Human recombinant monoamine oxidase B as reliable and efficient enzyme source for inhibitor screening

Article abstract of DOI:10.1016/j.bmc.2005.06.043

Interest in inhibitors of monoamine oxidase type B (MAO B) has grown in recent years, due to their therapeutic potential in aging-related neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. This study is devoted to the use of

Full text of DOI:10.1016/j.bmc.2005.06.043

Bioorganic & Medicinal Chemistry 13 (2005) 6212–6217
Human recombinant monoamine oxidase B as reliable
and efficient enzyme source for inhibitor screening
Laura Novaroli,a Marianne Reist,a Elisabeth Favre,a Angelo Carotti,b
Marco Cattob and Pierre-Alain Carrupta,*
a
´ ´ `
LCT-Pharmacochimie, Section des sciences pharmaceutiques, Ecole de Pharmacie Geneve-Lausanne, Universite de Geneve,
`
30 Quai Ernest Ansermet, CH-1211 Geneve 4, Switzerland
bDipartimento Farmaco-Chimico, University of Bari, I-70125 Bari, Italy
Received 3 May 2005; revised 14 June 2005; accepted 21 June 2005
Available online 27 July 2005
Abstract—Interest in inhibitors of monoamine oxidase type B (MAO B) has grown in recent years, due to their therapeutic poten-
tial in aging-related neurodegenerative diseases, such as ParkinsonÕs disease and AlzheimerÕs disease. This study is devoted to the
use of human recombinant MAO B obtained from a Baculovirus expression system (SupersomesTM MAO B, BD Gentest, MA,
USA) as reliable and efficient enzyme source for MAO B inhibitor screening. Comparison of inhibition potencies (pIC50 values)
determined with human cloned and human platelet MAO B for the two series of MAO B inhibitors, coumarin and 5H-inde-
no[1,2-c]pyridazin-5-one derivatives, showed that the difference between pIC50 values obtained with the two enzyme sources
was not significant (P > 0.05, StudentÕs t-test). Hence, recombinant enzyme is validated as convenient enzyme source for
MAO B inhibitor screening.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
posed to be involved in the formation of free radicals
and other neurotoxic species. In order to restore a suffi-
cient striatal dopamine level, MAO B inhibitors such as
l-deprenyl are currently coadministrated with L-DOPA
in the symptomatic treatment of PD.
Monoamine oxidase B (MAO B, EC 1.4.3.4) is one of two
flavin-adenine dinucleotide-depending isozymes playing
a key role in the regulation of endogenous monoamine
neurotransmitters as dopamine and serotonin.
Increased levels of MAO B have also been observed in
plaque-associated astrocytes of brains of patients suffer-
ing from AlzheimerÕs disease (AD).5 Studies have shown
that MAO B activity can increase up to 3-fold in the
temporal, parietal, and frontal cortex of AD patients
compared with controls. The increased MAO B activity
produces an elevation of hydroxyl radicals, which has
been correlated with the formation of Ab plaques. Cur-
rently, acetylcholinesterase inhibitors are the only drugs
approved for the treatment of cognitive dysfunction in
AD. Hence, MAO B inhibitors might offer an alterna-
tive in the therapy of AD.
A number of studies have shown that MAO B is in-
volved in aging-related neurodegenerative diseases,1,2
resulting in an increased interest in this enzyme as a tar-
get in the search of neuroprotective agents. Ontogenetic
studies have demonstrated that MAO B activity stays
unchanged until the 60th year of life and then increases
nonlinearly. This is in contrast to what has been
observed for most enzymes, as generally their activities
decrease with advancing years.3,4
MAO B plays a double role in the pathophysiology of
ParkinsonÕs disease (PD). It is the main enzyme impli-
cated in the metabolism of dopamine and is also sup-
These observations stress the need for new MAO B
inhibitors with antiapoptotic and neuroprotective prop-
erties for the therapy of neurodegenerative diseases.1 A
condition for the screening of new MAO B inhibitors
is a reliable screening system, that is easy to handle
and allows to obtain representative trustworthy inhibi-
tion values of human MAO B.
Keywords: Human recombinant MAO B; Human platelet MAO B;
Inhibitor screening; Neurodegenerative diseases.
*
Corresponding author. Tel.: +41 22 379 33 59; fax: +41 22 379 33
60; e-mail: pierre-alain.carrupt@pharm.unige.ch
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.043

L. Novaroli et al. / Bioorg. Med. Chem. 13 (2005) 6212–6217
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Since human platelets contain almost exclusively mono-
2. Results
amine oxidase type B, they became, in recent years, one
of the most used biological markers in MAO B-related
psychiatric diseases.6,7 Platelet MAO B has the same
amino acid sequence as human brain MAO B.8 Thus,
human platelets offer an excellent peripheral model to
indirectly assess MAO B activity in the CNS. Platelet
MAO B activity can be measured in whole blood,9 plate-
let rich plasma (PRP),10 platelet homogenate,11,12 or
even purified platelet mitochondrial fraction.13 The
higher the purity of the enzyme preparation, the smaller
the risk of erroneous results due to contaminants.
Indeed, substrates of MAO B being generally also
substrates of plasma amine oxidases, such as semicar-
bazide-sensitive amine oxidase (SSAO, EC 1.4.3.6), the
screening of MAO B inhibitors on whole blood or
PRP may lead to unfounded values.14 On the other
hand, since MAO B is located in the outer mitochondri-
al membrane of cells, its activity depends on the micro-
environment where the protein is inserted. As a
consequence, purification procedures risk to provide al-
tered enzyme with a very low specific activity, if not
done carefully. Hence, the platelet MAO B isolation
method used in this work is based on the simple separa-
tion of platelets from plasma, avoiding further purifica-
tion procedures.
2.1. Characterization of the enzymatic activity of Super-
somesTM MAO B and human platelet MAO B
To characterize the enzymatic activity of the two MAO B
sources used, Km values of kynuramine and pIC50 values
of three well-known MAO B inhibitors were determined
and compared with literature data.
The Km value of kynuramine, calculated by curve fitting
according to the classical Michaelis–Menten equation,
was 29 2 lM for SupersomesTM MAO B and 30 4 lM
for human platelet MAO B. The results obtained for
both the enzyme sources were in good agreement with
the Km values reported by Bembenek et al. (MAO B:
34 lM) on human liver mitochondria20 and McEntire
et al. (MAO B: 30 lM) on pooled platelet-rich plasma.10
Inhibition of SupersomesTM and blood platelet MAO B
by three-well known MAO B inhibitors, namely depre-
nyl, pargyline, and iproniazid, was determined (Table
1). The pIC50 values obtained corresponded well with
literature data.
2.2. Comparison of pIC50 values obtained with Super-
somesTM MAO B and human platelet homogenate for two
classes of inhibitors
Recently, expression systems permitting the produc-
tion of large quantities of purified human MAO B
have been developed. MAO B has been expressed
from human MAO B-cDNA using different transfer
vectors (e.g., plasmids15 and Baculovirus16) and
expression systems (e.g., Pichia Pastoris15). Up to
now, the characterization of the catalytic behaviour
of recombinant MAO B was mainly performed by
studying its interaction with different substrates and
by comparing the kinetic constants obtained with
those found for MAO B extracted from tissue prepa-
rations.15 Yan et al. also reported the sensitivity of re-
combinant enzyme toward a few known MAO B
inhibitors such as deprenyl and tranylcypromine.17
As MAO B is a membrane bound enzyme, the mito-
chondrial microenvironment is important for enzyme
activity and inhibitor specificity. Hence, an evaluation
of the influence on inhibitor specificities of any differ-
ences in the mitochondrial microenvironment of hu-
man platelets and insect cells with larger series of
compounds is needed.
To evaluate the reliability in using the cloned enzyme to
screen MAO B inhibitors, pIC50 values of the two series
of compounds, coumarin and 5H-indeno[1,2-c]pyrid-
azin-5-one derivatives, were determined for human
recombinant and human platelet MAO B.
pIC50 values determined for both the enzyme sources, as
well as DpIC50 values (difference between pIC50 Super-
somesTM and pIC50 human platelets), are reported in
Tables 2 and 3 for coumarin 5H-indeno[1,2-c]pyrida-
zin-5-one derivatives, respectively. As shown in Figures
1 and 2, for both the series of compounds, the correla-
tion between the inhibitory potencies determined with
the two MAO B sources, demonstrates a linear relation-
ship. Eqs. 1 and 2 describe this linear dependence for
coumarin and 5H-indeno[1,2-c]pyridazin-5-one deriva-
tives, respectively:
pIC50platelets ¼ 1.023ðꢀ0.054ÞpIC50Supersomese ꢁ0.20ðꢀ0.45Þ
n ¼ 19; r2 ¼ 0.99; s ¼ 0.10; F ¼ 1550
ð1Þ
In this study, the use of human recombinant MAO B as
a reliable and easily accessible enzyme source for inhib-
itor screening was confirmed with two larger series of
compounds, namely coumarin (n = 19) and 5H-inde-
no[1,2-c]pyridazin-5-one (n = 13) derivatives.18,19 The
reliability of the recombinant enzyme was validated by
comparison of inhibition potencies (pIC50 values) deter-
mined on human platelet and human recombinant
MAO B obtained from Baculovirus-infected insect cells
(SupersomesTM MAO B, BD Gentest, MA, USA) for
both the series of compounds. Convenience for inhibitor
screening and advantages of recombinant enzyme over
more traditional enzyme sources, as human platelets,
are discussed.
pIC50 platelets ¼ 1.12ðꢀ0.18ÞpIC50 Supersomese ꢁ 0.92ðꢀ1.35Þ
n ¼ 13; r2 ¼ 0.94; s ¼ 0.21; F ¼ 188
ð2Þ
Table 1. Comparison of pIC50 values obtained with literature data for
three known MAO B inhibitors
Literature dataa
SupersomesTM
Platelets
Deprenyl
Pargyline
Iproniazide
7.22
6.22b
4.80
7.25 0.05
6.33 0.08
4.28 0.11
7.44 0.05
6.56 0.09
4.69 0.18
a pIC50 values reported by BD Gentest using SupersomesTM as MAO B
source (posted on BD Gentest website as poster, 2002).
b The value differs from the old published value (pIC50 = 7.4).11

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L. Novaroli et al. / Bioorg. Med. Chem. 13 (2005) 6212–6217
Table 2. MAO B inhibitory activities of coumarin derivatives towards SupersomesTM and human blood platelets
a
Compound
R3
R4
R7
pIC50 human blood platelets
pIC50 SupersomesTM
DpIC50
C-2
C-4
H
H
OCH2C6H5
CH2NHC6H5
8.77 0.10
7.20 0.04
8.96 0.05
6.48 0.14
8.90 0.20
8.69 0.09
9.03 0.13
8.18 0.02
7.74 0.11
6.60 0.16
8.59 0.08
8.62 0.08
8.52 0.10
8.06 0.17
8.83 0.07
8.76 0.05
8.84 0.03
5.60 0.06
8.69 0.06
8.77 0.04
7.24 0.06
8.80 0.06
6.59 0.05
8.97 0.13
8.86 0.08
8.95 0.09
8.06 0.03
7.80 0.11
6.70 0.05
8.41 0.05
8.71 0.06
8.44 0.07
8.10 0.16
8.85 0.02
8.96 0.07
8.79 0.07
5.65 0.06
8.63 0.04
0.00
0.04
H
H
C-10
C-12
C-15
C-16
C-21
C-23
C-24
C-26
C-30b
C-50
C-54
C-56
C-60
C-61
C-71
C-75
C-85
H
CH3
C6H5
CH3
CH3
OCH2C6H5
OCH2C6H5
ꢁ0.16
0.11
0.07
H
CH3
CH3
OCH2C6H5
NHCH2C6H5
0.17
(ACH@CHA)2
OCH2C6H5
NHCOC6H5
ꢁ0.08
ꢁ0.12
0.06
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
OSO2C6H5
OSO2C6H4A40AOCH3
OCH2C6H5
OCH2C6H4A20ACN
OCH2C6H4A30AOCF3
OCH2C6H4 30ANHCOCH3
OCH2C6H4A30ACN
OCH2C6H4A30ANO2
OCH2C6F5
0.10
ꢁ0.18
0.09
ꢁ0.08
0.04
0.02
0.20
ꢁ0.05
0.05
OSO2C6H5
OCH2C(@O)C6H5
CH3
CH3
ꢁ0.06
a pIC50SupersomesTM-pIC50human blood platelets.
b 6-Hydroxycoumarin derivative.
Table 3. MAO B inhibition data for 5H-indeno[1,2-c]pyridazin-5-one derivatives towards SupersomesTM and human blood platelets
a
Compound
R
pIC50 human blood platelets
pIC50 SupersomesTM
Dp IC50
IP-12
IP-15
IP-17
IP-33
IP-34
IP-40
IP-43
IP-47
IP-48
IP-51
IP-53
IP-55
IP-57
CH2C6H5
t-CH@CHC6H5
5.81 0.15
7.57 0.12
7.89 0.22
7.68 0.11
7.02 0.14
7.31 0.17
6.75 0.13
8.64 0.21
8.35 0.29
8.26 0.10
7.57 0.12
6.09 0.13
7.98 0.14
6.12 0.12
7.58 0.06
8.20 0.03
7.37 0.04
7.13 0.13
7.37 0.07
6.72 0.13
8.25 0.16
8.09 0.04
8.27 0.13
7.48 0.11
6.28 0.22
8.08 0.05
0.31
0.01
20-Naphthyl
0.31
C6H4A30AOCH3
C6H4A40AOCH3
C6H4A30AF
ꢁ0.31
0.11
0.06
C6H4A20ACl
C6H4A30ABr
C6H4A40ABr
C6H4A40ACF3
C6H4A30ACN
C6H4A20ANO2
C6H4A40ANO2
ꢁ0.03
ꢁ0.39
ꢁ0.26
0.01
ꢁ0.09
0.19
0.10
a pIC50 SupersomesTM-pIC50 human blood platelets.
where n is the number of compounds investigated, r2 the
squared correlation coefficient, s the standard deviation
of the residuals, and F the Fischer test for significance of
the equation. 95% confidence limits are given in
parentheses.
The mean of the differences between the pIC50 values
obtained for SupersomesTM and those determined for
human platelets (DpIC50) is 0.01 ( 0.11) for the couma-
rin series and 0.002 ( 0.220) for 5H-indeno[1,2-c]-
pyridazin-5-one derivatives; 95% confidence limits are
given in parentheses. Statistical analysis (paired two-
tailed StudentÕs t-test) corroborates the assumption of
an identical inhibitor specificity of human recombinant
and human platelet MAO B. Indeed, the difference be-
tween the pIC50 values obtained for the two enzyme
sources resulted to be not significant (P > 0.05).
A closer look at Eqs. 1 and 2 shows that identical inhib-
itor specificity of the two MAO B sources can be as-
sumed. In fact, for both the series of compounds, the
slope is one and y-intercepts are zero within the limits
of experimental error.

L. Novaroli et al. / Bioorg. Med. Chem. 13 (2005) 6212–6217
6215
satisfies both of these requirements. Subsequent to the
isolation and publication of its gene sequence, expres-
sion systems permitting the production of large quanti-
ties of purified human MAO B have been developed.22
Human recombinant MAO B is now also commercially
available. On the other hand, the use of human blood
platelets or mitochondrial fractions of human tissue
homogenates as an enzyme source for medium- and
high-throughput inhibitor screening involves some
drawbacks; above all, the limited accessibility to outdat-
ed human platelet-rich plasma or human tissue.
Figure 1. Linear relationship between inhibition potencies (pIC50) of
coumarin derivatives determined with human recombinant and human
platelet MAO B.
Since MAO B is an integral protein of the outer mito-
chondrial membrane, tedious experimental procedures
are required to obtain purified enzyme with an intact
mitochondrial microenvironment. Therefore, the plate-
let MAO B isolation method used in this study was
based on a careful separation of platelets from plasma,
avoiding further purification procedures. An enzyme
fraction with unaffected specific activity was obtained,
the Km value of kynuramine being identical with liter-
ature data.10 However, the platelet homogenate ob-
tained was about 33 times less pure than the human
recombinant enzyme used, as can be illustrated by
the final protein concentration required in the assay
mixture, that was set to 0.5 mg/mL for human platelet
homogenate and to 0.015 mg/mL for human recombi-
nant MAO B.
In summary, human recombinant MAO B was shown to
be a reliable enzyme source, easy to handle, with repro-
ducible substrate and inhibitor specificities, suitable for
medium- and high-throughput inhibitor screening.
Figure 2. Linear relationship between inhibition potencies (pIC50) of
5H-indeno[1,2-c]pyridazin-5-one derivatives determined with human
recombinant and human platelet MAO B.
3. Discussion
4. Experimental
4.1. Materials
The present results provide unequivocal evidence that
human recombinant and human platelet MAO B show
the same inhibitor specificity. Within experimental
errors, the same inhibition potencies (pIC50 values)
were obtained with human cloned and human platelet
MAO B for the two series of MAO B inhibitors covering
IC50 values of more than three orders of magnitude
(pIC50 values of 5.6–9). Hence, any differences in the
mitochondrial microenvironment of human platelets
and insect cells have an insignificant influence on the
catalytic properties and inhibitor specificities of recom-
binant and natural MAO B.
Kynuramine, deprenyl, and iproniazide were obtained
from Sigma–Aldrich Chemical (St. Louis, MA, USA).
DMSO (microselect for molecular biology), 4-hydroxy-
quinolin, pargyline, potassium phosphate salts, potassi-
um chloride, and sodium hydroxide were obtained from
Fluka AG (Buchs, CH, Switzerland). The synthesis of
all compounds not commercially available was per-
formed in the Dipartimento Farmaco-Chimico (Univer-
`
sita di Bari, Bari, Italy). The details of the synthesis of
coumarin derivatives have been reported in Gnerre
et al.,18 except for compounds 75 and 85 whose prepara-
tion is described below. The synthesis of 5H-indeno[1,2-
c]pyridazin-5-one derivatives has been described by
Carotti et al.23 for compounds 17, 53 and 55, and by
Human recombinant MAO B is not only a reliable but
also an efficient human enzyme source for medium-
and high-throughput inhibitor screening, presenting
notable advantages over blood platelets and mitochon-
drial fractions of tissue homogenates. Principal demands
on an enzyme source for medium- and high-throughput
inhibitor screening are (i) prompt availability of suffi-
cient quantities for several hundreds of enzyme assays
per day, and (ii) well-defined, pure enzyme systems with
reproducible substrate and inhibitor specificities. In-
deed, the analyses of inhibition data from well-defined
systems such as recombinant enzymes, rather than crude
tissue homogenates, allow for a more detailed descrip-
tion of inhibitor binding.21 Human recombinant MAO B
Kneubuhler and co-workers19,24 for compounds 12, 15,
¨
33, 34, 40, 43, 47, 48, 51 and 57.
4.2. Synthesis of coumarin derivatives 75 and 85
Chemicals and reagents were obtained from Sigma–
Aldrich Chemical. The purity of compounds was
checked by microanalysis ( 0.40% of theoretical values
1
1
for C, H and N) and H NMR. IR and H NMR data
are listed below. For the former, only the most signifi-

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L. Novaroli et al. / Bioorg. Med. Chem. 13 (2005) 6212–6217
cant absorption bands (per cm) are reported, and for the
latter, chemical shifts are expressed in parts per millions
(d) and the coupling constants (J) in Hertz. Classical
abbreviations are used for the multiplicities.
(final DMSO concentration of 1% v/v), were preincubat-
ed at pH 7.4, 37 °C for 10 min. As positive control, 2 lL
of pure DMSO were used at the place of the inhibitor
solution. Diluted human recombinant enzyme (50 lL)
was then delivered to obtain a final protein concentra-
tion of 0.015 mg/mL in the assay mixture. Incubation
was carried out at 37 °C and the reaction was stopped
after 20 min by addition of 75 lL of NaOH (2 N).
Fluorescence emission at 400 nm was measured with
a 96-well microplate fluorescent reader (FLx 800,
Bio-Tek Instruments, Inc., Winooski, VT, USA).
4.2.1. 7-Benzensulfonyloxy-2H-chromen-2-one (75). 7-
Hydroxycoumarin (1.0 mmol) was dissolved in 5 mL
of anhydrous pyridine and 3.0 mmol of benzenesulfonyl
chloride were added. The mixture was refluxed for
30 min, then cooled, poured onto ice and acidified with
diluted HCl. A solid precipitate was formed. It was col-
lected by filtration and recrystallized (98% yield). Mp
134–135 °C from ethanol. IR (per cm) 1734, 1378,
1193. 7.85 (d, 2H, H(20), H(60), Jo = 7.4), 7.70 (t, 1H,
H(40), Jo = 7.4), 7.64 (d, 1H, H(4), Jo = 9.5), 7.55 (t,
2H, H(30), H(50), Jo = 7.8), 7.42 (d, 1H, H(5),
Jo = 8.4), 7.03 (dd, 1H, H(6), Jo = 8.4, Jm = 2.2), 6.86
(d, 1H, H(8), Jm = 2.2), 6.39 (d, 1H, H(3), Jo = 9.5).
Data analysis was performed with Prism V4.0 (Graph-
Pad Software Inc., CA, USA). The kinetic parameters
Km and Vmax were obtained by curve fitting according
to the classical Michaelis–Menten equation and the
degree of inhibition pIC50 (ꢁlogIC50) was assessed by
a sigmoidal dose–response curve. DpIC50 is the differ-
ence between the pIC50 values found for the same com-
pound using two MAO B sources.
4.2.2. 7-Benzoylmethyloxy-3,4-dimethyl-2H-chromen-2-
one (85). Ten millimoles of 7-hydroxy-3,4-dimethyl-
coumarin, a-bromoacetophenone, and potassium car-
bonate were heated in 10 mL of anhydrous
dimethylformamide at 120 °C for 3 h. After cooling,
the mixture was poured onto ice, and the formed precip-
itate was collected by filtration and recrystallized (51%
yield). Mp 188–189 °C from ethanol. IR (per cm)
4.3.2. Human platelets. Different batches of outdated hu-
man platelet-rich plasma (PRP) preparations were kind-
´
ly donated by the Laboratoire Central dÕHematologie,
Centre Hopitalier Universitaire Vaudois, Lausanne,
Switzerland. Each batch had an approximate volume
of 200 mL. Platelets were separated from PRP by centri-
fugation at 6000g for 5 min at 5–15 °C. Plasma was dis-
carded and the platelets were suspended in 0.1 M
potassium phosphate buffer, pH 7.4, made isotonic with
KCl. This platelet suspension was kept frozen at ꢁ20 °C
overnight. Samples were thawed by addition of a
volume of distilled water equal to half of the original
volume of PRP. Afterward, the platelets were homoge-
nized in a glass homogenizer with a Teflon pestle for
1 min at 600 rpm. The homogenate was separated in ali-
quots (1 mL) and kept frozen until use.
1
1695, 1615, 1235. H NMR 7.99–7.95 (m, 2H, H(20),
H(60)), 7.63 (tt, 1H, H(40), Jo = 7.4, Jm = 0.9), 7.53–
7.48 (m, 3H, H(5) + H(30) + H(50)), 6.92 (dd, 1H, H(6),
Jo = 8.9, Jm = 2.6), 6.76 (d, 1H, H(8), Jm = 2.6), 5.34
(s, 2H, CH2), 2.34 (s, 3H, CH3(4)), 2.16 (s, 3H, CH3(3)).
4.3. Biological assay
4.3.1. SupersomesTM. Human MAO B SupersomesTM, pur-
chased from BD Gentest (Woburn, MA, USA), are mem-
brane fractions of insect cells containing human
recombinant MAO B. SupersomesTM were stored at
ꢁ80 °C. After initial thawing, small aliquots were
refrozen.
The protein concentration of each batch was determined
according to Lowry et al., with bovine serum albumin as
standard.25 In the assay mixture, the final protein
concentration of the platelet homogenate was set to
0.5 mg/mL. To determine pIC50 values, the same fluores-
cent inhibitor-screening assay developed for human
recombinant enzyme was employed.
To determine pIC50 values, a fluorescence-based screen-
ing method (end point lecture) was adapted from a stan-
dard BD Gentest protocol. The substrate used for the
assay was kynuramine, which is nonfluorescent until
undergoing oxidative deamination by MAO B resulting
in the fluorescent metabolite 4-hydroxyquinolin
(kEx = 310 nm, kEm = 400 nm). Product formation was
quantified by comparing the fluorescence emission of
the samples to that of known amounts of authentic
metabolite 4-hydroxyquinolin.
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Reactions were performed in black, flat-bottomed poly-
styrene 96-well microtiter plates with enhanced assay
surface (FluoroNunc/LumiNunc, MaxiSorpTM Surface,
NUNCTM, Roskild, Denmark) using a final volume of
200 lL. The wells, containing 140 lL of potassium
phosphate buffer (0.1 M, pH 7.4, made isotonic with
KCl), 8 lL of an aqueous stock solution of kynuramine
(0.75 mM to get a final concentration corresponding to
its Km value), and 2 lL of a DMSO inhibitor solution

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