cloning, sequencing, and heterologous expression of the murine

Cloning, Sequencing, and Heterologous Expression of the Murine Peroxisomal

Flavoprotein, N 1-Acetylated Polyamine Oxidase*

Tianyun Wu, Victoria Yankovskaya and William S. McIntireI

From the Molecular Biology Division of the Department of Veterans Affairs Medical Center, San

Francisco, California 94121, the Northern California Institute for Research and Education, San

Francisco, California 94121, and the Department of Biochemistry and Biophysics, University of

California, San Francisco, California 94143

Running Title: Murine Polyamine Oxidase

*This study was funded by the Department of Veterans Affairs and the Heart, Lung, and

Blood Institute of the National Institutes of Health. The costs of publication of this article were

defrayed in part by the payment of page charges. This article must therefore be hereby marked

Aadvertisement@ in accordance with 18 U. S. C. Section 1734 solely to indicate this fact.

ITo whom correspondence should be addressed: Department of Veterans Affairs Medical

Center, Molecular Biology Division (151-S), 4150 Clement Street, San Francisco, CA 94121.

Tel.: 415-387-1431. Fax: 415-750-6959. Email: [email protected].

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1The abbreviations used are: N 1-acetyl-SPD, N 1-acetylspermidine; N 1-acetyl-SPM, N 1-acetyl-

spermine; BENSPM N 1,N 11-bis(ethyl)-nor-SPM; BESPM, N 1,N 12-bis(ethyl)-SPM; bPAO and

bpao, bovine N 1-acetylated polyamine oxidase and its gene; Cb Ac-SMO, N 1-acetylspermine

oxidase from Candida boidinii ; ESI-MS, electrospray ionization mass spectrometry; hPAO and

hpao, human murine N 1-acetylated PAO and its gene; hSMO, human spermine oxidase; MAO-A

and MAO-B, monoamine oxidase form A and form B from humans and other mammals; MAO-

N monoamine oxidase from Aspergillus niger ; MDL 72527, N 1, N 4-bis(butadienyl)-1,4-

diaminobutane; mPAO and mpao, murine N 1-acetylated PAO and its gene; mSMO, murine

spermine oxidase; PAGE, polyacrylamide electrophoresis; PAO and pao, N 1-acetylated

polyamine oxidase and its gene; PUT, putrescine; SPD, spermidine; SPM, spermine; SMO,

spermine oxidase.

2The cDNA sequences for murine and bovine N 1-acetylate polyamine oxidases were deposited in

GenBank on January 20, 2000, under accession numbers AF226656 and AF226658, respectively.

3SMO, referred to as PAO in reference 10 and 12, oxidizes SPM but not the N 1-acetylated poly-

amines. By definition, PAO, designated EC 1.5.3.11, is a flavoprotein that oxidizes specifically N

1-acetyl-SPM and N 1-acetyl-SPD. To date, an EC number for SMO has not been assigned. To

avoid further confusion concerning the identity of these two polyamine-oxidizing enzymes, the

acronyms SMO and PAO taken from reference 11 will be used.

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SUMMARY

The amino acyl sequences of three regions of pure bovine N 1-acetylated polyamine oxidase

(PAO) were obtained and used to search GenBank. This led to the cloning and sequencing of a

complete coding cDNA for murine PAO (mPAO), and the 5'-truncated coding region of the

bovine pao gene. A search of GenBank indicated that mpao maps to murine chromosome 7 as

seven exons. The translated amino acid sequences of mpao and bpao have a -P-R-L peroxisomal

targeting signal at the extreme C-termini. A �-�-� FAD-binding motif is present in the N-termin-

al portion of mPAO. This and several other regions of mPAO and bPAO are highly similar to

corresponding sections of other flavoprotein amine oxidases, although, the overall identity of

aligned sequences indicates that PAO represents a new subfamily of flavoproteins. A fragment of

mpao was used as a probe to establish the relative transcription levels of this gene in various

mature murine tissues, and murine embryonic and breast tissues at different developmental

stages. An Escherichia coli expression system has been developed for manufacturing mPAO at a

reasonable level. The mPAO so-produced was purified to homogeneity and characterized. It was

demonstrated definitively that PAO oxidizes N 1-acetylspermine to spermidine and 3-acetamido-

propanal, and that it oxidizes also N 1-acetylspermidine to putrescine and 3-acetamidopropanal.

Thus, this is the classical polyamine oxidase (EC 1.5.3.11) that is defined as the enzyme that

oxidizes these N 1-acetylated polyamines on the exo-side of their N 4-amino groups. This enzyme

is distinguishable from the plant polyamine oxidase that oxidizes spermine on the endo-side of

the N 4-nitrogen. It differs also from mammalian spermine oxidase (SMO) that oxidizes spermine

(but not N 1-acetylspermine or N 1-acetylspermidine) at the exo-carbon of its N 4-amino group.

This report provides details of the biochemical, spectral, oxidation-reduction, and steady-state

kinetic properties of pure mPAO.

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INTRODUCTION

Spermine (SPM1) and spermidine (SPD) are important polyamines required for numerous

fundamentally important cellular processes including wound healing, tissue growth, tissue differ-

entiation, and tumor growth (1-9). In mammalian cells, polyamine-pool homeostasis is main-

tained by a balance of enzymatic and transport processes. (A) Putrescine (PUT), derived from

ornithine via ornithine decarboxylase, (B) condenses with decarboxylate S-adenosyl-L-methio-

nine (SAMe) to yields SPD by the action of SPD synthase. (C) SPM is generated from SPD and

decarboxylate SAMe via SPM synthase. (Decarboxylated SAMe is the product of SAMe decar-

boxylase). (D) Acetyl-CoA:SPD/SPM N 1-acetyltransferase (SSAT) transforms SPM and SPD

into N 1-acetyl-SPM and N 1-acetyl-SPD. (E) N 1-acetylated polyamine oxidase (PAO; EC

1.5.3.11) converts N 1-acetyl-SPM to SPD (Fig. 1) and 3-acetamidopropanal, and it converts N 1-

acetyl-SPD to PUT and 3-acetamidopropanal. It also oxidizes inefficiently SPM to SPD and 3-

aminopropanal. (F) Spermine oxidase (SMO) oxidizes SPM to SPD and 3-aminopropanal (10-

12), but is unable to oxidize N 1-acetyl-SPM or N 1-acetyl-SPD. (G) The N 1-acetylated poly-

amines are transported from cells to the blood, and then to the kidneys for urine excretion. (H)

PUT, SPD, and SPM derived from ingested foods, are efficiently transported into cells (1).

[Figure 1]

Peroxisomal mammalian PAO contains noncovalently bound FAD as the redox cofactor.

When PAO oxidizes N 1-acetyl-SPM or N 1-acetyl-SPD, FAD is reduced, which is oxidized

subsequently by O2 to generate H2O2 (1) (Fig. 1). Thus, PAO has been proposed to play a role in

triggering and/or participating in the progression of apoptosis (13-17). Additionally, another

product of the oxidation of the N 1-acetylated polyamines, 3-acetamidopropanal, can be enzymat-

ically deacetylated to yield cytotoxic 3-aminopropanal (18) that is thought to contribute, either

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alone or in concert with H2O2, to tissue damage following traumatic injury (19-21).

Prior to the present study, the gene sequences of four other forms of polyamine oxidizing

flavoproteins were known. These are the N 1-acetyl-SPD oxidase from Candida boidinii peroxi-

somes (22), the SPM/SPD oxidase from corn (cPAO) (23), and (apparently) cytosolic human (10,

12) and murine SMO (11).3 These flavoproteins have similarities with the murine PAO (mPAO)

and bovine PAO (bPAO) described herein. In particular, all have an easily identified FAD-bind-

ing motif near their N-termini. This, and other features of the primary structures indicate that

these enzymes are members of a larger family of amine oxidases that includes mitochondrial

monoamine oxidase A and B (26), MAO-N from Aspergillus niger (27, 28), PUT oxidase from

Micrococcus rubens (29), and tyramine oxidase from Micrococcus luteus (30). [MAO-A and

MAO-B; both contain covalently bound FAD (24, 25), whereas other members of this family

harbor noncovalently bound FAD]. These amine oxidases belong to an even larger enzyme

superfamily (the GR [glutathione reductase] superfamily) that includes various disulfide reduc-

tases (GR1 family), glucose oxidase, fumarate reductase, cholesterol oxidase, D-amino acid oxid-

ase, (GR2 family, for which PAO is a member), protoporphyrinogen oxidase, and phytoene

desaturase (31, 32). Of the amine oxidases of this superfamily, only the structures of cPAO (26)

and MAO-B are known (33).

While PAO has significant clinical and pharmacological relevance pertaining to cancer,

ischemic tissue damage, apoptosis, etc., there is a paucity of solid data regarding the biochemical

properties, mechanism of substrate oxidation, mechanism of inhibition by highly selective com-

pounds such as N 1, N 4-bis(butadienyl)-1,4-diaminobutane (MDL 72527) and N 1-butadienyl-1,4-

diaminobutane (MDL 72521) (1), or structural information for any mammalian PAO. This situa-

tion prompted us to initiate a program to create a system to heterologously produce PAO. This

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paper reports the cloning and the complete sequencing of murine pao (mpao), the sequencing of

all but a small portion of bovine pao (bpao), and the production of active mPAO by Escherichia

coli at a reasonable level. Various aspects of UV-visible biochemical, spectral, redox, and steady-

state kinetic properties of the heterologously produced pure mPAO are presented below.

EXPERIMENTAL PROCEDURES

Materials. Chemicals (vendors): SPD trihydrochloride (CalBiochem, gold label); benzyl-

amine hydrochloride, Coomassie blue, SPM tetrahydrochloride, PUT dihydrochloride, 4-amino-

antipyrine, vanillic acid, horse radish peroxidase (type II), 3-(N-morpholino)propane (MOPS), N-

(2-hydroxyethyl)piperazine-N'-2-ethane sulfonic acid (HEPES), FAD (flavin adenine dinucleo-

tide), bovine liver catalase, dansyl chloride (Sigma Chem. Co.); N 1-acetyl-SPM trihydrochloride

and N 1-acetyl-SPD dihydrochloride (Fluka Chemika); Aspergillus niger glucose oxidase (Miles

Laboratories, Inc.); acrolein, 1-amino-3,3-diethoxypropane, proline (99+ %,), 2,4-dinitrophenyl-

hydrazine (moist solid, 30-35 % water) (Acros Organics); N-(3-aminopropyl)-1,10-diamindecane

trihydrochloride, N 1,N 12-bis(ethyl)-SPM dihydrochloride (BESPM) and N 1,N 11-bis(ethyl)-nor-

SPM dihydrochloride (BENSPM) (Tocris Cookson); 32P-ATP (Pharmacia). Silica gel (HETLC-

GHL) and Avicel (cellulose, 250 �) thin-layer chromatography (TLC) plates were from Analtech.

Silica gel, for Flash Chromatography (40 ��particle size) was from J. T. Baker, Inc. All other

chemicals, purchased from common vendors, were of reagent grade or better. Molecular Biology

reagents were from various vendors; for example, ethidium bromide (Amersham Pharmacia),

restriction enzymes (Life Technologies, and New England Biolabs), isopropyl thio-�-D-

galactoside (IPTG) (Amersham Pharmacia).

Analytical procedures. Electrospray ionization mass spectrometry (ESI-MS; positive-ion

and negative-ion mode) and elemental analyses of organic chemicals were done by HT Labora-

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tories, San Diego, CA. One-dimensional 300 MHz (Nicolet/GE NT 300) 1H NMR spectral were

obtained from Acorn NMR, Inc., Livermore, CA. The liquid chromatography-electrospray

ionization mass spectral (LC-ESI-MS) analysis of pure mPAO was done using a Finnigan LCQ

Deca XP (ion trap mass spectrometer). The work was performed at the State University of New

York Health Science Center’s Mass Spectrometry Facility (Brooklyn, NY). Uncorrected

capillary-tube melting points were determined using a Misco aluminum block device.

Synthesis of 1-acetamido-3,3-diethoxypropane. 1-Amino-3,3-diethoxypropane (the di-

ethyl acetal of 3-aminopropanal), 6.5 mL (5.9 g, 0.04 mol), was dissolved in 50 mL of dry pyri-

dine at 0 °C. Over a 25 min period, with stirring, 6.7 mL (7.25 g, 0.071 mol) of acetic anhydride

were added dropwise. The mixture was warmed to room temperature and stirred overnight. A

small spot of the reaction mixture, dried on an Avicel TLC plate, was sprayed with a solution of

ninhydrin (0.1 % w/v in n-butanol). Heating the plate for several minute at 200 °C indicated that

all of the material had been acetylated. The pyridine, acetic acid, and acetic anhydride were re-

moved in a rotary evaporator under high vacuum at 50-65 °C, to yield 7.86 g of material (expect-

ed yield 7.57 g). The recovered material was distilled under vacuum (0.45 mm Hg); bp 111 °C,

6.88 g, 91 % yield. 1H NMR, CDCl3 (ppm relative to tetramethylsilane) – 7.29 (0.98, s, amide-

NH), 4.57 (1.00, t, propyl-CH), 3.68 (2.02, m, ethyl-CH2), 3.52 (2.05, m, ethyl-CH2), 3.35 (2.07,

m, propyl-CH2), 1.95 (2.92, s, amide-CH3), 1.82 (2.04, m, propyl-CH2), 1.22 (6.00, t, ethyl-CH3).

To our knowledge this compound has not been described previously.

Synthesis of the 2,4-dintrophenylhydrazone of 3-acetamidopropanal. 1-Acetamido-3,3-

diethoxypropane, 250 mg (1.3 mmol), was mixed with 1.0 mL 1.5 N HCl. After 2-3 min, this

was added to a boiling solution of 5 mL ethanol/0.5 mL conc. HCl containing 295 mg of 2,4-

dintrophenylhydrazine (1.5 mmol). Heating was stopped immediately and 10 mL of room-temp-

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erature ethanol were added. In a few minutes, the 2,4-dinitrophenylhydrazone began to crystal-

lize. After 1 h at room temperature and 1 h on ice, the solid was filtered and washed with a small

volume of ice-cold ethanol. The yield of the 2,4-dinitrophenylhydrazone of 3-acetamidopropanal

was 335 mg (1.13 mmol, 86 % yield); mp 157.5-158 ºC. 1H NMR, d6-DMSO (ppm relative to

tetramethylsilane) – 11.35 (0.95, s, hydrazone-NH), 9.82 (0.86, d, aromatic-H), 8.33 (1.01, d-d,

aromatic-H), 7.97 (1.91, m , propyl-CH and amide-H), 7.86 (1.04, d, aromatic-H), 3.31 (~2, m,

propyl-CH2, overlaps with H2O peak), 2.49 (~2, m, propyl-CH2, overlaps with DMSO peaks),

1.81 (2.97, s, amide-CH3); ESI-MS, m/z, 294 [M - H]��negative-ion mode), 296 [M + H]�, 237

[M - acetamido]� (positive-ion mode).

Purification of bPAO and amino acid sequencing of segments of this enzyme. Bovine

livers were covered with ice and transported to the laboratory within 1 h after their removal from

live animals at a local slaughterhouse. Following a published procedure (34), bPAO was purified

from fresh liver, or tissue that had been cut into 1-inch cubes from fresh liver and immediately

frozen and stored at �80 �C. About 1mg of nearly pure bPAO was obtained from 1 kg of tissue.

The enzyme was purified further on a 10 % Tris-HCl SDS AReady Gel@ (Bio-Rad), then electro-

transferred onto an Immobilon-PSQ membrane (Millipore), and Coomassie blue-stained. The

membrane was submitted to the Biomolecular Resource Center (University of California, San

Francisco, CA) for the N-terminal sequence analysis.

Purified bPAO was electrophoresed as before, and the Coomassie-blue stained bPAO

band was excised and subjected to an in-gel tryptic digest at the Protein Sequencing Center at the

State University of New York, Brooklyn, NY. Two major internal peptides (Peptide-I and

Peptide-II; see Fig. 2) were purified and sequenced.

Cloning and sequencing of bpao and mpao.2 GenBank EST databases were searched

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using three bPAO peptide sequences (vide supra). Two murine ESTs (GenBank accession

numbers AA437705 and AI098814) were found to code for amino acyl sequences that were

~ 85% identical to that of bPAO Peptide-I (see Fig. 2). Both clones (IMAGE numbers 819909

and 1482295, respectively) were purchased from Genome Systems, Inc. (St. Louis, MO), and

plasmids were isolated using a QIAGEN kit and sequenced. (All DNA sequencing work was

done at the Biomolecular Resource Center, University of California, San Francisco, CA). The

AA437705 cDNA fragment (mpao1) was released from its plasmid by an Xba I/Sal I digestion.

This fragment was the template for generating a mixture of 32P-dATP-labeled probes, by using a

random-primed DNA labeling kit (Boehringer, Mannheim, GmbH, Germany) (35). The 32P-

labeled probes were used to screen a �gt 10-mouse 17-day embryo cDNA library (CLONTECH)

and a Uni-ZAP XR bovine liver cDNA library (Stratagen). Seventeen positive phage plaques

from the bovine cDNA library and 16 positives plaques from the mouse cDNA library were ob-

tained and rescreened. The final positive clones, containing different-length mpao and bpao

cDNA inserts were isolated and confirmed by standard Southern blotting (35) using the same 32P-

labeled probes. The largest mpao fragments from these clones were sequenced. A fragment of

bpao cDNA was excised from the Uni-ZP XR vector of the pBluscript phagmid. This 1.6-kb

fragment (bpao1) was sequenced using flanking primers (T3/T7), and it coded for all but a small

section of the N-terminal portion of bPAO. The 5'-end of the fragment coded for a sequence that

matched exactly the C-terminal portion of Peptide-I (see Fig. 2). A region near the 3'-end coded

for a protein sequence identical that of Peptide-II, which was determined later to be the C-termin-

us of the enzyme (see Fig. 2). The high similarity between the sequences of bpao1 and mpao1

confirmed that the mpao1 screening probe codes for a portion of mPAO.

DNA isolated from one plaque of the mouse-cDNA library was cloned into the Sal I sites

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of pUC19 to give the plasmid, pUC19_MPAO1 that was used for double-stranded sequencing.

The sequence of this fragment (mpao2) was missing the 5'-end of the complete mpao sequence

(see the RESULTS section). A 5'-extension (see Fig. 2) was obtained using the 5'-RACE PCR

method with mouse 17-day embryo Marathon Ready cDNA (CLONTECH) as the template, and

using a SMARTTM PCR cDNA Synthesis Kit (CLONTECH) for the PCR reactions. The mpao

gene-specific antisense primer, mpao1R (5'-GTTCTCTTCCGATAATTCTTTCTCC-3') spans

nucleotides 333 to 309 of mpao (see Fig. 2), and the CLONTECH AP1 universal sense adaptor

primer was specific for the Marathon Ready cDNA; 5 cycles for 30 sec at 94 �C and 3 min at 72

�C, 5 cycles for 30 sec at 94 �C and 3min at 70 �C, and 30 cycles for 20 sec at 94 �C and 3 min

at 68 �C. Using a 50-fold dilution of the resulting PCR product as template, and AP2 (an AP1

nested primer) and mpao1R as primers, a second PCR reaction was carried out under the same

conditions. The resulting cDNA fragment, about 400-bp long, was isolated from a 1% agarose

gel using a Gene Clean Kit (BIO 101). Melded with mpao2, the sequenced PCR product

provided a 1.7-kb section of mpao that coded for full-length mPAO.

Measuring the relative levels of mpao mRNA in different murine tissues. Murine Rapid-

Scan Gene Expression Panels were purchased from OriGene Technologies, Inc. Two gene-

specific primers were designed according to the manufacturer's instruction. The sense primer,

mpao2F (5'-TCGGAAGAGAACCAGCTTGTGG-3', 22mer), and the antisense primer, mpao2R

(5'-CAATGACATGATGTGCAGGCA-3', 22mer) generated a 570-bp long mpao cDNA frag-

ment by PCR. The 24 mouse cDNA samples, serially diluted over a 4-log range (1000x, 100x,

100x and 1x) by the manufacturer, were arrayed into a 96-well PCR plate. The first step of the

PCR reactions were carried out at 94 �C for 3 min, which was followed by 35 cycles: 94 �C for

30 sec, 55 �C for 1min and 72 �C for 2 min. The control-primer pair for detection of �-actin

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cDNA, provided by the manufacturer, was used for a PCR reaction that was carried out as just

described with 25 cycles. The amplified fragments were electrophoresed on an 1 % agarose gel,

and ethidium bromide-stained to provided a measure of mpao mRNA in each tissue.

Expression of mpao in E. coli. The pET 29 c(+) vector (Novagen) was used to construct a

mpao prokaryotic expression system, and E. coli DH5� was used for plasmid subcloning. First, a

5'-end fragment was generated by PCR using mpao1 as the template for the gene-specific anti-

sense primer mpao1R (vide supra), and a sense primer, mpao1F, which contains Sac I and Nde I

sites and an ATG start codon (5'-GCGAGCTCATACATATGGCGTTCCCTGGCCCGCGG-3').

The underlined regions indicate Sac I and Nde I sites, respectively. A Sac I/BamH I fragment of

the PCR product was subcloned into pUC19-MPAO1 to give pUC19-MPAO. This construct con-

tained the entire mpao gene. Next, the full-length mpao cDNA was ligated into Nde I and Hind

III sites of pET 29c to give a plasmid denoted pET-MPAO. E. coli BL21 GOLD (DE3) (Invitro-

gen) was transformed with this plasmid for mPAO production.

Growth of transformed bacteria. A culture of the E. coli transformant was grown on

Luria-Bertani (LB) agar plates containing 30 �g/mL kanamycin. A single positive colony was

inoculated into 3 ml of LB broth containing 30 �g/mL of kanamycin (LB-kan) for overnight

growth at 37 �C. This culture (500 �L) was transferred to 80 mL of fresh LB-kan medium for

overnight growth. Five milliliters of this culture were transferred to each of five 2-L flasks

containing 1 L of fresh LB-kan medium for overnight growth at 37 �C, with shaking. Each flask

was added to one of five 14-L New Brunswick FS-614 fermentors containing 12 L of LB-kan

media. The cell culture was incubated at 30 �C with rapid stirring and vigorous aeration. When

the OD600 of the culture reached 0.6 - 0.7, IPTG was added (final concentration = 50 �M).

Growth was continued until the OD600 reach 1.5-2.0. About 260 g of centrifuged cell paste were

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obtained from the 60 L of growth media. The paste was stored at �80 �C.

Purification of heterologously produced mPAO. Selected fractions for the various steps in

the purification were assayed for N 1-acetyl-SPM oxidase activities by a published method (36).

This assay measured the time-dependent formation of H2O2 (Fig. 1). The assay stock solutions

were: (A) 100 mM vanillic acid, pH 7.0 with KOH; (B) 50 mM 4-aminopyrine; (C) 400 units/mL

of horse radish peroxidase; (D) 50 mM N 1-acetyl-SPM; (E) 100 mM glycine/KOH, pH 9.5, the

pH for maximal activity (37). Thirty microliters each of solutions A-D were mixed with 2.88 mL

of solution E, and 50 �L of the resulting solution were pipetted into individual wells of a 96-well

plate. Anywhere from 1 to 50 �L of a particular fraction was added to a well. The relative

activities of different fractions were assessed visually from the time-dependent intensity change

of the developing pink color. The purity of various fractions were assessed by SDS-PAGE using

pre-cast 10-20 % Tris-HCl AReady Gels@ (Bio-Rad), following the manufacturer=s instructions.

Unless noted otherwise, all purification steps were carried out at 4 �C. Frozen E. coli cell

paste (260 g) was thawed in a beaker with 10 mM MOPS buffer, pH 7.25. (The pH was adjusted

at 21 �C; the estimated pH at 4 �C is 7.35). The 800-mL suspension was homogenized with a

large glass/teflon piston (Potter/Elvehjem) tissue grinder, and then twice passed through an

Avestin Emulsiflex C5 Homogenizer at 15-20,000 psi. Next, 15 mg of solid FAD were dissolved

in the suspension, and it was centrifuged (50,000 � g, for 30 min). The supernatant was dialyzed

in the dark against 13 L of 10 mM MOPS buffer, pH 7.25 for 4 h, and then, overnight, against 13

L of fresh buffer. The resulting solution was diluted to 2 L with this buffer and applied to a 14 �

25 cm DEAE cellulose (Whatman, DE-53) column with a flow rate of 20 mL/min. The column

was then washed with 2 L of the buffer, and eluted with an 8-liter gradient from 0 to 400 mM

KCl in the 10 mM MOPS, pH 7.25 buffer. Activity eluted from 4.7-7.8 L after the gradient was

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initiated. The 3.1-liter volume was reduced to ~ 500 mL using 350 mL Amicon pressure concen-

trators fitted with Amicon YM-10 membranes. After dissolving 15 mg of FAD, the resulting

solution was dialyzed in the dark for 4 h against 13 L of 10 mM HEPES buffer, pH 7.8 (pH

adjusted at 21 �C; estimated pH at 4 �C was 8.05), and then overnight against 13 L of fresh

buffer. The resulting sample was applied to a 5 � 39 cm DEAE Spherodex LS column (100-300

�m bead size; Ciphergen) already equilibrated with the 10 mM HEPES buffer. The column was

washed with 500 mL of this buffer before starting a 2.4-liter gradient from 0 to 500 mM KCl in

the same buffer. The column, with a 7 ft pressure-head, was run at the maximum flow rate. Once

the gradient was started, 26-mL fractions were collected. The majority of the activity eluted in

tubes 82-108, which were combined (~ 700 mL) and concentrated to ~ 50 mL as described

earlier. This solution was dialyzed for 4 h, against 7 L of 10 mM KH2PO4/KOH buffer, pH 7.2,

and then overnight against 7 L of fresh buffer.

The sample was chromatographed on a MONO P HR 5/20 column (Amersham/Pharma-

cia), at room temperature. After injecting 2 mL of the sample at a flow rate of 1 mL/min with

solution I (H2O), proteins were eluted with the following gradient: 0 to 1 % II (1 M

KH2PO4/KOH, pH 7.2) in 4 min; 1 to 30 % II in 125 min. mPAO, eluting from 38-41 min, was

collected as a single fraction and immediately put on ice. This procedure was repeated until the

entire sample had been processed. The mPAO fractions from all of the MONO P runs were com-

bined, and then concentrated and washed into 1 mM KH2PO4/KOH buffer, pH 7.2, using 2-mL

Centricon-10 centrifuge concentrators (Amicon). The final volume was 2 mL in the 1 mM buffer.

This sample was chromatographed on a 1 � 10 cm ceramic hydroxyapatite (HAP; Bio-

Rad, type II) column (Amersham/Pharmacia HR 10/10 column), at room temperature. The

mPAO sample (100 �L), diluted to 1 mL with H2O, was injected immediately onto the HAP

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column with a flow rate of 2 mL/min. The elution was carried out as follows: 0 % solution II for

7 min; 0 to 1 % II in 2 min; hold at 1 % II for 10 min. mPAO eluted as a broad peak from 14 to

17 min. This step was repeated until the entire sample had been processed. The combined frac-

tions were concentrated as for the MONO P fraction. The solution was washed into 10 mM

KH2PO4/KOH buffer, pH 7.2, to give a solution that was 3.68 mg/mL of mPAO (based on an ε458

= 10,400 M�1 cm�1 and a Mr = 56,101 Da for the enzyme; see the RESULTS section). The

enzyme was judged pure by SDS-PAGE, by ion-exchange chromatography on an analytical TSK

DEAE 2SW column (0.4 � 25 cm; a 0.75 mL/min flow rate, with a gradient from 1 % to 50 %

solution II in 30 min; a single sharp peak eluted at 23 min), and gel-filtration chromatography on

a TSK 3000SW column (0.7 � 30 cm; 0.5 mL/min flow rate; 250 mM KH2PO4/KOH buffer, pH

7.2). The purity and integrity of the protein was confirmed also by the electrospray ionization

(ESI) mass spectral analysis, which provided a single peak of 55,311 ��mass unit (the mass of

apo-mPAO based on the sequence is 55,316 mass units). The yield of pure mPAO was 36.8 mg.

Using the conditions for the steady-state kinetic assay described below, it was found that

the enzyme, at 2 - 4 mg/mL was stable when frozen at �20 �C or �80 �C and thawed through

several cycles. However, at a concentration of 30 �g/mL, activity was lost quickly after several

freeze/thaw cycles, with more rapid loss occurring at �80 �C. When 33 % (v/v) ethylene glycol

was added, mPAO was stable for several of freeze/thaw cycles for solutions containing 20 �g/mL

to 4 mg/mL, regardless of the storage temperature. It was decided to store the enzyme at �20 �C

in the presence of 33 % (v/v) ethylene glycol. The enzyme maintains full activity and measured

biochemical, redox, Mr and kinetic properties after 16-months storage under these conditions.

Ethylene glycol removal and buffer exchange is accomplished easily by several

concentration/dilution cycles using Centricon-10 centrifuge concentrators.

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Binding of FAD to mPAO. The spectrum of a 0.1 mg/mL (0.8 mL) solution of mPAO in

10 mM KH2PO4/KOH buffer, pH 7.2 indicated that the sample contained 7.4 nmol of FAD. This

solution was treated with 80 �L of 55 % trichloroacetic acid (38), and centrifuged to give a clear

yellow supernatant and a white pellet. Overnight incubation of the isolated solution at room

temperature in the dark, resulted in the conversion of the liberated FAD to FMN. Fluorescence

analysis with a Hatachi F-4010 Fluorescence Spectrophotometer (450 nm excitation, 525 nm

emission; reference - authentic FMN) indicated that FAD was released quantitatively from

mPAO. Thus, FAD is noncovalently bound to mPAO.

Spectral characterization and redox properties of mPAO. All UV-visible spectra were

recorded with a Hewlett-Packard 8452A diode array spectrophotometer. mPAO, in 50 mM

KH2PO4/KOH buffer, pH 7.6, at 25 �C, was titrated anaerobically with a solution of sodium

dithionite. This solution was standardized by using it to titrate anaerobically a FAD solution of

known concentration. The anaerobic cuvette and other details of this procedure are described

elsewhere (39-41). The anaerobic mPAO solution contained also 50 mM D-glucose, 3 �g of

catalase, and 50 �g of glucose oxidase to scavenge trace dissolved O2. The spectral data were

subjected to AFactor Analysis@ using the SPECFIT program (Spectrum Software Associates,

Chapel Hill, NC) as described earlier (40, 41).

A 1.20 �M solution of mPAO was titrated anaerobically with a solution of 0.5 mM

N 1-acetyl-SPD in 50 mM KH2PO4/KOH buffer, pH 7.6, at 25 �C. The enzyme and substrate

solutions contained 50 mM D-glucose, 3 �g of catalase, and 50 �g of glucose oxidase.

Steady-state kinetic experiments. Spectrophotometric assays were done at 30 �C in 50

mM KH2PO4/KOH buffer, pH 7.6 following a published procedure (36). This method provided a

continuous monitor of the H2O2 produced in the reactions. The assays were done in 1-mL, 1 cm-

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path length cuvette with 0.8 mL of solution containing varying amounts of substrate, 0.1-0.2 �g

of mPAO, 1 mM vanillic acid, 0.5 mM 4-aminopyrine, and 4 units of horseradish peroxidase. By

varying each of the last three components, it was found that none were inhibitory. The reactions

were monitored at 498 nm with a UVIKON 840 spectrophotometer (KONTRON Instruments) for

the formation of the quinoneimine dye ( = 4,650 M�1cm�1 at pH 7.6; ref. 36), the condensation

product of vanillic acid and oxidized 4-aminopyrine. The latter was produced from 4-amino-

pyrine by its interaction with horseradish peroxidase that had been oxidized by H2O2 (36). Assays

were done by varying the concentration of the amine substrate while the dissolved [O2] was

constant at the air-saturated level (237 �M) in the buffer at 30 �C. The data were fit by nonlinear

regression (42) to the appropriate steady-state kinetic equations.

The value for the apparent dissociation constant, KD (= KI) for each inhibitor was estim-

ated by measuring the rates of N 1-acetyl-SPM oxidation as its concentrations and that of the

inhibitor were varied. It was assumed that these substances, which are either very poor or non-

substrates, are competitive inhibitors for the oxidation of the substrate. These data were analyzed

also by nonlinear regression using the appropriate equation.

Steady-state kinetic assays were done also by varying the [N 1-acetyl-SPM] in buffer

saturated with pure O2 (1.12 mM). After several minute of bubbling a cuvette solution with a

stream of pure O2 gas, a small aliquot of substrate was added followed by the enzyme. Once the

enzyme was added, the bubbling was terminated and the absorbance change recorded.

Some (oxygraph) assays were carried out by monitoring directly the dissolved-O2 con-

sumption in air-saturated buffer (dissolved [O2] = 0.237 mM), or in buffer saturated with pure O2

at 30 �C (dissolved [O2] = 1.12 mM). The dissolved [O2] was measured with a Yellow Springs

Instruments, Inc. Model 53 Oxygen Monitor equipped with a Clark electrode, in a glass 1.4 mL-

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reaction chamber. The true kcat and KO values for the oxidation of N 1-acetyl-SPM and N 1-acetyl-

SPD were determined by progress curve analyses of reactions that were allowed to go to comple-

tion (dissolved [O2] = 0 at t = ). Using a published procedure (43), the data were fitted to the

intergrated Mechaelis-Menton equation. The analyses were done using Maple VI (Windows

2000) software (Waterloo Maple, Inc) running on a PC computer. Using KI values (see Table 1)

as a gauge, the saturating concentrations of N 1-acetyl-SPM and N 1-acetyl-SPD were made high

enough (3.7 mM) so that product inhibition by SPD or PUT, respectively, was insignificant at all

times during the reaction (see Table 1). Inhibition by the H2O2, formed as a product of polyamine

oxidation by mPAO, was assessed by addition of 2 �L of a 30 mg/mL (30,000 units/mg)

solutions of catalase before a reaction was started. The catalase converted immediately each mol

of H2O2 formed to 0.5 mol of dissolved O2. After correcting the data by a factor of 2, the rate of

O2 consumption was the same as in the absence of catalase. This indicated a lack of inhibition by

the H2O2 formed in the assays lacking catalase.

Analyses of the aldehyde produced when N1-acetyl-SPM and N1-acetyl-SPD are oxidized

by mPAO. mPAO, 29 �g, was dissolved in 2 mL of 50 mM KH2PO4/KOH buffer, pH 7.6 con-

taining 0.8 mM N 1-acetyl-SPM and 30 �g of catalase (30 units). An identical solution containing

0.8 mM N 1-acetyl-SPD in the place of N 1-acetyl-SPM was also prepared. Each solution was stir-

red at room temperature for 2 h. After dansylating a small aliquot, HPLC analysis (vide infra)

indicated that the substrates had been oxidized completely for both solutions. Each solution (100

�L) was mixed separately with 100 �L of a 2,4-dinitrophenylhydrazine solution (100 mg in 94

mL ethanol/6 mL of concentrated HCl). Each of the resulting solutions (25 �L) was injected onto

a Prodigy octadecylsilyl silica gel HPLC column (5 ��particle size, 0.46 ��5.0 cm; Phenomenex):

flow rate, 1 mL/min; gradient elution – 0 % B for 0.5 min, 0 to 35 % B from 0.5 to 1.5 min, hold

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at 35 % B from 1.5 to 5.0 min, 35 to 100 % B from 5.0 to 9.0 min; solutions A and B were H2O

and acetonitrile, respectively, both containing 0.5 % (v/v) trifluoroacetic acid. The HPLC system

used SpetraSYSTEM P2000 gradient pumps, a UV6000LP Diode Array Detector, and the

ThermoQuest ChromQuest Chromatography Data System (Thermo Separation Products). The

368-nm chromatograms were used for quantitative analyses.

Authentic 3-acetamidopropanal, the expected product of mPAO oxidation of N 1-acetyl-

SPM and N 1-acetyl-SPD, was generated by treating 10 �L (~10 mg, 53 �mol) of 1-acetamido-

3,3-diethoxypropane with 100 �L of 1.5 N HCl for 1 min and then diluting immediately to 0.8

mM with 50 mM KH2PO4/KOH buffer, pH 7.6. A 0.8 mM solution of 3-aminopropanal was

produced similarly after treating 10 �L (9.1 mg, 62 �mol) of 1-amino-3,3-diethoxypropane with

100 �L of 1.5 N HCl for 1 min. A 0.8 mM solution of commercial acrolein was also prepared.

Each of these solutions was mixed immediately, 1:1 (v/v), with the 2,4-dintrophenylhydrazine

reagent, and analyzed by HPLC as describe in the previous paragraph. Another stock solution

containing 0.8 mM of all three aldehydes was treated and analyzed in the same manner. These

analyses provided the retention times and reference-peak areas for unreacted 2,4-dinitrophenyl-

hydrazine and the 2,4-dinitrophenylhydazones of each aldehyde (see Fig. 6).

The following experiment was carried out in order to obtain an analytical amount of the

aldehyde produced by mPAO oxidation of N 1-acetyl-SPM. A 10 mL solution of 50 mM

KH2PO4/KOH buffer, pH 7.5, containing 5.0 mM N 1-acetyl-SPM (50 �mol or 17.7 mg total),

7.25 �g/mL mPAO and 7.5 �g/mL catalase was stirred at room temperature. The reaction was

monitored by treating 10 �L of the solution with dansyl chloride (vide infra) and analyzing by

TLC; silca gel plates using cyclohexane/ethyl acetate, 2:3; Rf values, 0.04 for N 1-acetyl-SPM and

0.77 for SPD. After 23 h, there remained no N 1-acetyl-SPM. The solution was filtered using

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Centricon-10 centrifuge concentrators (Millipore), and 1.25 mL of concentrated HCl was added

with stirring. Next, 23 mg (115 �mol) of 2,4-dinitrophenylhydrazine in 0.625 mL of tetrahydro-

furan were added to the filtrate. After stirring for 1-2 min, the solution became slightly cloudy.

The mixture was put on ice for 2h, filtered, and the solid material washed with 1-2 mL of ice-

cold 1.5 M HCl followed by 6-7 mL of ice-cold water. The yield of the dry yellow solid (Sample

I) was 13.2 mg (yield, 89 % based on Mr of the 2,4-dintrophenylhydrazone of 3-acetamidopro-

panal). 1H NMR, d6-DMSO (ppm relative to tetramethylsilane) – 11.36 (0.98, s, hydrazone-NH),

9.82 (0.83, d, aromatic-H), 8.34 (1.01, d-d, aromatic-H), 7.97 (1.95, m , propyl-CH and amide-

H), 7.87 (1.04, d, aromatic-H), 3.31 (~2, m, propyl-CH2, overlaps with H2O peak), 2.49 (~2, m,

propyl-CH2, overlaps with DMSO peaks), 1.81 (2.90, s, amide-CH3); ESI-MS, m/z, 294 [M -

H]��negative-ion mode), 296 [M + H]�, 237 [M - acetamido]� (positive-ion mode). The NMR

and mass spectral data indicate that this material is identical to the 2,4-dintrophenylhydrazone of

3-acetamidopropanal made by chemical synthesis (vide supra).

Analyses of the polyamine products of mPAO oxidation of N 1-acetyl-SPM and N 1-acetyl-

SPD. The 2-h N 1-acetyl-SPM and N 1-acetyl-SPD enzyme-reaction solutions described at the

beginning of the previous section were used for these analyses. To 0.5 mL of each solution was

added 10 �L of a 15 mM solution of N-(3-aminopropyl)-1,10-diaminodecane trihydrochloride

(the internal standard; 0.3 mM final conc.), which was determined to be a mPAO non-substrate.

Separate solutions of 0.8 mM N 1-acetyl-SPM, N 1-acetyl-SPD, SPM, SPD, and PUT, and 0.3

mM N-(3-aminopropyl)-1,10-diaminodecane were prepared. A solution containing 0.8 mM N 1-

acetyl-SPM, N 1-acetyl-SPD, SPM, SPD, and PUT, and 0.3 mM N-(3-aminopropyl)-1,10-

diaminodecane was also prepared. The reference solutions established retention times and signal

intensities for each compound and the internal standard.

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A literature procedure for dansylation with dansyl chloride and sample preparation was

used with minor modifications (44). To 50 �L of an unknown or reference solution, in a 1.5 mL

screw-cap plastic vial, was added 200 �L of a saturated Na2CO3 solution and 200 �L of a 10

mg/mL dansyl chloride solution in acetone. Each sample was vortex-mixed for 20 sec, before

incubation at 65 °C for 10 min. After cooling on ice for several minutes, 100 �L of a proline sol-

ution (250 mg/mL) was added, and the sample vortex-mixed for 10 sec. The phases were separ-

ated by centrifugation and the upper organic phase (~ 350 �L) was removed. Ten microliters of

this phase were injected onto a Prodigy HPLC column (octadecylsilyl silica gel, 5-��particle

size, 0.46 ��5.0 cm), using a flow rate of 1 mL/min, and the following elution gradient – 0 to 45

% B from 0 to 0.1 min, 45 to 80 % B 0.1 to 8 min, hold at 80 % B from 8 to 11 min, 80 to 90 %

B from 11 to 12 min. Detection was accomplished with a Gilson Spctra/Glo fluorescence detec-

tor using a 7-51X excitation filter (330-400 nm) and a 3-72M emission filter (460-600 nm). The

retention times (min) were (data not shown): N 1-acetyl-SPD, 10.4; PUT, 11.5; N 1-acetyl-SPM,

13.4; SPD, 14.8; N-(3-aminopropyl)-1,10-diaminodecane, 16.4; SPM, 17.9. Baseline separation

of all peaks was achieved.

RESULTS

Cloning and sequencing of mpao, and sequence analysis. With the hope of studying the

properties of a mammalian PAO, this enzyme was purified from bovine liver following a pub-

lished protocol (34, 45). Although it was reported that ~ 20 mg of PAO could be obtained from 1

kg of bovine liver, we obtained ~ 1 mg from this mass of tissue in two separate attempts. In order

to carry out careful biochemical and kinetic studies, larger amounts of PAO are required. There-

fore, we decided to clone and sequence the gene for a mammalian PAO, and attempt to produce

the enzyme in a heterologous system. Since a mammalian peroxisomal pao gene was unavail-

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able, in order to clone such a gene, amino acid sequence information was needed. The amino acid

analysis of purified bPAO provided the N-terminal sequence, EAEAPGRGPRVLVVGGGIAGL.

The underlined segment identifies bPAO as a member of family of FAD-containing oxidases

(31) that includes human MAO-A, human MAO-B, corn (maize) polyamine oxidase (cPAO) and

an N 1-acetyl-SPM oxidase from Candida boidinii (Cb Ac-SMO) (22, 23, 26). The GXGXXG

sequence is a flavoprotein fingerprint. This motif is present in members the GR1 (glutathione

reductase) and GR2 family of flavoproteins, which have known structures (32). MAO-A, MAO-

B, cPAO and several non-amine oxidizing enzymes are known to be of the GR2-structural type.

The sequencing of 2 tryptic bPAO peptides resulted in the internal protein sequences

SEHSFGGVVEVGAHWIHGPS (Peptide-I) and LMTLWDPQAQWPEPR (Peptide-II). The

underlined segment of Peptide I aligns with the end of the FAD-containing enzyme superfamily

motif near the N-termini of these proteins (31).

Translated EST GenBank sequences were screened using these bPAO sequences, and two

mouse GenBank EST sequences (accession numbers AA437705 and AI098814) containing a

translated sequence very similar to Peptide I (85% identity) were identified. The plasmid DNA

for AA437705 and AI098814 were purified and sequenced. Both clones were truncated at the 5'-

end. The DNA from EST clone AA437705, with a longer mpao insert, was subjected to restric-

tion-enzyme digestions and purified. A 968-bp segment (mpao1) was excised from this clone and

sequenced. The mpao1 cDNA fragment was used as a library-screening probe (EXPERIMENT-

AL PROCEDURES section). This process allowed us to clone a nearly complete mpao cDNA

(mpao2; 1710 bp) from the mouse embryo library. The sequence of the full-length mpao (1770

bp; Fig. 2) was obtained by combining the sequence information obtained for mpao2 and from a

5'-RACE PCR procedure. However, we failed to obtain the missing 5'-end of bpao. A search of

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GenBank revealed that mpao maps to murine Chromosome 7 (cytogenic position 7F4) as 7 exons

(GenBank accession number NW_000335).

[Figure 2]

During the review of the current work, a paper by Vujcic, et al. (46) appeared that

reported the cDNA sequences for mPAO and human PAO (hPAO). These sequences were found

by BLAST searching GenBank using the SMO cDNA sequence. Although the details are not

provided herein, we have also cloned and sequenced the hpao gene and submitted its sequence in

GenBank (accession number AF312698) on October 11, 2000. Vujcic, et al. (46) made no

attempt to sequence the mpao and hpao cDNA from the clones that they obtained commercially.

Compared to our cDNA sequences, their sequences differ at numerous positions. We are

confident that our bPAO, mPAO and hPAO sequences are correct because we sequenced each at

least twice; ambiguous regions were sequenced three or four times.

Vujcic, et al. (46) reported also the transient transfection of HEK-293 cultured human

kidney cells with mpao and hpao cDNA. Although these cells expressed the enzymes from the

transfected genes, no effort was made to purify the proteins for biochemical characterization (46).

A comparison of mPAO and bPAO with each other and with other known amine

oxidases, and the identification of these as peroxisomal proteins. The full-length mpao cDNA

(1770 bp) (GenBank accession number AF226656) and deduced mPAO amino acid sequences

are presented in Fig. 2. Upstream from the ATG start codon, there is a single TAA stop codon.

No other possible translational start sites were found upstream from this ATG codon. The mpao

coding region terminates with a single TGA stop codon. The gene contains a 1512-bp open

reading frame that encodes for 504 amino acids. The mature apo-protein (minus the N-terminal

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Met) has a Mr = 55,316 Da. The incompleted bpao nucleotide (1625 bp) and deduced amino acid

(452 aa) sequences can be found at GenBank (accession number AF226658). Its 5'-end nucleo-

tide sequence is missing. The coding region of bpao terminates with by a single TGA stop codon.

Both mpao and bpao have an ATAAA sequence as polyadenylation signals that are near to the

poly-A tails.

An inspection of the bPAO and mPAO sequences (3) indicates the presence of the peroxi-

somal targeting signal sequence, -P-R-L, at the C-termini of these proteins. This consensus

sequence -(S/A/C/P)-(K/H/R)-(I/L/M) (47), which is not cleaved after protein import into the

peroxisome, is seen also in the Cb Ac-SMO and MAO-N sequences (i.e. -S-K-L and -A-R-L,

respectively) (Fig. 3), indicating that these enzymes reside also in the peroxisomes of the

respective host yeast cells. Like many oxidases, PAO is localized in the peroxisomes where its

oxidation product H2O2 can be degraded by catalase.

[Figure 3]

A CLUSTALW (version 1.8) alignment of many (but not all) known flavoprotein amine

oxidase amino acid sequences is provided in Fig. 3. Among these sequences, there are two re-

gions of high similarity. One, near the N-termini, is clearly a ��consensus domain that inter-

acts with the ADP moiety of FAD (32, 33, 49). The second conserved region, near the C-termini,

is involved also in FAD binding. The C-terminal region contains a conserved region that harbors

the Cys residues that are covalently linked to FAD in the human monoamine oxidases: Cys-406

(MAO-A) and Cys-397 (MAO-B) (24, 25). For bPAO and mPAO, a Ser (Ser429 of mPAO)

aligns with these Cys residues. We have determined that mPAO bind FAD noncovalently (see

the EXPERIMENTAL PROCEDURES section).

The CLUSTALW analysis (Fig. 3) provided the following percent identities (percent

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similarities) between mPAO and the other flavin-containing amine oxidases: bPAO, 73 % (82

%); human and murine SMO, 37 % (53 %); Micrococcus rubens PUT-Ox, 18 % (32 %); cPAO,

17 % (34 %); Salmo gairdneri MAO, 16 % (30 %); and Mycobacterium tuberculosis amine

oxidase, 17 % (30 %); human MAO-B, 15 % (30 %); human MAO-A, 14 % (30 %); Candida

boidinii N 1-acetyl-SPD oxidase, 13 % (32 %); Micro luteus tyramine oxidase, 13 % ( 28 %);

Aspergillus niger MAO-N, 12 % (25 %). Overall, the amino acid sequences identity between

mPAO and the other protein is low, generally less than 20 %, except for the 37 % identity with

the newly discovered human and murine SMO. This indicates that peroxisomal PAO represents a

new subfamily of mammalian amine oxidases.

The distribution of mpao mRNA in murine tissues. The availability of mpao cDNA

allows, for the first time, the determination of the transcription level of this gene in mammalian

tissues. We probed PCR-amplified mRNA of murine tissue from numerous organs and murine

tissues at different developmental stages (see the EXPERIMENTAL PROCEDURES section).

mpao mRNA is detected in all the murine tissues tested (Fig. 4), with the liver and stomach

having the highest levels. This is in accord with earlier finding of large levels of mPAO in the

liver of various mammals (34, 37, 50, 51). Lesser, but significant levels of mpao were detected in

heart, spleen, thymus, small intestine, muscle, pancreas, uterus, and breast at various develop-

mental stages. Relatively lower levels of mpao mRNA are expressed in brain, kidney, lung,

testis, skin, adrenal gland, and prostate gland. The 100x panel (Fig. 4) clearly shows that the

mRNA level increases during embryonic development; there is a gradual increase in the tissues

on going from 8.5-day to 19-day embryos. mRNA levels change also with breast development.

Fig. 4 shows that level of mpao mRNA is very low in the virgin breasts, is quite high in the

pregnant breasts, but is decreased in lactating and involuting breasts. These findings for breast

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and embryo were confirmed by repeating this analysis with a murine multiple-tissue panel from a

different lot (Origene). Apparently, high mpao transcription, and presumably translation is

important in tissue growth and development (1). These data suggest that mpao expressions are

regulated by growth hormones. It has been proposed that PAO, via its participation in the poly-

amine interconversion pathway, is an important regulator for maintaining cellular polyamine and

tissue homeostasis.

[Figure 4]

Currently, we do not know how these mRNA levels relate to the amount of the mPAO

protein in different murine tissues. However, a study measuring PAO enzyme activity in various

rat tissues has been reported (1). High activity was found in the pancreas and liver. Lower, but

significant activity was seen in spleen, kidney, small intestines, testes, prostate tissue, thymus,

brain, heart, and lung, and very low activity was observed in skeletal muscle. It was found that

PAO activity increased from a low level at birth to quite high levels at 70 days postnatal and

beyond in rat brain and liver.

It is important to note that Vujcic, et al. (46) reported the relative levels of the transcripts

for pao and smo in numerous normal and neoplastic human tissues. For the few normal tissues

probed, the agreement with our findings for murine tissues is fair, except that no pao mRNA was

detected in normal human spleen.

Heterologous production of mPAO by E. coli and characterization of the pure enzyme.

The cloned mpao gene was heterologously expressed in E. coli, and active mPAO was purified to

homogeneity and characterized. PAGE, gel-filtration and ion-exchange chromatography, and

mass spectral analysis indicated that the highly purified, homogeneous enzyme is a monomer of

the expected molecular mass. Fig. 5 displays the UV-visible spectrum of the pure oxidized

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enzyme. The �max values (and relative absorbances) for the protein are 274 (1.0), 377 (0.09), and

456 (0.11). The calculate 274 = 66,000 M�1 cm�1 for the protein component, based on the amino

acid composition (52), and an estimated 274 = 26,000 M�1 cm�1 for the bound FAD (assuming

1mol of FAD/mol of protein) (53), provide an 274 � 89,000 M�1 cm�1. Using this value, and

assuming that the 458 is the same as free FAD, i.e., 11,300 M�1 cm�1, the estimated molar ratio of

protein to FAD is 1:0.90. This supports the contention of 1 mol of noncovalently bound

FAD/mol of enzyme. The pI and the Mr values for apo-mPAO (minus the N-terminal Met),

calculated from the amino acid composition, are 4.84 and 55,316 Da, respectively, while the

calculated Mr of the holo-enzyme (FAD-containing) is 56,101 Da. (Because of the negative

charges of the phosphate groups of FAD, the pI value is expected to be somewhat lower than

4.84). ESI mass spectral analysis of purified mPAO gave a Mr = 55,311 �� mass units, in perfect

agreement with that deduced from the cDNA-translated protein sequence.

[Figure 5]

mPAO was titrated anaerobically with a standardized solution of sodium dithionite (Fig.

5). A significant amount of the one-electron reduced flavin radical formed in the initial phase of

the titration, and disappeared in the final phase as it converted to the two-electron fully reduced

form of bound FAD. The intermediate one electron-reduced species was predominantly the

anionic (so-called Ared@) radical, but a trace of the neutral (so-called Ablue@) radical was evident

also by the low absorbance in the 500-650 nm region of the spectrum (Fig. 5) (54). From this

titration, the 458 for the bound FAD was found to be 10,600 M�1cm�1, while the 274 was

determined to be 99,200 M�1cm�1.

mPAO was titrated anaerobically with N 1-acetyl-SPD (data not shown). We chose N 1-

acetyl-SPD as the mPAO reductant rather than the better substrate N 1-acetyl-SPM because we

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want to avoid the possible slow reduction of the high-concentration enzyme by SPD, the N 1-

acetyl-SPM-oxidation product. Since mPAO oxidizes N 1-acetyl-SPM, N 1-acetyl-SPD or SPM at

the exo-carbon of secondary amino groups (vide infra), there is no chance that the N 1-acetyl-SPD

oxidation product PUT (which does not have a secondary amino group) would be oxidized

during the titration. Based on the A458, the concentration of enzyme was 1.20 �M, whereas, a

concentration of 1.16 �M was determined from the N 1-acetyl-SPD titration; as expected 1mol of

substrate reduced 1 mol of FAD. Thus, all enzyme molecules in the preparation are capable of

oxidizing the substrate. No trace of a flavin radical was detected during this titration. This

indicates rapid transfer of 2 electrons from enzyme-bound substrate to enzyme-bound FAD.

The steady-state kinetic properties of mPAO. It was assumed that steady-state mechan-

ism for the oxidation of the various polyamine derivatives listed in Table 1 is of the ping-pong

type. This is supported by the fact that kcat /KS values for assay done in air-saturated (0.237 mM

O2) and pure O2-saturated (1.2 mM O2) buffers were approximately equal. Further support for

this contention is provided by the kcat /KO values for N 1-acetyl-SPM and N 1-acetyl-SPD. These

values are equal (Table 1), as expected for a ping-pong type mechanism (42).

[Table 1]

The steady-state kinetic studies indicated that N 1-acetyl-SPM is the best substrate for

mPAO, although N 1-acetyl-SPD is also a good substrate (Table 1). The kcat /KS value (the so-

called Aspecificity constant@) for the former substrate is over an order-of-magnitude higher than

for the latter. While SPM can be oxidized by the enzyme, it is much less efficient than for the

oxidation of N 1-acetyl-SPM or N 1-acetyl-SPD; the kcat /KSPM value is four orders-of-magnitude

lower than that for N 1-acetyl-SPM. It was found that SPD, PUT, N 8-acetyl-SPD and benzyl-

amine were not oxidized by mPAO.

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Vujcic, et al. (46) expressed cloned hpao and mpao genes in a human kidney cell line and

determined that these cells oxidized substrates with the following preference: N 1-acetyl-SPM ��

N 1-acetyl-SPD > N 1,N 12-diacetyl-SPM >> spermine. These findings are basically the same as

those reported herein.

Interestingly, both BESPM and BENSPM are fairly good mPAO substrates, both being

better than SPM (Table 1). This is an important finding because BENSPM has been used for

Phase II cancer clinical trials (55). These N-ethylated polyamines have been used widely also to

study the physiological effects of polyamine metabolizing enzymes. They down-regulate

polyamine biosynthetic enzymes, but dramatically up-regulate SSAT synthesis (13, 14), which

results in mammalian cells becoming apoptotic.

It has been reported that terminally alkylated polyamine analogs like BESPM and

BENSPM are oxidatively dealkylated by PAO; for BESPM and BENSPM this would result in

the formation of N 1-ethyl-SPM and N 1-ethyl-nor-SPM, respectively, and acetaldehyde (56-59).

In contrast, it was reported recently that the lysates of cultured human cells transiently transfected

with mpao or hpao cDNA did not dealkylated BESPM and BENSPM, but converted them to N 1-

ethyl-SPD and N 1-ethyl-nor-SPD ({3-[(3-aminopropyl)amino]propyl}ethylamine), respectively,

and N-ethyl-3-aminopropanal. It was not possible for us to resolve this dilemma by analyzing the

products formed when BESPM or BENSPM are oxidized by pure mPAO, as was done for N 1-

acetyl-SPM and N 1-acetyl-SPD (vide infra). The appropriate reference compounds (i.e., N 1-

ethyl-SPM, N 1-ethyl-nor-SPM, N 1-ethyl-SPD, N 1-ethyl-nor-SPD or N-ethyl-3-aminopropanal)

are not available commercially and are not conveniently synthesized.

Inspection of KI values (Table 1) indicates that SPM and PUT (but not benzylamine) are

weak inhibitors for the oxidation of N 1-acetyl-SPM (Table 1), whereas, N 8-acetyl-SPD and SPD

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are somewhat better inhibitor. All of these compounds are competitive inhibitors, since in assay

with each inhibitor at levels that would produced significant inhibition, the inclusion of N 1-

acetyl-SPM at a saturating concentration eliminated the inhibition.

Ideally, we would like to compare the biochemical and kinetic properties of the E. coli-

produced mPAO with that isolated from a natural source. However, this was not feasible for

several reasons. First, it is not be prudent to compare the E. coli-produced mouse enzyme with

the enzyme purified from bovine liver because of the species difference. Furthermore, the specif-

ic activities obtained from two different purifications of the bPAO were not the same, and were

lower than expected for a fully functional enzyme. Since only 1 mg of PAO could be obtained

from 1 kg of bovine liver, it would be difficult to obtain sufficient quantities of PAO from mouse

liver for comparative studies. We attempted to obtain a full-length bpao coding cDNA fragment.

Unfortunately, we could not identify the appropriate clones from any cDNA library that was

screened. Therefore, we were not able to heterologously produce bPAO for comparison with the

enzyme obtained from bovine liver. Furthermore, a kcat/KS value of 2.54 � 106 M-1 s-1 for

oxidation of N 1-acetyl-SPM by mPAO is that expected for a native, fully active enzyme (60).

Additionally, recombinant mPAO is a highly stable, monomeric protein that maintains its

biochemical, redox and kinetic properties even after prolonged storage. Finally, 1 mol of enzyme

FAD is reduced efficiently by 1 mol of substrate in the reductive titration (vide supra). Thus, we

are confident that this E. coli-produced mPAO is in its native, fully active form.

The nature of the product resulting from the oxidation of N 1-acetyl-SPM and N 1-acetyl-

SPD by mPAO. Using the HPLC method describe in the EXPERIMENTAL PROCEDURES

section, it was found that complete oxidation of N 1-acetyl-SPM and N 1-acetyl-SPD by mPAO

yielded 96 and 94 % (based on 1 mol of aldehyde/mol of substrate), respectively, of the 2,4-dini-

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trophenylhydrazone of 3-acetamidopropanal (Fig. 6). There was no trace of any other 2,4-dinitro-

phenylhydrazone such as would be seen if the enzyme oxidized the substrates on the endo-side of

the substrates N 4-nitrogens. We expect that the phenylhydrazone of this aldehyde, because of its

positive charge would have very short HPLC retention times; the retention time of the 2,4-

dintritophenylhydrazone of 3-aminopropanal (a reference compound that is positively charged in

the HPLC solutions containing trifluoroacetic acid) has a much shorter retention times than the

same derivatives of 3-acetamidopropanal and acrolein, which are uncharged.

[Figure 6]

The acetyl group of 3-acetamidopropanal did not hydrolyzed during the enzymic reaction

or during the workup preceding the analyses. This hydrolysis would produce 3-aminopropanal,

which can spontaneously convert to acrolein (18). However, the 2,4-dinitrophenylhydrazones of

neither 3-aminopropanal nor acrolein were detected in the HPLC analyses of the enzyme reaction

solutions (Fig. 6).

To prove definitively that 3-acetamidopropanal was the true product of these enzymic

oxidations, a larger scale reaction between N 1-acetyl-SPM and mPAO was carried out, and the

2,4-dintorphenylhydrazone of the product aldehyde was isolated in 89 % yield. The chemical

properties of this compound and the 2,4-ditrophenylhydrazone of 3-acetamidopropanal generated

by organic synthesis were compared. The two substances were identical in all respects, and 1H

NMR and mass spectral analyses prove incontrovertibly that 3-acetamidopropanal is the enzymic

oxidation product.

Using an HPLC method to analyze dansylate polyamines, it was found that complete

mPAO oxidation of N 1-acetyl-SPM and N 1-acetyl-SPD produced 95 % SPD and 91 % PUT

(based on 1 mol/mol of substrate), respectively (data not shown). Another research group

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exposed these substrates to lysates from HEK-293 cultured human kidney cells transiently

transfected with the genes for hpao or mpao (46). By using an HPLC method similar to that

describe herein for analyzing dansylated polyamines, they found also that mPAO and hPAO

converted these substrates to SPD and PUT, respectively.

These observations indicate that N 1-acetyl-SPM and N 1-acetyl-SPD are always oxidized

at the carbon on the exo-side of their N 4-nitrogens. Thus, there can be no doubt that mPAO is the

classical polyamine oxidase that has been described and studied over the past few decades.

DISCUSSION

Peroxisomal PAO, an integral component of polyamine interconversion pathway, is an

important player in regulating cellular polyamine levels. Thus, understanding the precise bio-

chemical and structural properties of PAO are essential for a deeper understanding of its partici-

pation in many fundamental cellular processes. With this in mind, we set out to develop a system

that would provide, in good yield, a highly purified preparation of a mammalian peroxisomal

PAO. In the course of accomplishing this goal, we clone and sequenced the entire mpao (Fig. 2)

gene and most of the bpao gene (GenBank accession number AF226658). Based on a

comparison of primary structures (Fig. 3), the sequence identity with other flavin-containing

amine oxidases is less than 40 %. This indicates that PAO represents a new subfamily of

flavoproteins.

Inspection of the translated mPAO and bPAO sequences indicated the presence of a

�1��2 FAD-binding fingerprint motif (Fig. 3) that interacts with the ADP moiety of the enzyme-

bound FAD (26, 33). This motif along with numerous other conserved regions are found in the

“FAD-binding domain” and are elements of the Rossmann fold (Fig. 3). Two other conserved

regions are located in the “substrate-binding domain” (26, 33).

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Cys406, Cys397, and Cys399, the residues that are covalently attached to the 8�-carbon

of the isoalloxazine ring of FAD in of MAO-B, MAO-A (24, 25, 33), and fMAO, respectively,

are pointed out in the aligned sequences of Fig. 3. These Cys residues align with Ser residues in

bPAO, mPAO (Ser429) and the murine and human SMO (Ser481 in both). However, except for

the mitochondrial monoamine oxidases, FAD is noncovalently bound to all known amine

oxidases of this family.

As with MAO-A, MAO-B (61, 62) and MAO-N (63), mPAO forms an intermediate

anionic (Ared@) radical when titrated with dithionite. This indicates that there is either a positively

charged amino acyl group (i.e., Arg or Lys), or the positive end of an �-helix dipole is near the

N1 position of the flavin=s isoalloxazine ring. This positively charged environment stabilizes the

red radical=s negative charge, which is localized at the N1/C2/C2O locus of the isoalloxazine ring

of the flavin. In the MAO-B and cPAO structures, the positive end of an �-helix (Met438-

Met454 for MAO-B, Fig. 3; His469-Gln487 for cPAO). In the MAO-B and cPAO structures,

these helices interact with the N1/C2/C2-O locus of FAD (26, 33). These segments of cPAO and

MAO-B align with the highly conserved region near the C-termini (25) of the other flavoproteins

amine oxidases (Fig. 3; for mPAO, Thr475 - Gln496), and the secondary structure prediction

program APsi-Pred@ (version 2, at the web site - http://insulin.brunel.ac.uk/psipred/) indicated that

this region of mPAO forms a �-helix.

While SPM can be oxidized by peroxisomal mPAO, it is a poor substrate when compared

to N 1-acetyl-SPM and N 1-acetyl-SPD. SPM and SPD are acetylated for transport from the cells

and eventual excretion from the body (1). High PAO levels could prevent transport of N 1-acetyl-

SPM and N 1-acetyl-SPD from the cell, and increase the SPD and PUT levels of the cell’s poly-

amine pool (Fig. 1). In contrast, hSMO and mSMO oxidize SPM (KS = 18 �M) (10) but not N 1-

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acetyl-SPM or N 1-acetyl-SPD (10, 11). In addition to the absence a peroxisomal transport signal

at the C-termini of hSMO and mSMO (they also do not have a transport signal at their N-termini,

suggesting that they are cytosolic enzymes), there are other significant sequence differences

between these and mPAO (Fig. 3).

Polyamine interconversion involving peroxisomal PAO helps maintains the intracellular

balance of these substances. It has been proposed that in some cells under stress, polyamine

oxidation generates the toxic byproducts H2O2 and 3-aminopropanal (generated by enzymatic

deacetylation of 3- acetamidopropanal, the product of N 1-acetyl-SPM oxidation by PAO; Fig. 1),

which can initiate cell death (15-17, 64-69). In addition 3-aminopropanal can spontaneously

convert to the extreme cytotoxin acrolein (18). H2O2 can be inactivated by catalase in peroxi-

somes, unless the levels of N 1-acetyl-SPM and PAO are extremely high (or the catalase level

low), which seems to be the case in some pre-apoptotic cells. It is believed also that the level of

SSAT, which produces N 1-acetyl-SPD and N 1-acetyl-SPM from the polyamine pool, is elevated

in these cells (15-17, 64-68). However, in some cultured cancer cells, the levels of SSAT, and

thus, the N 1-acetylated polyamines are high, but PAO is low. In fact, the level of PAO activity

decreases as the histological grade of breast cancer tumors increases (70). From these observa-

tions, it can be proposed that a PAO-dependent apoptosis-initiation mechanism is intact in some

precancerous cells. However, events take place whereby PAO activity is interrupted, shutting

down cell death, and cellular proliferation ensues.

In contrast, for tissue damaged by ischemia/reperfusion, the level of SSAT, N 1-acetylate

polyamines, and PAO increase (19-21). This results in the production of high levels of H2O2 and

3-aminopropanal (and probably acrolein), which contributes to tissue damage.

With the work described herein, a program has been initiated to study the detailed chemi-

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cal, biochemical, structural, kinetic, inhibition and mechanistic properties of a mammalian per-

oxisomal PAO. Hopefully, this will lead to a richer appreciation of its involvement in apoptosis,

cellular proliferation, cell signaling, tissue damage, wound healing, tissue development and

differentiation, etc, and aid in the development of clinically relevant approaches to treat cancer

and ameliorate ischemic tissue damage.

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Table 1. Steady-state kinetic parameter for the reaction of various amines (S) and O2 (O) with pure mPAO.a

Compound (S)

kcat�

b (sec�1)

KS �

b (µM)

kcat�/KS�

b (M�1sec�1)

KI = KD (µM)

N 1-Acetyl-SPM

4.53 ��0.05 1.78 � 0.10 (2.54 � 0.01)��106

NAc

N 1-Acetyl-SPD

4.85 � 0.03 36.8 � 1.1 (1.32 � 0.03)��105

NDc

N 8-Acetyl-SPD

0 0 0

70 �7 Benzylamine

0 0 0

SPM 0.175 � 0.005 716 � 33 (2.47 � 0.01)��102

750�80

SPD

0 0 0

190�20 PUT

0 0 0

1,000�100

BESPM 0.415 ± 0.012 150 ± 10 (2.77 ± 0.13) x 103 ND BENSPM 1.93 ± 0.03 157 ± 7 (1.27 ± 0.03) x 104 ND Compound (S)

kcat

d (sec�1)

KS

e (µM)

kcat/KO

d (M�1sec�1)

KO

d (µM)

N 1-Acetyl-SPM

8.0 � 0.8 3.1 � 0.3 (4.4 � 0.4) ��104

180 � 20

N 1-Acetyl-SPD

13 � 1 83 � 8 (4.3 � 0.4) ��104

301 � 30

a Assays were done at 30 �C, in 50 mM KH2PO4/KOH, pH 7.6. b These apparent values were determined

from assay done with air-saturated buffer, for which the dissolved [O2] (= 237 mM) is not saturating.

Since the reactions of the N 1-acetylated polyamines and O2 with mPAO obeys a ping-pong type mechan-

ism, the apparent values, kcat�/KS�, are equal to the true kcat /KS values. c NA indicates not applicable, and

ND indicates not determined. d The true kcat , KO, and kcat /KO values determined from by progress-curve

analysis of dissolved O2 consumption in the presence of saturating N 1-acetylated polyamine substrate.

The concentration of the substrate was assumed to be high enough to overcome any inhibition by the

polyamine product formed during the reaction. The buffer was saturated with pure O2 gas (= 1.12 mM).

The errors were estimated to be about 10 %. e The Atrue@ KS values calculated from the apparent values

determined in air-saturated buffer. The KS values were calculated from the equation KS =

KS�(1 + KO /[O2]).

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FIGURE LEGENDS

Figure 1. The oxidation of N 1-acetyl-SPM and N 1-acetyl-SPD by the mammalian peroxisomal

flavoprotein, PAO. The figure shows that the oxidation of enzyme-bound N 1-acetyl-SPM pro-

duces 2-electron reduced FAD and 1 mol/mol each of 3-acetamidopropanal and SPD. The oxida-

tion of reduced FAD by O2 produces and 1 mol/mol of H2O2. N 1-Acetyl-SPD is oxidized similar-

ly to produce 1mol/mol each of PUT, 3-acetamidopropanal and H2O2. The structures of N 1-

Acetyl-SPD/PUT differ from those of N 1-acetyl-SPM/SPD by the lack of 3-aminopropyl groups.

(The dashed line differentiates the structures and reactions for the two substrates). The

numbering system used in this paper is shown with the upper-left structure. The endo- and exo-

carbon centers discussed herein are indicated in the middle-left structure. R represents the ribityl-

ADP portion of FAD.

Figure 2. The DNA and translated protein sequences for mPAO. The overlined segment repre-

sents the 5'-segment that was missing from the original truncated mpao1 clone. This sequence

was obtained by using the 5'-RACE PCR method. The underlined nucleotide sequence denotes

the region corresponding to the antisense primer (bases 333 to 309) used for the PCR 5'-

extension experiment. The underlined portions of the amino acid sequence correspond to the

regions of bPAO that were sequenced by the Edman degradation method, and the asterisk

denotes the stop codon.

Figure 3. Alignment of the protein sequences of various flavoprotein amine oxidases. The

alignment and (default) shading was accomplished using CLUSTALW (version 1.8) within the

BioEdit8 Program, version 5.0.9 (Dept. of Microbiology, North Carolina State University) (48).

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Aligned are bovine (peroxisomal) PAO (bPAO), murine (peroxisomal) PAO (mPAO), human

SMO (hSMO; GenBank accession number AY033889) (10, 12), murine SMO (mSMO;

GenBank # BC004831) (11), human MAO-A (hMAO-A; GenBANK # M69226) (24), human

MAO-B (hMAO-B; GenBank # M69177) (24), Salmo gairdneri (trout liver) MAO (fMAO;

GenBank # L37878), Mycobacterium tuberculosis amine oxidase (Mt AmOx: GenBank #

AL021646), PUT oxidase from Micrococcus rubens (Put-Ox; GenBank # D12511) (29), Zea

mays (corn) PAO (cPAO; GenBank # AJ002204) (23, 26), Micrococcus luteus tyramine oxidase

(Ml TyrOx; GenBank # 3298360) (30), Aspergillus niger MAO (MAO-N; GenBank # L38858),

and Candida boidinii N 1-acetyl-SPD oxidase (Cb Ac-SPMO; GenBank #AB018223) (22). The

question marks for the bPAO sequence indicate a region of unknown composition. The

composition of the segment preceding this region was obtained by protein sequencing, while the

sequence following this region was deduced from the translated-cDNA sequence. The cPAO

sequence is the only one with a recognizable N-terminus transport signal sequence, which is

underlined. At the C-termini of bPAO, mPAO, MAO-N and Cb Ac-SMO, the tripeptide

peroxisomal-transport signals are indicated by asterisks. The position of Cys residues that are

covalently linked to the FAD in MAO-A, MAO-B (24, 25) and fMAO are indicated by the I

symbol. The = symbols above the sequences indicate regions that are highly conserved in this

alignment. For example, the regions labeled Beta-1, Alpha and Beta-2 are components of the

�1��2 motif near the N-termini that interacts with the ADP portion of FAD. The positive end of

the alpha helix of this motif interacts with the diphosphoryl group of the ADP moiety. The

regions labeled “Fl” or “Flx” are in the flavin-binding domains of MAO-B and cPAO (26, 33).

These regions constitute elements of the Rossmann fold. The helix that has its positive end

interacting with the N1/C2/C2-O locus of FAD is labeled “Flx” (near the C-ternimi) (33). The

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regions labeled “Sub” are conserved regions in the substrate-binding domain, which are remote

from the FAD and seemingly remote from the substrate/inhibitor-binding site (26, 33). The

extended C-terminal regions of MAO-A, MAO-B and fMAO anchor these proteins to the outer

surface of mitochondrion (33).

Figure 4. Agarose electrophoresis of ARapid-Scan Gene Expression Panel@ PCR-amplified mpao

cDNA samples for 24 major mouse tissues and developmental stages. The right frame presents

the results for the PCR-amplified cDNA of a 540-bp portion of the �-actin gene for each tissue,

which is the control. The middle frame presents the results for the PCR-amplified cDNA of a

570-bp portion of mpao for each tissue using a high level of first-strand cDNA for the PCR

reaction (the 100x panel). The right frame displays the results for the panel using 100-times

lower first-strand murine cDNA (the 1x panel). The total mRNA of each tissue was subjected to

oligo-dT selection, and the first-strand cDNA used for the PCR reactions for each tissue were

generated from the poly-A+ mRNA using oligo-dT primers and MMLV reverse transcriptase.

The amplified fragments were electrophoresed on an agarose gel, and the intensity of the

ethidium bromide-stained bands provided a measure of the level of mpao mRNA in each tissue.

Figure 5. The anaerobic dithionite (DT) titration of pure mPAO. The titration was done in a one-

mL, one-cm path anaerobic cuvette, in 50 mM KH2PO4/KOH buffer, pH 7.6, at 21 �C. The

concentration of the standardized sodium DT solution was 0.541 �M. Panel A shows the spec-

trum of the oxidized enzyme (- - - - - -), those obtained at the beginning of the titration (solid

lines; 2.16 and 4.33 nmol of DT added), and that of fully reduce enzyme (─ ─ ─ ; 17.3 nmol DT

added). The arrows indicate the direction of the absorbance changes that occurred as more DT

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was added. In the 380-nm region, the increase in absorbance indicates the formation of the red

radical, while the small increase in the 550 - 700 nm region indicates the formation of a small

amount of the blue radical (54). Panel B displays the spectral changes that occurred in the latter

phase of the titration. The arrows indicate the direction of the absorbance changes that took place

as progressively more DT was added: 4.33, 6.49, 8.66, 10.8, 13.0, 15.1 and 17.3 nmol. Although

impossible to see in this reproduction, the absorbance in the 550 - 700 nm region increased

slightly then decreased during this phase of the titration. The inset the Panel B shows a graph of

A377, A458 and A590 vs the amount of DT added. From this plot, it was determined that 15.2 nmol

of DT were required to fully reduce the enzyme sample. Panel C displays the spectra of the fully

oxidized (─ ─ ─ ─), the radical (──────), and the fully reduced (- - - -) forms of FAD bound

to mPAO that resulted from the Factor Analysis of the titration data presented in Panels A and B.

Figure 6. HPLC analyses of the aldehyde products generated when N 1-acetyl-SPM and N 1-

acetyl-SPD are oxidized completely by mPAO. The details of the 2,4-dinitrophenylhydrazine

derivatizations and the analyses are presented in the EXPERIMENTAL PROCEDURES section.

The upper chromatogram is for the analysis of a standards solution that contained 0.8 mM each

of 3-aminopropanal, 3-acetamidopropanal and acrolein (each peak represents 10 nmol of the 2,4-

dinitrophenylhydrazone of each aldehyde). The middle and lower chromatograms are for the

analyses of the N 1-acetyl-SPM and N 1-acetyl-SPD reactions, respectively; 0.8 mM substrate at t

= 0. Unreacted 2,4-dinitrophenylhydrazine eluted at 4.5 min. The peaks at 8.4 min are due to the

2,4-dinitrophenylhydrazone of acetaldehyde, which is a trace contaminant of the ethanol solution

used to dissolve 2,4-dinitrophenylhydrazine. The other minor peaks are of unknown origin.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Tianyun Wu, Victoria Yankovskaya and Wiliam S. McIntire-acetylated polyamine oxidase1flavoprotein, N

Cloning, sequencing, and heterologous expression of the murine peroxisomal

published online March 26, 2003J. Biol. Chem.

10.1074/jbc.M302149200Access the most updated version of this article at doi:

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