Y. Shen et al. / Biochimie 92 (2010) 530e537
531
Intrinsic disulfide bridges are an important mechanism to
maintain protein secondary structures; and protein disulfide
isomerase is a major catalyst of the S-thiolation in physiological
conditions [4,26,27]. However, intramolecular S-thiolation is also
evoked by oxidative stress, temporally affecting the protein struc-
ture and function, such as enzymatic activity [27,28].
sodium salicylate, 2 ml of ROS mixture was mixed with 30
m
l 10.0 M
HCl and 4 ml of chilled diethylether. Samples were mixed for 30 s
and then 2 ml of the upper ether layer was withdrawn and evap-
orated to dryness in a water bath at 50 ꢁC. After cooling down,
residue was dissolved in 1 ml of cold water, followed by addition of
0.5 ml of 10% (w/v) trichloroacetic acid in 0.5 M HCl, 1 ml of 10%
(w/v) sodium tungstate, and 1 ml of 0.5% (w/v) NaNO2. After being
incubated for 5 min at room temperature, 2 ml of 0.5 M KOH was
added and absorbance at 510 nm was measured.
Protein disulfide
isomerase
SH
S
Protein
+ 2GSH
+ GSSG
Protein
SH
S
2.4. Measurements of sulfhydryl (ꢀSH) groupsdEllman's test
Mixed disulfides with physiological thiols, GSH or Cys, and
intrinsic disulfide bridges within a protein are both reversible and
thus are an important regulatory/protective mechanism of
a protein in response to oxidative stress [29]. AKR1B10 contains Cys
residues, and like the Cys298 in aldose reductase (AR), Cys299 in
AKR1B10 is a key residue for its enzymatic activity; a substitute of
Cys299 to serine (C299S) significantly impacts its affinity and
catalytic activity and sensitivity to inhibitors [30]. Therefore,
AKR1B10 activity may be modulated by ROS-evoked S-thiolation,
which is evaluated in this current study.
Reduced thiols were determined using Ellman's test [38,39].
Briefly, reaction mixture (1 ml) consisted of 50
Dithio-bis(2-nitrobenzoic acid)) in 50 mM sodium acetate, 100
1 M Tris (pH 8.0), and 10 l samples. After incubation at room
m
l of2 mM DTNB (5,50-
ml of
m
temperature for 5 min, absorbance at 412 nm was measured. Pure
cysteine (Sigma, MO) was used as a standard; and the reaction
mixture without thiols was applied as a blank control. The eSH
contents were calculated as: eSH ¼ 100 ꢂ OD412/13,600, in which
13,600isextinctioncoefficient(Mꢀ1 cmꢀ1), and100denotesdilution.
AKR1B10 is similar in the structure and substrate specificity to
AR [1,31]. In past two decades, AR has been used as a target for the
prevention and treatment of diabetic complications, and numerous
aldose reductase inhibitors (ARIs) have been developed and used in
diabetic patients [32e36]. Previous reports have demonstrated the
inhibitory activity of some ARIs to AKR1B10, including tolrestat,
sorbinil, zopolrestat, EBPC, fenofibrate, Wy 14,346, gemfibrozil and
ciprofibrate [13,30]. This study recognized the ARIs epalrestat and
statil as new AKR1B10 inhibitors and revealed for the first time that
thiol/disulfide exchanges modulate the sensitivity of AKR1B10 to
the ARIs. This finding is invaluable for the application of ARIs in
diabetic clinic and the design of new inhibitors.
2.5. AKRB10 protein purification, S-thiolation, and activity assays
Recombinant AKR1B10 protein was prepared using a pQE
prokaryotic protein expression system (Qiagen, CA) as previously
described [8]. AKR1B10 S-thiolation was carried out in a mixture
(600
ml) containing 1
mg protein, 50 mM potassium chloride,
125 mM sodium phosphate (pH 7.0), and 120
ml of ROS products.
Herein, ROS products include ROS, ROS with Cys at 0.1, 0.5 or
1.0 mM, and ROS with GSH at 0.1, 0.5 or 1.0 mM. After incubation at
35 ꢁC for 15 min, 0.2 mM NADPH and appropriate substrates were
added to initiate the enzymatic reactions at 35 ꢁC for 10 min.
Oxidized NADPH was monitored at 340 nm. Mixtures without
proteins or substrates were used as blank controls. Kinetic
constants were calculated by fitting the MichaeliseMenten func-
tion directly in hyperbolic form to data provided with GraphPad
Prism 4 (Graph Pad Software, CA) [40,41].
2. Materials and methods
2.1. Reagents
b
-Nicotinamide adenine dinucleotide 20-phosphate reduced
tetrasodium (NADPH), DL-Cysteine, -Glutamyl- -cysteinyl-
glycine
-GSH reduced), 5,50-Dithio-bis(2-nitrobenzoic acid),
g-
L
L
v ¼ Vmax*S=ðKm þ SÞ
(1)
(
L
sodium salicylate, and sodiumtungstatewere purchased from Sigma
Chemical Co. (St. Louis, MO). (Z, E)-5-(2-Methyl-3-phenyl-2-prope-
nylidene)-4-oxo-2-thioxothiazolidine-3-acetic acid (Epalrestat)
and Ethyl 1-benzyl-3-hydroxy-2(5H)-oxopyrrole-4- carboxylate
(EBPC) were from BIOTREND Chemical AG (Zurich, Switzerland).
[3-(4-Bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetic acid
(Statil) was from Tocris Cookson Ltd. (Bristol, UK).
In equation, v is the enzyme velocity, Vmax is the maximum initial
velocity, Km is the MichaeliseMenten constant, and S denotes
substrate concentrations in micromolar.
AKR1B10 inhibition was tested using DL-glyceraldehyde at
0e20 mM as a substrate, in the presence of inhibitors indicated.
Inhibition constants (Kii and Kis) and LineweavereBurk plots of
1/velocity versus 1/substrate concentrations were obtained using
the GraphPad Prism 4 (Graph Pad Software, CA). Data conforming
to linear competitive and non-competitive inhibition were fitted to
equation (2)e(4) below.
2.2. Database and AKR1B10 structure
used for the analyses of AKR1B10 protein 3D structure and Cys
residue location. Distance between Cys residues was determined
using the measurement function in the database.
v ¼ Vmax*S=½Km þ Sð1 þ I=KiiÞꢃ
(2)
(3)
(4)
v ¼ Vmax*S=½Kmð1 þ I=KisÞ þ Sð1 þ I=KiiÞꢃ
v ¼ Vmax*S=½Kmð1 þ I=KisÞ þ Sꢃ
2.3. ROS production and hydroxyl radical measurements
where I is the inhibitor concentration, Kii and Kis are the intercept
and slope inhibition constants.
ROS was produced at 35 ꢁC for 5 h in a reaction mixture con-
taining 125 mM sodium phosphate (pH 7.0), 0.3 mM FeSO4 and
0.9 mM EDTA in the presence or absence of GSH or cysteine at
0.1e1.0 mM as indicated. Hydroxyl radicals were determined by
measuring the formation of 2,3-dihydroxybenzoate from salicylate
to indicate ROS production [37]. Briefly, in the presence of 2.5 mM
IC50, the inhibitor concentration leading to 50% inhibition of
AKR1B10 activity, was determined by constructing the dose-
response curve (sigmoidal doseeresoponse) using the formula:
Y ¼ Bottom þ (Top ꢀ Bottom)/{1 þ 10 * [(LogEC50-X) * HillSlope]},
where X is the logarithm of concentration; and Y is the response