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S. Endo et al. / Biochemical Pharmacology 86 (2013) 1366–1375
cDNA was deposited in DDBJ database with the accession number
AB821295.
In the RT-PCR analysis of the expression of mRNA for AKR1C29
in rabbit tissues, the preparation of total RNA samples, RT and PCR
0.1-M potassium phosphate, pH 7.4. The substrate and products
were extracted into 4-mL ethyl acetate 30 min after the reaction
was started at 37 8C. The products of oxidoreduction of steroids
[25] and reduction of PGD2 [28], farnesal [29] and 4-oxo-2-
nonenal [18] were analyzed by TLC, as described. The reduced
products of TBE were identified by the HPLC methods [23]. The
were carried out as described above. The cDNA for rabbit
b-actin
was also amplified as an internal control with a pair of sense and
antisense primers, 50-ccggcttcgcgggcgacg-30 and 50-tcccggccagc-
caggtcc-30, respectively. The PCR products were separated by
agarose gel electrophoresis, and revealed with ethidium bromide.
products of 3HB oxidation, 3OB reduction,
5b-androstane-
3
a
,17 -diol oxidation and 5 -androstan-3 -ol-17-one reduc-
b
b
a
tion were analyzed by the liquid chromatography–electrospray
ionization-mass spectrometry (LC–ESI-MS) using a Hewlett-
Packard HP 1100 Series LC/MSD system attached with a diode
2.3. Enzyme purification
array detector and
a column (Mightysil RP-18 GP 5 mm,
The expression construct was transfected into Escherichia coli
BL21 (DE3) pLysS, and the recombinant AKR1C29 fused to the N-
terminal 6-His tag was expressed and purified to homogeneity
from the cell extracts using the nickel-charged Sepharose 6FF resin
(GE Healthcare, Chalfont St Giles, UK) as described previously [25].
3HBD was purified to homogeneity from the rabbit liver (50 g) by
the methods of Takenoshita and Toki [5], except that Q-Sepharose
(GE Healthcare) was used instead of TEAE-cellulose. The protein
concentrations of the enzymes were determined with Bradford
reagent using bovine serum albumin as the standard.
4.6 mm ꢀ 250 mm, Kanto Chemical Co., Tokyo, Japan). Separa-
tions were carried out at a flow rate of 0.5 mL/min and 40 8C
using the following mobile phases: 25% acetonitrile aqueous
solution containing 0.1% formic acid for 3OB and
80% acetonitrile aqueous solution containing 0.1% formic acid
for the two steroids. 3OB, -3HB, -3HB, 5 -androstan-3 -ol-
17-one and -androstane-3 ,17 -diol were detected by
a/b-3HBs, and
a
b
b
a
5
b
a
b
monitoring their total ions (m/z 249.1, 251.1, 251.1, 289.4
and 291.4, respectively) in the negative ESI mode, and eluted at
the retention times of 20.1, 17.6, 16.8, 14.9 and 12.7 min,
respectively. The detection limits of 3OB,
a/b-3HBs and the two
2.4. Mass spectrometry
steroids were 0.1, 0.1 and 1 nmol, respectively.
The identity of the purified hepatic 3HBD with AKR1C29 was
examined by the mass spectrometry method. The purified protein
(0.1 mg) was digested by lysylendopeptidase, and then the digest
was analyzed using a Bruker-Franzen Mass Spectrometry System
2.7. Cell culture experiments
Bovine aortic endothelial cells (BAECs) were generous gift from
Taisho Pharmaceutical Co. (Saitama, Japan), and cultured in
Dulbecco’s modified Eagle medium supplemented with 10% fetal
bovine serum, penicillin (100 units/mL) and streptomycin (0.1 mg/
mL) at 37 8C in a humidified incubator containing 5% CO2. The
construction and transfection of the pGW1 plasmids harboring the
cDNA for AKR1C29 into BAECs were performed as described
previously [30]. Briefly, the cDNA was amplified by PCR using the
primers. The sense primer, 50-ccccgaattcGCCACCatggatcccaagcat-
cagcg-30, contains an EcoRI site, a Kozak sequence and a start
codon, which are shown in underlined, capital and italic letters,
respectively. The antisense primer was the same as that used for
the cDNA cloning. After the sequences of the PCR products were
verified, they were subcloned at the EcoRI and SalI sites of the
pGW1 expression vector. Using a Lipofectamine 2000 reagent (Life
Technologies), the cells were transfected with the expression
plasmids. The transfected cells were maintained in the medium
(2 mL) containing 2% fetal bovine serum for 24 h, and then
Ultraflex TOF/TOF with a saturated
a-cyano-4-hydroxycinnamic
acid matrix as described previously [26].
2.5. Assay of enzyme activity
The dehydrogenase activities for the enzymes were assayed by
measuring the rate of change in fluorescence (at 455 nm with an
excitation wavelength of 340 nm) or absorbance (at 340 nm) of
NAD(P)H. The corresponding standard reaction mixture consisted
of 0.1-M potassium phosphate buffer, pH 7.4, 0.25-mM NADP+ or
2.0-mM NAD+, substrate and enzyme, in a total volume of 2.0 mL.
The reductase activities, except for all-trans-retinal reductase
activity, were determined by measuring the rate of change in
NAD(P)H absorbance in the phosphate buffer, pH 7.4, containing
0.1-mM NADH or NADPH and an appropriate amount of carbonyl
substrate. The assay of all-trans-retinal reductase activity was
´
`
performed according to the method of Pares and Julia [27], except
that 0.1-M potassium phosphate, pH 7.4, containing 0.01% Tween
80 was added as the buffer. One unit of enzyme activity was
defined as the amount of enzyme that catalyzes the formation or
incubated with
a
-3HB, 3OB (each 200
m
M), 5
b
-androstan-3
a-ol-
17-one or 5 -androstane-3
b
a
,17 -diol (each 100 m
b
M) at different
times. The culture medium was collected by centrifugation, and
the lipidic fraction of the medium was extracted twice by 4-mL
ethyl acetate. The extract was evaporated to dryness, and the
oxidation of 1
mmol NAD(P)H per min at 25 8C.
The apparent Km and kcat values for coenzymes and substrates
were determined by fitting the initial velocities to the Michaelis–
Menten equation. The IC50 values for inhibitors were determined
with 2-mM b-ionol as the substrate in the above standard reaction
mixture. The inhibitor constant, Ki, was estimated from the Dixon
residue was dissolved in methanol (0.1 mL). The portion (10 mL) of
the sample was analyzed by the LC–ESI-MS method as described
above. The amounts of the eluted substrates and metabolites were
quantified from their peak areas using the standard curves
constructed with the authentic samples. The effects of the
AKR1C29 overexpression on the cytotoxicity of 4-oxo-2-nonenal
were examined using the above BAECs as described previously
[25].
plot and/or Cornish–Bowden plot of the velocities obtained in the
b
-ionol range (0.5–20 ꢀ Km) with three concentrations of the
inhibitor. The kinetic constants and IC50 values are expressed as the
means of at least three determinations. The standard deviations of
the determinations were less than 15%, unless otherwise noted.
3. Results
2.6. Product identification
3.1. Amino acid sequence and cDNA cloning of 3HBD
The reaction was conducted at 37 8C in a 2.0-mL reaction
mixture, containing coenzyme (1-mM NADP+ or 0.1-mM
NADPH), substrate (0.05–0.1 mM), enzyme (0.1–0.3 mg), and
3HBD was purified from rabbit liver and its 13 lysylendopepti-
dase-digested peptides were subjected to amino acid sequence
analysis by the mass spectrometry (Fig. 2). The sequences
Endo, Satoshi
