

contain, in addition to metal, a second organic cofactor, which reacts with

semicarbazide and phenylhydrazine. This organic cofactor was identified as

trihydroxyphenylalanine quinone (TPQ) (Janes et al. 1990, Klinman et al. 1991),

13

produced by the copper self-catalyzed posttranslational oxidation of a tyrosine

residue. The catalytic reaction follows a ping-pong mechanism, consisting of

enzyme reduction by the amine and re-oxidation by molecular oxygen (see Eq. (1)).

Cu-AOs proteins have been implicated in leukocyte adhesion and glucose uptake,

and their activities altered have been associated with inflammatory bowel disease,

chronic liver disease, and diabetes (O’Sullivan et al 2004).

Cu-AOs have been found in many different organisms ranging from bacteria to

mammals. In bacteria, the Cu-AOs have a well-defined role in the metabolism of

primary amines as alternate sources of carbon and nitrogen to support growth. In

contrast, the role of Cu-AOs in higher organisms has not been clearly established,

although they have been implicated as key components in complex processes such

as leukocyte trafficking involving the Cu-AO, vascular adhesion protein-1 (Salmi

and Jalkanen 2002). Despite these functional differences, Cu-AOs from every

known source are dimers, having subunit masses ranging from 70 to 95kDa, with

33 fully conserved residues near the catalytic site, and share fundamentally

identical chemistry.

In plants, Cu-AOs have been detected in rice (Chaudhuri and Ghosh 1984),

barley (Cogoni et al. 1990), maize (Suzuki and Hagiwara 1993), millet (Awal and

14

Hirasawa 1995), white lupine (Siepaïo and Meunier 1995), and were isolated from

several sources, but only some of them, i.e. Pisum sativum (Kumar et al. 1996),

Lens esculenta (Medda et al. 1995), Triticum aestivum (Suzuki 1996), Euphorbia

characias (Padiglia et al. 1998) and Vigna radiate (Choudhary et al. 1999) have

been highly purified and characterized. It is interesting to note that Hordeum

vulgare and Zea mays contain both Cu-AO (Cogoni et al. 1990, Suzuki and

Hagiwara 1993) and FAD-AO (Smith and Bickley 1974, Suzuki and Yanagisawa

1980 ). The term ‘‘SSAO’’, semicarbazide-sensitive AOs, can easily be found in

reference to AOs containing a carbonyl group in the cofactor. Cu-AOs usually

have a similar molecular organization of their strongly interacting subunits

(Klinman et al. 1991). The active site, consisting of one Cu2+

and one TPQ,

connected by a water molecule, is located inside each subunit and communicates

with the solvent through a hydrophobic channel. In the last few years the structure

of several Cu-AOs has been solved by X ray crystallography and cloned (Brazeau

et al. 2004). Crystal structure of bovine serum AO was recently published (Lunelli

et al. 2005). Under anaerobic conditions, the TPQ bond is fully cleaved in both

bovine serum AO and lentil Lens culinaris AO, but the reaction rates are different.

In lentil AO, a fast release of aldehyde occurs and the formed Cu2+

-quinolamine is

15

in rapid equilibrium with a radical intermediate and designated

Cu+-semiquinolamine (Bellelli et al. 1991), while bovine serum AO forms the

Cu2+

-quinolamine at a slower rate. No radical in this enzyme is detected in the

UV/VIS spectrum, and the very low intensity signal detected in the EPR spectrum

does not appear to be related to the catalytic reaction (Pietrangeli et al. 2000).

These results indicate that the reaction mechanism and the role of copper have not

yet been definitively defined (Morpurgo 2001, Pietrangeli et al. 2003). However,

enzymes from different sources may behave in a different way: bovine serum AO

and lentil AO offer an example. The titration of TPQ with phenylhydrazine shows

the binding of one mole of inhibitors per dimer in bovine serum AO, and one mole

per subunit in plant AOs. The lower value was thought to arise from half of the site

reactivity (Biase et al. 1996). The reaction of oxygen appears to be different for

plant and mammalian enzymes, i.e., copper is involved in lentil AO redox

reactions, while bovine serum AO appears to have a structural function and/or a

Lewis acid role (De Matteis et al. 1999).

In this paper, I report a detailed study on the new MAO in Narcissus tazetta. The

objectives of this doctoral research are,

a) To find out the better method to purify MAO from the seedlings

16

homogeneity of Narcissus tazetta.

b) To characterized the properties of MAO of Narcissus tazetta and compare

its properties with other AOs. And to examine if the enzyme is involves in

flower-scent producing pathways.

17

Chapter ΙI

Purification and molecular analysis of a monoamine oxidase isolated from

Narcissus tazetta

Introduction

AOs have been divided into two categories depending on the cofactor, FAD

or a copper-containing quinone. Enzymes containing FAD (EC.1.4.3.3) are

further subdivided into MAOs and PAOs, depending on substrate specificity. In

the second category, AOs (EC 1.4.3.6) are characterized by the presence of TPQ

or lysyl tyrosylquinone (Mure 2004), cupric Cu-containing quinone cofactors.

The copper AOs include DAO and semicarbazide-sensitive, mammalian MAO.

In plants, there are two well-established types of AOs: copper-containing DAOs

that act on the primary amine of many amine compounds, and flavin-containing

PAOs that act function on the secondary amine of polyamines (Smith 1991,

Suzuki et al. 1990). Plant DAO is one of the best known enzymes involved in

amine catabolism. Spermidine and spermine are specific substrates for PAO.

Additionally, MAO activity has been reported in several plants (Werle et al.

1950, Tsushida and Takeo 1985). In the plant species reported, Tsushida and

18

Takeo used tea leaves to purify an AO that catalyzes the oxidation reaction on

monoamines including some alkylamines, ethanolamine, and benzylamine,

although this last was not classified as an MAO (Tsushida and Takeo 1985).

The cofactor in the tea-derived AO was not identified. Recently we reported a

semicarbazide-insensitive MAO from Avena sativa (Zhang et al. 2012). This

FAD-containing enzyme had high substrate specificity for benzylamine and

2-phenethylamine, which are oxidized to benzaldehyde and 2-phenylactaldehyde

respectively. Many aromatic and flavor compounds including fatty acid

derivatives, benzenoids, and isoprenoids are found in many parts of the plant

(Knudsen et al. 1993). The flavor volatiles 2-phenylacetaldehyde and

2-phenylethanol are presumably 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

(Tieman et al. 2006). 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 (Suzuki Y, personal

communication). In this study, we purified an AO from Narcissus tazetta and

examined its properties to determine whether it is involved in the

19

flower-scent-producing pathway.

20

Materials and Methods

Plant material

Flowering shoots of narcissus (Narcissus tazetta L. var. chinensis) were

perchased from Kansaikako (Osaka, Japan). The shoots were used for enzyme

purification.

Chemicals

The procedures of MAO purification have utilized several columns. Sepharose

G-25, DEAE Sephacel and Phenyl Sepharose were obtained from GE Healthcare

and hydroxylapatite was obtained from Nacalai, Japan. Lower molecular marker

proteins and bovine serum albumin (BSA) were purchased from Takara, Japan.

Other chemicals for pure grade were purchased from Wako, Japan.

Enzyme activity and concentrations

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 (Awal and Hirasawa 1995).

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

21

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 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 s−1

).

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 × 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

22

supernatant at 10,000 × 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 × g for 25 min.

The supernatant was applied to a Sephadex G-25 column (3.5 × 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 × g for 10 min, and the supernatant was applied to a DEAE-Sephacel (GE

Healthcare, Little Chalfont, UK) column (2.5 × 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 × 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 × 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

23

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.

The protein concentration of the pooled fraction from each purification step

was estimated by the micro-assay method of Bradford (1976) 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 using a 7.5% (w/v) gel without SDS by the method described by

Davis (Davis 1964). 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 (Laemmli

1970). Full-Range Rainbow Molecular Weight Markers (GE Healthcare) were

used as protein standards.

24

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

μL) 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 × 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 min−1

. 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 × 300 mm) was used to determine

the molecular mass of the narcissus MAO. The column was calibrated by the

25

following protein standards: ovalbumin, α-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.

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, 2,2′-bipyridyl, 2-bromoethylamine, 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 monoamines 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,

26

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. (1991).

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 μm 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.

27

Results and Discussion

Purification of the narcissus MAO

AO activity was detected in all organs, especially 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 in 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 stable 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

28

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 mg−1

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 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.

3a), indicating that the enzyme was purified to homogeneity and that the

active-stained band is an MAO protein.

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.3b). Many studies

indicate that Cu-DAOs in plants are typically homodimers (Medda et al. 1995).

Tea AO is also a homodimer, with a molecular mass of 80 kDa per subunit

(Tsushida and Takeo1985). The product of GS-520 HQ column chromatography

29

had an apparent molecular mass of 135 kDa (Fig. 4). The two bands visualized

on SDS-PAGE, which represented narcissus MAO, indicated that the

narcissus-derived enzyme is comprised of two different subunits, suggesting a

heterodimer. There is another possibility that a 75kDa-homodimer of MAO was

partially cleaved by an endogenous protease(s) during purification. However,

despite the addition of protease inhibitors (0.5 mM phenylmethylsulfonyl

fluoride 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. 3b (data not shown).

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. 2). 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

30

(Tsushida and Takeo1985), not 70°C as for the narcissus MAO. Additionally, the

optimum pH of the narcissus MAO was 7.0 (Fig. 5), similar to that of other

Cu-AOs (Suzuki et al. 1990).

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-Propylamine was found to be the best substrate for the amines. Other

short-aliphatic monoamines, ethanolamine 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. 6). I concluded from these

results that the enzyme from Narcissus tazetta was an MAO. The Km values for

n-propylamine and benzylamine were 5.9 × 10−5

M and 4.1 × 10−4

M respectively.

The enzyme did not catalyze the oxidation of hydroxylated benzylamines,

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 enzyme was also inhibited by a metal-chelating

31

reagent, 2, 2′-bipyridyl. In the inhibition experiments, diethyldithiocarbamate 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 (Tsushida and Takeo1985), 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 μM. Medda et al. indicated that

2-bromoethylamine (an irreversible inhibitor of lentil DAO) is a poor substrate

and combines directly with TPQ in the enzyme (Medda et al. 1995). 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.

Cofactor Identification

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

32

(Mn)/0.3419 μM 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 (Paz et al. 1991).

Fenugreek Cu-AO contains Mn, and its contents is 0.2 moles per mole of native

enzyme as determined by atomic absorption spectrometry (Šebela et al. 1997).

When manganese ions were added to the reaction mixture for MAO, enzyme

activity was 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 8, 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 Figure 8 shows the electroblot seen in panel A

counterstained red for protein with Ponceau S. The MAO consists of a

33

heterodimer containing 75-kDa and 65-kDa subunits (Fig. 3b). As panel B (lane

2) 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 (Suzuki et al. 2009). 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 x 10-5

M enzyme c)

(Medda et al. 1995) and 0.05 in tea MAO solution (8.6 x 10

-6 M) (Tsushida and

Takeo1985). The spectrum of pink was shifted to that of yellow at 346, 432, and

462 after the addition of putrescine (Medda et al. 1995). No obvious spectrum at

about 470-490 nm was detectable in the narcissus enzyme solution (6.8 x 10-7

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.

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

34

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 (Matsumoto et al. 2011), the 75 kDa subunit had no

homology anywhere with the barley AO (Fig. 7). A comparison of the

quinone-containing 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 (Jones et al.1992).

Researchers should examine hereafter the sequencing of the two monomers to

determine whether narcissus MAO has two different monomers containing TPQ.

In mammals, three main functions have been postulated 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, proliferation, and survival dramatically; and

to produce hydrogen peroxide as a signal molecule rather than a harmful side

product on-site (Boyce et al. 2009). In prokaryotes and fungi, AOs allow for

the use of primary amines as sole sources of nitrogen for growth. In Aspergillus

35

niger, two AOs were induced in cells in a growth medium containing

benzylamine as nitrogen source (Schilling and Lerch 1995). In plants, enzymes

containing DAOs are important to the synthesis of alkaloids, cell-wall formation,

and wound healing (Medda et al. 2009), 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 phenethylamine, resulting in the decarboxylation of

phenylalanine, which results in the hydrolysis of storage proteins in oat grain

during germination (Zhang et al. 2011). 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 (Ueno et al. 2003). Tryptamine-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 (Tsushida and

Takeo 1984), but there is no report on monoamines in the narcissus species. The

36

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

(Dobson et al. 1997), 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). I intend to conduct

further studies of endogenous substrates of MAOs in plant species.

37

ChapterΙII

General discussion

The heterogeneous superfamily of AOs includes enzymes found in all living

systems, ranging from prokaryotes to eukaryotes (Agostinelli et al. 2004). These

enzymes are involved in cellular and extracellular metabolism of amines

(monoamines, diamines and polyamines) whose oxidation may generate other

biologically active substances like aldehydes, ammonia, and hydrogen peroxide.

In prokaryotes and fungi, AOs allow using primary amines as sole sources of

nitrogen for growth. In Aspergillus nigar, two AOs were induced in cells in a

growth medium containing benzylamine as nitrogen source (Schilling and Lerch

1995), whereas the regulatory mechanisms of AOs have not yet understood in the

metabolism of amines in plants.

MAO appears to be ubiquitously expressed in higher eukaryotic origanisms.

MAO-like activity has been reported in the Ascarida galli, Nippostrongylus

braziliensis (Mishra et al. 1983; Agarwal et al. 1985), cockroach (Subhashni et al.

1983), fish, birds, amphibians, echinoderms, carp (Kinemuchi et al. 1983;

Yoshino et al. 1984), ring-dove (Hall et al. 1985) and frog (Kobayashi et al. 1981).

38

MAOs are found in all mammals where MAOs have been looked for, and the

enzymes are bound to the outer mitochondrial membrane and have a FAD

molecule covalently bound to the protein via an 8α-thioether linkage to a cysteinyl

residue (Kearney et al. 1971). In plants, MAO that was purified from oat, like

mammal and other MAOs, is a FAD-containing MAO (EC.1.4.3.4). In this report,

I have successfully purified it from Narcissus tazetta and examined its properties,

especially determined copper as cofactor in Narcissus MAO.

In plants, the enzymes containing DAOs were important for the synthesis of

alkaloid, cell wall formation and wound healing (Medda et al. 2009). On the other

hand, a physiological role of plant MAO has not yet been determined clearly. As

far as we know, MAO-A and MAO-B catalyze the oxidation of the principle

biogenic amines are serotonin, adrenaline, epinephrine, dopamine and tyramine

(Waldmeier 1987). It was not similar with MAO-A and MAO-B of mammals’ on

substrate specificity. The amines catalyzed by narcissus MAO were aliphatic and

aromatic short-chain monoamines. The classification of copper-containing amine

oxidases (Cu-AOs) including mammalian, microbial, and plant AOs takes into

account their origins and substrate specificities. The enzymes of plant origin

especially are subjected to intensive studies of the interface of biochemistry and

39

plant physiology since play key roles in essential metabolic pathway in plants

(Bachrach, 1985). Cu-AOs have been shown to contain a covalently bound

cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that derives from the

modification of an endogenous tyrosine residue (Janes et al 1990). In addition to

TPQ, Cu-AOs also contain a single copper ion in the active site that is involved in

the biogenesis of TPQ as well as the catalytic cycle involving oxidation of primary

amine substrates. Thus, Cu-AOs contain both an inorganic and organic cofactor,

and, in particular, a detailed understanding of the nature of the interaction of

molecular oxygen with the cofactors is likely to shed new light on oxygen

activation in biology.

The enzyme was also inhibited by a metal-chelating reagent, 2,

2’-bipyridyl.Using an ICP-MS, narcissus MAO was found to be a

copper-containing protein having 2.45 atoms of copper per one molecule of

enzyme protein. It can be considered that the enzyme is a Cu-AO. Medda et al.

(1995) indicated that 2-bromoethylamine, the irreversible inhibitor to lentil DAO,

is poor substrate and combines directly with TPQ in the enzyme. The results by

NBT/glycinate method which was used as a specific stain for quinoprotein show

that each 75kDa and 65kDa subunit of narcissus MAO has a qunone, respectively.

40

The quinone in narcissus MAO may be TPQ, comparing with other copper AOs

(Suzuki et al. 2009).

In fact, Cu-AOs generally play a nutritional role in microorganisms, utilizing

primary amines as nitrogen or carbon sources, and in mammals are involved in

detoxification and metabolic and vascular diseases. In mammals, three main

functions have been put forward concerning the biological importance of Cu-AOs,

namely, (1): to remove biologically active molecules like primary amines, (2): the

formation of the corresponding aldehydes that in some case were found to affect

dramatically cell differentiation, proliferation and survival, (3): the on-site

production of hydrogen peroxide as a signal molecule rather than a harmful side

product (Boyce et al. 2009).

Although physiological roles of Cu-AOs in plants is not precisely known, they

are implicated in wound healing, detoxification, and cell growth by regulating

intracellular diamine and polyamine levels. The aldehyde produces may have a

key role in the biosynthesis of some alkaloids and the hydrogen peroxide released

may be involved in the formation of lignin and suberin. The strong and specific

inhibitor may be useful for a probe as searching physiological roles on narcissus

MAO, especially doing if the enzyme involves any fragrant aldehydes in flower of

41

narcissus.

42

Summary

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.

43

References

Abell CW, Kwan SW (2001) Molecular characterization of monoamine oxidases

A and B. Progress in Nucleic Acid Research and Molecular Biology, 65:

129-156.

Agarwal A, Mishra A, Mishra SK and Ghatak S. (1985) Monoamine oxidase

in Nippostrongylus braziliensis. Ind. J. Parasitol. 9: 221-224.

Agostinelli E, Arancia G, Dalla L, Belli VF, Marra M, Salvi M, Toninello A.

(2004). The biological functions of polyamine oxidation products by amine

oxidases. Perspectives of clinical applications. Amino Acids, 27: 347-358.

Agostinelli E, Condello M, Molinari A, Tempera G, Viceconte N, Arancia G

(2009) Cytotoxicity of spermine oxidation products to multidrug resistant

melanoma M14 ADR2 cells: sensitization by MDL 72527 lysosomotropic

compound. Int J Oncol, 35:485–498.

Alain Bouchereau, Pierre Guenot (2000) Analysis of amines in plant materials.

Journal Chromatography B, 747:49-67.

Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C,

Carrasco P, Tiburcio AF(2010) Polyamines: molecules with regulatory

functions in plant abiotic stress tolerance. Planta, 231: 1237-1249.

44

Alcázar R, Marco F, Cuevas JC, Patrón M, Ferrando A, Carrasco P,

Tiburcio AF, Altabella T (2006) Involvement of polyamines in plant response

to abiotic stress. Biotechnol Lett, 28:1867–1876.

Amendola R, Cervelli M, Fratini E, Polticelli F, Sallustio DE, Mariottini

P( 2009) Spermine metabolism and anticancer therapy. Curr Cancer Drug

Targets, 9:118–130.

Arena ME, Manca de Narda MC (2001) Biogenic amine production by

Lactobacillus. Journal of Applied Microbiology, 90:158–162.

Averill-Bates DA, Ke Q, Tanel A, Roy J, Fortier G, Agostinelli E (2008)

Mechanism of cell death induced by spermine and amine oxidase in mouse

melanoma cells. Int J Oncol, 32:79–88.

Awal HMA and Hirasawa EJ (1995) Diamine oxidase from millet catalyzes the

oxidation of 1, 3-diaminopropane. J Plant Res 108:395–397.

Bachrach U (1985) In: B. Mondovi (eds), Structure and Functions of Amine

Oxidases, CRC Press, Boca Raton, FL, p. 5.

Bagni N, Tassoni A (2001) Biosynthesis, oxidation and conjugation of aliphatic

polyamines in higher plants. Amino Acids, 20:301–317.

45

Bell C (1988) Dopamine release from sympathetic nerve terminals, Progress in

Neurobiology, 30: 193-208.

Bellelli A, Finazzi Agro A, Floris G, Brunori M (1991) On the mechanism and

role of substrate oxidation by amine oxidase from lentil seedlings, J.Biol.Chem.

266:20654– 20657.

Biase D.De, Agostinelli E, Matteis G.De, Mondovı` B, Morpurgo L (1996)

Half of the sites reactivity of bovine serum amine oxidase. Reactivity and

chemical identity of the second site, Eur. J. Biochem. 237:93– 99.

Binda C, Coda A, Angelini R, Federico R, Ascenzi P, Mattevi A (1999) A

30-angstrom-long U-shaped catalytic tunnel in the crystal structure of

polyamine oxidase, Struct. Fold Des. 7:265–276.

Binda C, Newton-Vinson P, Hubalek F, Edmunson DE, Mattevi A (2002),

Structure of human monoamine oxidase B, a drug target for the treatment of

neurological disorders, Nat. Struct. Biol., 9:22– 26.

Blier P, De Montigny C (1998) Possible serotonergic mechanisms underlying the

antidepressant and anti-obsessive–compulsive disorder responses. Biol

Psychiatry, 44: 313-323.

Bouchereau A, Aziz A, Larher F, Martin-Tanguy J (1999) Polyamines and

46

environmental challenges: recent development. Plant Sci, 140:103–125.

Bowden K, Brown BG and Batty JE (1954) 5-Hydroxytryptamine: its

occurrence in cowhage. Nature, 13:925-926.

Bowman WH, Tabor CW, Tabor H (1973) Spermidine biosynthesis.

Purification and properties of propylamine transferase from Escherichia coli. J

Biol Chem, 248:2480–2486.

Boyce S, Tipton KF, O’Sullivan M, Davey GP, Gildea MM, McDonald AG,

Olivieri A, O’Sullivan J (2009) Nomenclature and Potential Functions of

Copper Amine Oxidases. Floris G, Mondovi B eds. Copper Amine Oxidases.

Structure, Catalytic Mechanisms, and Role in Pathophysiology. CRC Press

Boca Raton FL.

Bradford MM (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye binding.

Anal Biol Chem 72:248–254.

Brazeau BJ, Johnson BJ, Wilmot CM (2004) Copper-containing amine

oxidases. Biogenesis and catalysis; a structural perspective, Arch. Biochem.

Biophys. 428:22–31.

Brickman TJ, Armstrong SK (1996) The ornithine decarboxylase geneodc is

47

required for alcaligin siderophore biosynthesis in Bordetella spp: putrescine is

a precursor of alcaligin. J Bacteriol , 178:54–60

Cabib S, Puglisi-Allegra S (2012) The mesoaccumbens dopamine in coping with

stress. Neuroscience and Biobehavioral Reviews, 36: 79-89.

Cassan F, Maiale S, Masciarelli O, Vidal A , Luna V, Ruiz O (2009 )

Cadaverine production by Azospirillum brasilense and its possible role in plant

growth promotion and osmotic stress mitigation. European Journal of Soil

Biology, 45: 12-19.

Cervelli M, Polticelli F, Federico F, Mariottini P( 2003) Heterologous

expression and characterization of mouse spermine oxidase. J Biol

Chem,278:5271–5276.

Chaudhuri MM and Ghosh B (1984) Purification and characterization of

diamine oxidase from rice embryos. Phytochemistry, 23: 241-243.

Choudhary A, Singh I, Singh RP (1999). A thermostable diamine oxidase from

Vigna radiata seedlings. Phytochemistry, 52: 1-5.

Cogoni A, Piras C, Farci R, Melis A, Floris G (1990) Hordeum vulgare

seedlings amine oxidase. Plant Physiol., 93: 818-821.

Dai YR, Michaels PJ, Flores HE (1993) Stimulation of ethylene production by

48

catecholamines and phenylethylamine in potato cell suspension cultures. Plant

Growth Regul, 12:219-222.

Dannenburg WN and Liverman JL (1957) Conversion of Tryptophan-2-C14 to

Indoleacetic Acid by Watermelon Tissue. Plant Physioogyl, 32:263-269.

Davis BJ (1964) Disc electrophoresis II Methods and application to human serum

proteins. Annu N Y Acad Sci 121:404–427

De Matteis G, Agostinelli E, Mondovì B, Morpurgo L (1999) The metal

function in the reactions of bovine serum amine oxidase with substrates and

hydrazine inhibitors. J. Biol. Inorg. Chem., 4:348– 353.

Dobson HEM, Srroyo J, Bergström G and Groth I (1997) Interspecific

variation in floral fragrances within the genus Narcissus (Amaryllidaceae).

Biochemical Systematics and Ecology, 25, 685-706.

Docherty JR, Green AR (2010) The role of monoamines in the changes in body

temperature induced by 3,4-methylenedioxymethamphetamine (MDMA,

ecstasy) and its derivatives. British Journal of Pharmacology, 60: 1029

Dobson HEM, Srroyo J, Bergström G and Groth I (1997) Interspecific

variation in floral fragrances within the genus Narcissus (Amaryllidaceae).

Biochemical Systematics and Ecology, 25, 685-706.

49

Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C,

Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L, Fussi

H, Deszcz L, Hartl R, Schraml E, Criollo A, Megalou E, Weiskopf D, Laun

P, Heeren G, Breitenbach M, Grubeck-Loebenstein B, Herker E,

Fahrenkrog B, Fröhlich KU, Sinner F, Tavernarakis N, Minois N,

Kroemer G, Madeo F (2009) Induction of autophagy by spermidine promotes

longevity. Nat Cell Biol, 11:1305–1314

Flugge G, van Kampen M, Mijnster MJ (2004) Perturbations in brain

monoamine systems during stress. Cell and Tissue Research, 315: 1-14.

Foster JW (2004) Escherichia coli acid resistance: tales of an amateur acidophile.

Nat Rev Micro, 2:898–907.

Galston AW, Kaur-Sawhney R (1990) Polyamines in plant physiology. Plant

Physiol., 94:406–410.

Gibson RA, Barrett G and Wightman F (1972) Biosynthesis and Metabolism of

Indol-3yl-acetic Acid: III. PARTIAL PURIFICATION AND PROPERTIES OF

A TRYPTAMINE-FORMING L-TRYPTOPHAN DBCARBOXYLASE

FROM TOMATO SHOOTS. J.Exp.Botany., 23:775-786.

Guinaudeau H, Bruneton J (1993) Isoquinoline alkaloids. In Alkaloids and

50

Sulphur Compounds. Methods in Plant Biochemistry Volume 8. Edited

by:Watermann PG, Dey PM, Harborne JB. London Academic Press, 373-419.

Hall TR, Lea RW, Harvay S , Vowles DM (1985) Biochemistry and physiology

of monoamine oxidase (MAO) activity in the ring dove (Streptopelia

risoria)—II. Distribution of MAO in different tissues. Comparative

Biochemistry and Physiology Part C: Pharmacology, Toxicology and

Endocrinology. 82: 423-426.

Heby O (1981) Role of polyamines in the control of cell proliferation and

differentiation. Differentiation, 19: 1–20.

Igarashi K and Kashiwagi K (1999) Polyamine transport in bacteria and yeast.

Biochem J, 344: 633-642

Illingworth C, Mayer MJ, Elliott K, Hanfrey C, Walton NJ, Michael AJ

(2003) The diverse bacterial origins of the Arabidopsis polyamine biosynthetic

pathway. FEBS Lett., 549:26–30.

Janes SM, Mu D, Wemmer D, Smith A.J, Kaur S, Maltby D, Burbingame

AL, Klinman JP (1990) A new redox cofactor in eucariotic enzymes:

6-hydroxy-dopa at the active site of bovine serum amine oxidase. Science, 248:

981-987.

51

Jänne J, Pösö H and Raina A (1978) Polyamines in rapid growth and cancer.

Biochim. Biophys, Acta., 473: 241–293.

Jung IL, Oh TJ, Kim G (2003) Abnormal growth of polyaminedeficient

Escherichia coli mutant is partially caused by oxidative stress-induced damage.

Arch Biochem Biophys , 418:125–132.

Kakinuma Y, Hoshino K, Igarashi K (1988) Characterization of the inducible

polyamine transporter in bovine lymphocytes. European Journal of

Biochemistry, 176: 409-414.

Kearney EB, Selach JI, Walker WH, Seng RL, Kenney W, Zeszotek E,

Singer TP (1971) The covalently-bound flavin of hepatic monoamine oxidase.

Isolation and sequence of a flavin peptide and evidence for binding at 8-alpha

position. Eur. J. Biochem., 24: 321– 327.

Kinemuchi H, Sudo M, Yoshino M, Kawaguchi T, Sunami Y and Kamijo K

(1983) A new type of mitochondrial monoamine oxidase distinct from type-A

and type-B. Life Sci., 32:517-524.

Klinman JP, Dooley DM, Duine JA, Knowles PF, Mondovi B, Villafranca JJ

(1991) Status of the cofactor identity in copper oxidative enzymes. FEBS

Letters, 282:1-4.

52

Knudsen JT, Tollsten L, Bergstrom G (1993) Floral scents – a checklist of

volatile compounds isolated by head-space techniques. Phytochemistry, 33:

253–280.

Kobayashi S, Takahara K, Kamijo K (1981) Monoamine oxidase in frog liver

and brain. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol.,

69:179-183.

Kuklin AI and Conger BV (1995) Catecholamines in plants. Journal of Plant

Growth Regulation, 14:91-97.

Kumar A, Altabella T, Taylor MA, Tiburcio AF (1997) Recent advances in

polyamine research. Trends Plant Sci., 2:124–130.

Kumar V, Dooley DM, Freeman HC, Guss JM, Harvey I, McGuirl MA,

Wilce MCJ, Zubak V (1996) Crystal structure of a eukaryotic (pea seedling)

copper-containing amine oxidase at 2.2 angstrom resolution. Structure, 4:

943-955.

Kurihara S, Kato K, Asada K, Kumagai H, Suzuki H (2010) A

putrescine-inducible pathway comprising PuuE-YneI in which

gamma-aminobutyrate is degraded into succinate in Escherichia coli K-12. J

Bacteriol., 192:4582–4591.

53

Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines:essential

factors for growth and survival. Planta, 228:367–381.

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the

head of bacteriophage T4. Nature 227:680–685.

Lunelli M, Di Paolo ML, Biadene M, Calderone V, Battistutta R, Scarpa M,

Rigo A, Zanotti G (2005) Crystal structure of amine oxidase from bovine

serum. J. Mol. Biol., 346:991–1004.

Malmberg RL, Watson MB, Galloway GL, Yu W (1998) Molecular genetic

analyses of plant polyamines. Crit Rev Plant Sci., 17:199–224

Marczynski B (1985) The binding of spermine to polynucleotides and

complementary oligonucleotides at near physiological ionic strength.

International Journal of Biochemistry, 17:107-111.

Margherita SB (2001) Biotransformation of xenobiotics by amine oxidases.

Fundamental & Clinical Pharmacology, 15:75-84.

Matsumoto T, Tanaka T, Sakai H, Amano N, Kanamori H, Kurita K, Kikuta

A, Kamiya K, Yamamoto M, Ikawa H, Fujii N, Hori,K, Itoh T, Sato K

(2011) Comprehensive Sequence Analysis of 24,783 Barley Full-Length

cDNAs Derived from 12 Clone Libraries. Plant Physiol, 156:20-28.

54

McEwen BS (2000) The neurobiology of stress: from serendipity to clinical

relevance. Brain Res., 886:172–189.

Medda R, Bellelli A, Pec P, Federico R, Cona A, Floris G (2009) Copper

Amine Oxidases from Plants. Floris G, Mondovi B eds. Copper Amine

Oxidases. Structure, Catalytic Mechanisms, and Role in Pathophysiology. CRC

Press Boca Raton FL.

Medda R, Padiglia A, Floris G (1995) Plant copper-amine oxidases.

Phytochemisty, 39:1-9.

Medda R, Padiglia A, Pedersen JZ, Agro AF, Rotilio G, and Floris G (1997)

Inhibition of copper amine oxidase by haloamines: a killer product mechanism.

Biochemistry, 36:2595-2602.

Minguet EG, Vera-Sirera F, Marina A, Carbonell J, Blazquez MA (2008)

Evolutionary diversification in polyamine biosynthesis. Mol.Biol.Evol.,

25:2119–2128

Mishra Sk, Sen R, Ghatak S (1983) Monoamine oxidase in adult Ascaridia-gall.

Journal of Helminthology, 57 : 313-318.

Miyamoto S, Kashiwagi K, Ito K, Watanabe S and Igarashi K (1993)

Estimation of polyamine distribution and polyamine stimulation of protein

55

synthesis in Escherichia coli. Arch. Biochem. Biophys, 300:63–68.

Morpurgo L (2001) Structure and function of copper-containing amine oxidases,

Inflamm. Res., 50:126–127.

Moschou PN, Wu J, Cona A, Tavladoraki P, Angelini R,

Roubelakis-Angelakis KA (2012) The polyamines and their catabolic products

are significant players in the turnover of nitrogenous molecules in plants.

JOURNAL OF EXPERIMENTAL BOTANY, 63: 5003-5015.

Mure M (2004) Tyrosine-derived quinone cofactors. Acc Chem Res.,

37:131-139.

Nutt DJ, Forshall S, Bell C, Rich A, Sandford J, Nash J, Argyropoulos S

(1999) Mechanisms of action of selective serotonin reuptake inhibitors in the

treatment of psychiatric disorders. Eur Neuropsychopharmacol, 9:S81-S86.

O’Sullivan J, Unzeta M, Healy J, O’Sullivan M, Davey G, Tipton KF (2004)

Semicarbazide-sensitive amine oxidases: enzymes with quite a lot to do.

NeuroToxicol. 25:303–15.

Padiglia A, Medda R, Lorrai A, Murgia B, Pedersen JZ, Finazzi Agró A,

Floris G (1998) Characterization of Euphorbia characias latex amine oxidase.

Plant Physiol., 117: 5193-5198.

56

Patel CN, Wortham BW, Lines JL, Fetherston JD, Perry RD, Oliveira MA

(2006) Polyamines are essential for the formation of plague biofilm. J

Bacteriol., 188:2355–2363.

Percival FW, Purves WK (1974) Multiple amine oxidases in cucumber

seedlings. Plant Physiol., 54:601–607.

Pietrangeli P, Nocera S, Fattibene P, Wang X, Mondovì B, Morpurgo L

(2000) Modulation of bovine serum amine oxidase activity by hydrogen

peroxide. Biochem. Biophys. Res. Commun., 267: 174– 178.

Pietrangeli P, Nocera S, Mondovì B and Morpurgo L (2003) Is the catalytic

mechanism of bacteria, plant and mammalian copper-TPQ amine oxidase

identical? Biochim. Biophys. Acta., 1647:152– 156.

Regula I (1972) Kromatografska identifikacija alkoloide bufotenina uliutoj

koprivi (Urtica pilulifera L). Acta Bot. Croat., 31:109-112.

Robertson PA and Macfarlane WV (1957) Pain-producing substances from the

stinging bush Laportea moroides. Australian J. Exp. Biol. Med. Sci.,

35:381-393.

Salmi M and Jalkanen S(2002) Enzymatic control of leukocyte trafficking:

Role of VAP-1. Adv Exp Med Biol., 512:57-63.

57

Saxena PR, Tangri KK, Bhargava KP (1966) Identification of acetylcholine,

histamine, and 5-hydroxytryptamine in Girardinia heterophylla (Decne). Can. J.

Physiol. Pharmacol., 44:621-627.

Schilling B and Lerch K (1995) Amine oxidases from Aspergillus nigar;

identification of a novel flavin-dependent enzyme. Biochem Biophy Acta.,

1243:529-537.

Šebela M, Luhova L, Frebort I, Hirota S, Faulhammer HG, Stuzka V, Pec P

(1997) Confirmation of the presence of a Cu(II)/topa quinone active site in the

amine oxidase from fenugreek seedlings. J.EXP.BOT., 48:1897-1970.

Siepaïo MP and Meunier JCF (1995). Diamine Oxidase and Transglutaminase

Activities in White Lupine Seedlings with Respect to Cross-Linking of

Proteins. J. Agric. Food Chem., 43: 1151-1156.

Singer TP (1985) Inhibitors of FAD-containing monoamine oxidases. In:

Structure and Functions of Amine Oxidases. Mondovi, B.(Ed.). CRC Press,

Boca Raton, Florida, pp. 219–229.

Smith TA (1977a) Recent advances in the biochemistry of plant amines. In:

Reinhold L, Harborne JB and Swain T, eds. Progress in Phytochemistry,

4:27-82. Oxford: Pergamon Press.

58

Smith TA (1977b) Phenethylamine and Related Compounds in Plants.

Phytochemistry, 16: 9-18

Smith TA (1977c) Tryptamine and Related Compounds in Plants. Phytochemistry,

16: 171-175.

Smith TA (1980) Plant amines. In Secondary Plant Products, Encyclopedia of

Plant Physiology New Series, Volume 8. Edited by: Bell EA, Charlwood BV.

Berlin, Springer Verlag; pp:433-440.

Smith TA (1991) A Historical Perspetive On Research in Plant Polyamine

Biology, in Biochemistry and Physiology of Polyamines in Plants, Slocum RD

and Flores HE ed., CRC Press Boca Raton, FL,1-22.

Smith TA and Bickley DA (1974) Further properties of the polyamine oxidase of

barley leaves. Phytochemistry, 13:2437-2443.

Subhashni K, Prameela M, Murali MP and Sasira BK (1983) Effects of

scorpion venom on monoamine oxidase activity in cockroach tissues. Indian J.

Physiol. Pharmac. 27: 261-263.

Suzuki S, Okajima T, Tanizawa K, Mure M (2009) Cofactor of Amine Oxidase:

Copper Ion and its Substitution and the 2,4,5-Trihydroxyphenylalanine Quinone.

Floris G, Mondovi B ed. Copper Amine Oxidases. Structure, Catalytic

59

Mechanisms, and Role in Pathophysiology. CRC Press Boca Raton FL.

Suzuki Y (1996). Purification and characterization of diamine oxidase from

Triticum aestivum shoots. Phytochemistry, 42: 291-293.

Suzuki Y and Hagiwara M (1993). Purification and characterization of diamine

oxidase from Zea mays shoots. Phytochemistry, 33: 995-998.

Suzuki Y, Hirasawa E, Yanagisawa H, Matsuda H (1990) The enzymes of

polyamine metabolism in higher plants. - In: Flores HE, Arteca RN, Shannon

JC (ed.) Polyamines and Ethylene; Biochemistry, Physiology and Interaction.

pp.73–90. American Society of Plant Physiologists, Rockville, MD

Suzuki Y and Yanagisawa H (1980) Purification and properties of maize

polyamine oxidase: a flavoprotein. Plant and Cell Physiology, 21:1085-1094.

Sturgill G and Rather PN (2004) Evidence that putrescine acts as an

extracellular signal required for swarming in Proteus mirabilis. Mol Microbiol ,

51:437–446.

Szopa J, Wilczyński G, Fiehn O, Wenczel A, Willmitzer L(2001) Identification

and quantification of catecholamines in potato plants (Solanum tuberosum) by

GC-MS. Phytochemistry, 58:315-320.

Tabor CW and Tabor H (1984) Polyamines. Annual Review of Biochemistry,

60

53:749-790.

Tait GH (1976) A new pathway for the biosynthesis of spermidine. Biochem Soc

Trans., 4:610–612

Takatsuka Y and Kamio Y (2004) Molecular dissection of the Selenomonas

ruminantium cell envelope and lysine decarboxylase involved in the

biosynthesis of a polyamine covalently linked to the cell wall peptidoglycan

layer. Biosci Biotechnol Biochem, 68:1–19.

Thomas T and Thomas TJ (2001) Polyamines in cell growth and cell death:

molecular mechanisms and therapeutic applications. Cell Mol Life Sci.,

58:244–258.

Tieman D, Taylor M, Schauer N, Fernie AR, Hanson AD, and Klee HJ

(2006) Tomato aromatic amino acid decarboxylases participate in synthesis of

the flavor volatiles 2-phenylethanol and. 2-phenylacetaldehyde. Proc. Nat.

Acad. Sci., 103:8287-8292.

Tsushida T and Takeo T (1984) Ethylamine content of fresh tea shoots and

made tea determined by high performance liquid chromatography. J. Sci. Food

Agri., 35:77-83

Tsushida T and Takeo T (1985) Purification and some properties of tea leaf

61

amine oxidase. Agric Biol Chem., 49:319–326.

Ueno M, Shibata H, Kihara J, Honda Y, Arase S (2003) Increased tryptophan

decarboxylase and monoamine oxidase activities induce Sekiguchi lesion

formation in rice infected with Magnaporthe grisea. The plant Journal,

36:215–228.

Ungless MA, Argilli E, Bonci A (2010) Effects of stress and aversion on

dopamine neurons: Implications for addiction. Neuroscience and Biobehavioral

Reviews, 35:151-156.

Vujcic S, Diegelman P, Bacchi CJ, Kramer DL, Porter CW (2002)

Identification and characterization of a novel flavin-containing spermine

oxidase of mammalian cell origin. Biochem J., 367:665–675.

Walden R, Cordeiro A, Tiburcio AF (1997) Polyamines: Small molecules

triggering pathways in plant growth and development. Plant Physiol.,

113:1009–1013.

Waldmeier PC (1987) Amine oxidases and their endogenous substrates (with

special reference to monoamine oxidase and the brain). J Neural Transm

Suppl., 23:55-72.

Wallace HM, Fraser AV, Hughes A (2003) A perspective of polyamine

62

metabolism. Biochem J., 376:1–14.

Wang Y, Devereux W, Woster PM, Stewart TM, Hacker A, Casero RA Jr

(2001) Cloning and characterization of a human polyamine oxidase that is

inducible by polyamine analogue exposure. Cancer Res., 61:5370–5373.

Welsh, J. H. (1970) In Biogenic Amines as Physiological Regulators (Blum, J. J.

ed.) p. 75. Prentice-Hall, New Jersey.

Werle E and Roewer F (1950) Monaminoxydase in Planzen. Biochem Z.,

320:298-301.

Werle E and Roewer F (1952) Uber tiersche and pflanzliche Monaminoxydasen.

Biochem Z., 322:320-326.

Widrych A, Stachowiak J, Szopa J (2004) The catecholamine potentates starch

mobilization in transgenic potato tubers. Plant Physiol Biochem, 42:103-109.

Wouters J (1998) Structural aspects of monoamine oxidase and its reversible

inhibition, Curr. Medic. Chem., 5: 137-162.

Yoshino M, Obata T, Sho S, Kinemuchi H (1984) Enzymic and molecular

characteristics of a new form of monoamine oxidase, distinct from form-A and

form-B. Jpn J Pharmacol., 35:105-115.

Zhang Y-M, Livingstone JR, Hirasawa E (2012) Purification and

63

characterization of monoamine oxidase from Avena sativa. Acta Physiologiae

Plantarum, 34:1411-1419.

64

Tables

Table 1. Summary of the Purification of Monoamine Oxidase

from Narcissus Shoots

Purification

Step

Total volume

(ml)

Total protein

(mg)

Total activity

(nKat)

Specific activity

(nKat mg-1

prot.)

Purification

(fold)

Yield

(%)

Crude extract 500 718 37.8 0.053 1 100

(NH4)2SO4 precipitate 70 728 25.87 0.036 0.68 68

Heat treatment 70 242 23.52 0.097 1.83 62

DEAE- Sephacel 60 30.3 19.15 0.632 11.9 51

Hydroxyapatite 20 0.54 11.08 20.52 387 29

Phenyl-Sepaharose 4 0.092 7.53 81.85 1544 20

65

Table 2 Substrate specificity of narcissus MAO

Substrate Relative activity (%) Km (M) k(s-1

)

n-Propylamine 100 5.9 x 10-5

53.4

n-Butylamine 98 1.1 x 10-4

Ethylamine 84 1.3 x 10-4

Benzylamine 74 4.1 x 10-4

n-Pentylamine 56 5.0 x 10-4

Ethanolamine 50 2.0 x 10-3

Methylamine 34 1.1 x 10-4

Phenethylamine 9

Hexylamine 7

Tryptamine 0

Serotonine 0

Dopamine 0

Tyramine 0

Histamine 0

Putrescine 0

Cadaverine 0

Diaminodecane 0

Spermidine 0

Spermine 0

66

Table 3 Effects of inhibitors and manganese chloride on narcissus MAO

activity

Inhibitor Concentrations (mM) Inhibition (%)

Semicarbazide

0.01 100

0.001 51

2-Bromoethylamine

0.01 100

0.001 71

0.0005 42

2,2’-Bipyridyl 1 50

o-Phenanthroline 1 4

Pargyline 1 2

Clorgyline 1 1

MnCl2

1 90

0.1 122

0.05 105

67

Figures

Fig. 1 Polyamine metabolism and interaction with other metabolic

routes. Biosynthetic pathways for polyamines and related metabolites

are indicated by continuous lines. Dashed lines show the formation of

putrescine-derived alkaloids, polyamine conjugation and catabolic

processes.

Numbers refer to the following enzymes: 1 nitrate reductase, 2 nitrite reductase,

3 nitrogenase, 4 glutamine synthetase, 5 glutamate synthase, 6 glutamate

N-acetyltransferase, 7 acetylglutamate kinase, 8 N-acetyl-_-phosphate reductase,

9 acetylornithine transaminase, 10 acetylornithine deacetylase, 11

ornithine-carbamoyl transferase, 12 arginosuccinate synthase, 13 arginosuccinate

lyase, 14 arginine decarboxylase, 15 agmatine iminohydrolase, 16

N-carbamoylputrescine amidohydrolase, 17 ornithine decarboxylase, 18 SAM

synthetase, 19 SAM decarboxylase, 20 spermidine synthase, 21 spermine

68

synthase, 22 thermospermine synthase, 23 glutamate decarboxylase, 24 diamine

oxidase, 25 putrescine hydroxycinnamoyl transferase, 26 polyamine

oxidase, 27 _-aminobutyrate aminotransferase, 28 succinic semialdehyde

dehydrogenase, 29 arginase, 30 ornithine aminotransferase, 31

nitric oxide synthase, 32 ACC synthase, 33 ACC oxidase, 34 nitrate

reductase, 35 lysine decarboxylase.

69

Fig. 2. Thermal inactivation curve of narcissus monoamine oxidase.

The purified monoamine oxidase solution (●) or the desalted solution after

ammonium sulfate precipitation (▲) was incubated at the specified temperature

for 10min and each activity was measured. For other details, see Materials and

Methods.

70

a b

Fig. 3. Polyacrylamide gel electrophoresis of narcissus monoamine oxidase.

(a)SDS-free gel of narcissus monoamine oxidase. L; Coomassie-stainined

SDS-free gel, R; N-propylamine-positive spot on SDS-free gel, (b) SDS-gel of

narcissus monoamine oxidase. L; Marker, R; Coomassie-stainined SDS-gel. For

other details, see Materials and Methods.

71

Fig. 4. Molecular mass determination of narcissus monoamine oxidase.

Gel filtration (GS-520 HQ, HPLC) column; 1 ovalbumin (45kDa), 2

α-glucosidase from yeast (52 kDa), 3 lactate dehydrogenase 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.

72

Fig. 5. Effect of pH on narcissus monoamine oxiadse activity.

73

Fig. 6. Detection of propionaldehyde as DBD-H derivative using HPLC.

A: authentic propionaldehyde solution, B: the reacted mixture with

n-propylamine as substrate, C: before reacting, 2 -bromoethylamine (0.1 mM)

was added into the mixture. The arrows are peak of propionaldehyde-DBD-H.

For other details, see Materials and Methods.

74

75 kDa subunit 1 NPNPDHESETLLP 13 Current study

65 kDa subunit 1 ALPPRRAASVVQF 13 Current study

Barley predicted

AO 132 ALPPRRAVAVVRF 144 Matumoto et al. 2011

Fig.7. Comparison of the amino acid sequence of narcissus monoamine

oxidase with homologous sequence of barley predicted amine oxidase.

N-terminal narcissus MAO was sequenced by the Edman degradation method.

NCBI GenBank accession number for barley is BAJ85075. Amino acids of

barley AO identical to that of narcissus MAO are shade in gray. For other details,

see Materials and Methods.

75

A B

Fig. 8. NBT/glycine staining of quinoproteins on an SDS-polyacrylamine gel

electrophoresis electroblot. A and B; lane 1, marker; lane 2, purified narcissus

monoamine oxiadse; lane 3. bovine serum amine oxidase; lane 4, pig kidney

diamine oxidase. The electroblot was first stained with NBT/glycine (A) and then

counterstained with Ponceau (B). For other details, see Materials and Methods.

76

LIST OF ABBREVIATIONS

ADC arginine decarboxylase

AO amine oxidase

Cu-AO Cu-containing amine oxidase

DAO diamine oxidase

MAO monoamine oxidases

ODC ornithine decarboxylase

PAO polyamine oxidase

SAMDC S-adenosylmethioninamine decarboxylase

SAO serum amine oxidase

SMO spermine oxidase

77

SSAO semicarbazide-sensitive amine oxidase

TPQ trihydroxyphenylalanine quinone

78

Acknowledgments

First, I would like to express my hearty thanks to my supervisor Prof. Eiji

Hirasawa, who give me the opportunity to research in this lab. Without his

guidance, encouragement, and continuous emotional support, I can’t finish my

doctoral research. I am also very grateful to Ms. Satomi Shimonaka and Prof.

Harue Masuda for the metal analyses by ICP-MS.

I would also thank the members of my lab for their friendships, supports and

encouragements. Especially, I heartfelt gratitude Hironori Inue, who helped

me with important experiments in this study.

Finally, I would like to very thank my family and friends who give me

continuous emotional and economic supports.