Available online at www.sciencedirect.com
`
`Bioorganic & Medicinal Chemistry 16 (2008) 3580–3586
`
`Catechin gallates are NADP+-competitive inhibitors
`of glucose-6-phosphate dehydrogenase and other enzymes
`that employ NADP+ as a coenzyme
`Eui Seok Shin,a Jiyoung Park,b Jae-Min Shin,c,d Dooho Cho,d Si Young Cho,a
`Dong Wook Shin,a Mira Ham,b Jae Bum Kimb and Tae Ryong Leea,*
`aR&D Center, AmorePacific Corporation, 314-1 Bora-Dong, Giheung-Gu, Yongin, Gyeonggi 446-729, Republic of Korea
`bSchool of Biological Sciences, Seoul National University, San 56-1, Sillim-Dong, Kwanak-Gu, Seoul 151-742, Republic of Korea
`cDepartment of Bioinformatics, Soongsil University, 1-1 Sangdo-Dong, Dongjak-Gu, Seoul 156-743, Republic of Korea
`dSBscience Inc., 3rd Fl. Sungok-Building, 4-1 Sunae-Dong, Bundang-Gu, Gyeonggi 463-825, Republic of Korea
`
`Received 9 January 2008; revised 1 February 2008; accepted 5 February 2008
`Available online 14 February 2008
`
`Abstract—Recent studies have shown that glucose-6-phosphate dehydrogenase (G6PD) is an effectual therapeutic target for meta-
`bolic disorders, including obesity and diabetes. In this study, we used in silico and conventional screening approaches to identify
`putative inhibitors of G6PD and found that gallated catechins (EGCG, GCG, ECG, CG), but not ungallated catechins (ECG,
`GC, EC, C), were NADP+-competitive inhibitors of G6PD and other enzymes that employ NADP+ as a coenzyme, such as
`IDH and 6PGD.
`Ó 2008 Elsevier Ltd. All rights reserved.
`
`1. Introduction
`
`Oxidative stress and associated inflammatory processes
`are believed to play important roles in the pathogenesis
`of metabolic syndromes as well as major age-related dis-
`eases.1–3 NADPH is an essential coenzyme for several
`enzymes that generate oxygen-free radicals, including
`NADPH oxidase, nitric oxide synthase, and the cyto-
`chrome P450 monooxygenases.4 Thus, a reduction in
`NADPH production could result in a significant change
`in cellular oxidative stress. In addition, NADPH is an
`essential element in lipogenesis5,6 and contributes to
`fatty acid and cholesterol synthesis by supplying reduc-
`ing power. Therefore, NADPH-producing enzymes
`might be closely associated with oxidative stress, chronic
`inflammatory signals, and lipid metabolism disorders.
`
`NADPH is produced by reduction of NADP+ in bio-
`chemical
`reactions
`catalyzed by several
`enzymes,
`including malic enzyme (ME), isocitrate dehydrogenase
`
`Keywords: Catechin gallate; Glucose-6-phosphate dehydrogenase; Iso-
`citrate dehydrogenase; 6-Phosphogluconate dehydrogenase; NADP.
`* Corresponding author. Tel: +82 31 280 5961; fax: +82 31 899 2595;
`e-mail: trlee@amorepacific.com
`
`0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
`doi:10.1016/j.bmc.2008.02.030
`
`(IDH), and glucose-6-phosphate dehydrogenase (G6PD)
`and 6-phosphogluconate dehydrogenase (6PGD), which
`are the first two enzymes of the pentose phosphate path-
`way (PPP).6 Among the four NADPH-producing
`enzymes, G6PD is the rate-limiting enzyme of PPP,
`and is highly conserved in most mammalian species.7
`G6PD, which is expressed ubiquitously, is implicated
`in various cell functions, including cell growth, survival,
`and redox regulation, and its deficiency causes hemolytic
`anemia and neonatal jaundice.8 Interestingly, hormonal
`or nutritional regulation of G6PD was restricted to liver
`and adipose tissues.6 Hepatic G6PD is regulated by
`nutritional signals, including a high-carbohydrate diet,
`polyunsaturated fatty acids, and hormonal signals such
`as insulin, glucagon, thyroid hormone, and glucocorti-
`coids.6,7 Furthermore, G6PD-deficient patients show a
`decrease in lipogenic rate and serum lipoprotein concen-
`trations, implying the importance of G6PD in fatty acid
`synthesis.9,10 Recent studies have elucidated novel roles
`of adipose tissue G6PD in the etiology of metabolic dis-
`orders. G6PD expression is highly increased in obese
`subjects including ob/ob, db/db, and diet-induced obese
`mice, and high expression of G6PD in adipocytes is
`tightly associated with lipid dysregulation, oxidative
`stress, and the chronic inflammation found in obese or
`
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`
`3581
`
`Table 1. IC50 values of G6PD inhibitors
`
`Structure
`
`IC50 (lmol/L)
`
`diabetic subjects.11,12 These observations indicate that
`G6PD is a potential therapeutic target for obesity and/
`or diabetes-related diseases.
`
`is a well-known,
`Dehydroepiandrosterone (DHEA)
`uncompetitive inhibitor of G6PD.13–15 It has anti-oxida-
`tive, anti-carcinogenic, anti-obesity, and anti-aging
`properties.16,17 Use of DHEA as an anti-obesity drug
`is hampered by the requirement of high oral dosage
`and its easy conversion into various active androgens.
`Thus, it is expected that finding more efficient inhibitors
`of G6PD could lead to potent therapeutic drugs against
`obesity and/or diabetes.
`
`In this study, both in silico and conventional screening
`approaches targeting the coenzyme (NADP+) and sub-
`strate (glucose-6-phosphate, G6P) binding sites of
`G6PD were performed in an effort to discover candidate
`G6PD inhibitors, and catechin gallates were identified as
`potent NADP+-competitive inhibitors of G6PD.
`
`2. Results and discussion
`
`OH
`
`OH
`
`OH
`
`OH
`
`OH
`
`OH
`
`HO
`
`O
`
`O
`
`O
`
`OH
`
`OH
`
`OH
`
`N
`
`NH
`
`HN
`
`N
`
`S
`
`0.25
`
`56.37
`
`21.76
`
`2.1. Identification of EGCG as a potent inhibitor of
`G6PD through virtual screening approach
`
`HO
`
`OH
`
`N
`
`HN
`
`O
`
`HO
`
`NH
`
`N
`
`O
`
`From a virtual screening experiment targeting the coen-
`zyme (NADP+) and substrate (G6P) binding sites of
`G6PD, 250 candidate compounds were selected from a
`collection of three million commercially available com-
`pounds, and purchased from several chemical library
`distribution companies. These candidate compounds
`were used in an in vitro G6PD inhibition assay and eight
`compounds showed more than 50% inhibition at less
`
`than 100 lmol/L. Among eight compounds, ()-epigal-
`
`locatechin gallate (EGCG) was identified as the most
`potent inhibitor of G6PD (Table 1 and Fig. 1). EGCG
`is the most abundant polyphenolic catechin isolated
`from green tea, which exhibits profound pharmacologi-
`cal activities including anti-oxidant activity, inhibition
`of cell proliferation, inhibition of ultraviolet B (UVB)-
`induced inflammatory responses, modulation of cell cy-
`cle regulation, anti-cholesterolemic activity, suppression
`of angiogenesis, and anti-carcinogenic effect.18–21
`
`2.2. Galloyl moiety of catechins is an essential structural
`feature in the inhibition of G6PD
`
`To elucidate the structure–activity relationship of the
`inhibitory effects of EGCG on G6PD, the inhibition
`kinetics of EGCG and other green tea catechins were
`investigated. Interestingly,
`the inhibitory activity of
`green tea catechins on G6PD was restricted to gallated
`catechins, such as EGCG,
`
`()-gallocatechin gallate
`(GCG), ()-epicatechin gallate (ECG), and ()-catechin
`such as ()-epigallocatechin (EGC), ()-gallocatechin
`()-epicatechin (EC), and ()-catechin (C)
`(GC),
`(Fig. 1 and Table 2). All gallated catechins exhibited
`similar IC50 values (0.18–0.25 lmol/L) for the inhibition
`of G6PD (Table 2). Therefore, the galloyl moiety of cat-
`echins is an essential structural feature in the inhibition
`
`gallate (CG) when compared to ungallated catechins,
`
`Figure 1. Dose–response curves for EGCG and EGC on the rate of
`G6PD catalysis. The activity of G6PD was measured in the presence of
`various concentrations of EGCG (j) and EGC (d).
`
`0
`
`of G6PD, while the 5
`-hydroxyl group on the B ring and
`the stereochemistry of the 2-position of the catechin
`skeleton are not necessary for inhibition of G6PD. Sev-
`eral supportive studies have shown that the galloyl moi-
`ety has active biological features. Tian and co-workers
`reported that EGCG is a potent inhibitor of fatty acid
`
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`
`Table 2. G6PD, 6PGD, and IDH IC50values for gallated catechins
`OH
`
`HO
`
`H
`
`O
`
`OH
`
`R1
`
`HO
`
`H
`
`O
`
`OH
`
`R1
`
`OH
`
`OR2
`
`G6PD
`
`1000
`1000
`
`0.18 ± 0.01
`0.25 ± 0.02
`0.24 ± 0.01
`0.23 ± 0.02
`
`1000
`1000
`
`IC50 (lmol/L)
`6PGD
`
`1000
`1000
`
`1.21 ± 0.13
`0.72 ± 0.07
`1.28 ± 0.08
`1.45 ± 0.08
`
`1000
`1000
`
`IDH
`
`1000
`1000
`
`10.8 ± 1.66
`6.44 ± 1.12
`6.62 ± 0.63
`2.72 ± 0.21
`
`1000
`1000
`
`OH
`
`H
`
`OR2
`
`OH
`A B
`
`H
`
`Compound
`
`General structure
`
`R1
`
`R2
`
`EC
`EGC
`ECG
`EGCG
`CG
`GCG
`GC
`C
`
`A
`A
`A
`A
`B
`B
`B
`B
`
`H
`OH
`H
`OH
`H
`OH
`OH
`H
`
`H
`H
`3,4,5-Trihydroxybenzoyl
`3,4,5-Trihydroxybenzoyl
`3,4,5-Trihydroxybenzoyl
`3,4,5-Trihydroxybenzoyl
`H
`H
`
`synthase (FAS), and that the galloyl moiety is the criti-
`cal structural feature in the inhibition of the b-ketoacyl
`reductase activity of FAS via a reversible association
`with the NADPH-binding site or with an adjacent area
`of the b-ketoacyl reductase of FAS.22,23 In addition,
`EGCG and ECG, but not EGC and EC, are potent
`inhibitors of glutamate dehydrogenase (GDH) with
`EC50s in the nanomolar range. EGCG is a non-compet-
`itive inhibitor of both GDH substrates (NADH and 2-
`oxoglutarate), but acts in an allosteric manner.24
`
`2.3. Catechin gallates are NADP+-competitive inhibitors
`of G6PD and other enzymes that employ NADP+ as a
`coenzyme
`
`To examine the manner in which gallated catechins inhi-
`bit G6PD activity, various concentrations of EGCG and
`CG were added to reactions containing various concen-
`trations of NADP+ and G6P. Both EGCG and CG are
`competitive inhibitors of NADP+, but are uncompeti-
`tive inhibitors of G6P (Fig. 2). Based on the NADP+-
`competitive inhibition patterns of gallated catechins, it
`was proposed that gallated catechins could act as gen-
`eral inhibitors of enzymes that employ NADP+ as a
`coenzyme. Gallated catechins were indeed potent inhib-
`itors of 6PGD and IDH, enzymes which both employ
`NADP+ as a coenzyme (Table 2).
`
`It is well known that green tea catechins affect the reduc-
`tion of body weight and prevent obesity-related meta-
`bolic disorders such as diabetes, hyperlipidemia, and
`hypertension in various animal models and in hu-
`mans.25,26 Although catechins have been shown to be
`effective inhibitors of G6PD,
`it has not been clearly
`determined whether the therapeutic effects of catechins
`on metabolic disorders are directly associated with
`G6PD inhibition. Gallated catechins are not only inhib-
`itors of G6PD, but are also inhibitors of 6PGD and
`IDH, which use NADP+ as a coenzyme. In addition,
`gallated catechins are good inhibitors of FAS and
`GDH, which play important roles in lipid synthesis
`
`and insulin secretion, respectively. Green tea catechins
`are also reported to be inhibitors of pancreatic phospho-
`lipase A2 (PLA2), and were found to inhibit the intesti-
`nal absorption of lipids in ovariectomized rats.27 Thus,
`although gallated catechins are effective inhibitors of
`G6PD in vitro and show good anti-obesity effects
`in vivo, the extent of the effects that are directly associ-
`ated with the inhibition of G6PD by catechins remains
`unclear.
`
`2.4. Effects of EGCG on endogenous dehydrogenase
`activity in 3T3-L1 adipocytes lysates
`
`To further investigate the inhibitory effects of EGCG,
`ECG, GCG, and CG on the production of NADPH
`by G6PD and 6PGD in adipocytes, measurement of
`NADPH production was performed using cell lysates
`from differentiated 3T3-L1 adipocytes. As shown in Fig-
`ure 3A, EGCG, ECG, GCG, and CG effectively sup-
`pressed NADPH production in 3T3-L1 adipocytes
`with IC50 values near 25 lmol/L. However, EGC, EC,
`GC, and C did not suppress NADPH production in
`3T3-L1 adipocytes (Fig. 3B). We also compared the
`inhibitory effect of EGCG with that of DHEA, a well-
`known uncompetitive inhibitor of G6PD activity.13–16
`As shown in Figure 3C, both DHEA and EGCG inhib-
`ited NADPH production. EGCG inhibited NADPH
`production in a dose-dependent manner, but DHEA
`showed only 40% maximum inhibition at concentrations
`above 100 lmol/L. This difference in inhibition patterns
`between EGCG and DHEA represents a difference in
`inhibition mechanisms. Consistent with the observation
`of differing inhibition mechanisms, EGCG also inhibited
`6PGD activity in a dose-dependent manner, while
`DHEA did not inhibit 6PGD activity (Fig. 3D).
`
`EGCG, ECG, GCG, and CG were significantly better
`inhibitors of endogenous NADPH production when
`compared to DHEA. However, considering the very
`low IC50 values (0.18–0.25 lmol/L) in activity assay
`using purified yeast G6PD, it was somewhat disappoint-
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`3583
`
`Figure 2. Inhibition kinetics of EGCG and CG with respect to G6P and NADP+. G6PD was analyzed with respect to NADP+ at three
`concentrations of EGCG and CG. (A) EGCG: 0 lmol/L (h), 0.5 lmol/L (n), and 1.0 lmol/L (s). (B) CG: 0 lmol/L (h), 5.0 lmol/L (n), and
`10.0 lmol/L (s). G6PD was analyzed with respect to G6P at three concentrations of EGCG and CG. (C) EGCG: 0 lmol/L (h), 1.0 lmol/L (n), and
`2.0 lmol/L (s). (D) CG: 0 lmol/L (h), 5.0 lmol/L (n), and 10.0 lmol/L (s).
`
`ing that catechin gallates showed 100-fold higher IC50
`values in the NADPH production assay using cell ly-
`sates of differentiated 3T3-L1 adipocytes. This difference
`in IC50 values may be partially due to the presence of
`other proteins, such as IDH, FAS, GDH, and others,
`which bind with EGCG and consequently reduce the
`amount of available free EGCG in the 3T3-L1 adipocyte
`cell lysate. The total plasma concentration after 50 mg
`EGCG oral intake (estimated amount of EGCG in a
`cup of green tea) in human is approximately 0.3 lmol/
`L.28 Catechin gallate IC50 values of approximately
`25 lmol/L seem too high to suggest any physiological
`relevance to known in vivo activities of green tea cate-
`chins. However, unlike DHEA, catechin gallates are
`NADP+-competitive inhibitors of enzymes that employ
`NADP+ as a coenzyme. Therefore, the in vivo EC50 val-
`ues of catechin gallates will vary depending on the con-
`centration of free NADP+ in the target organ. The
`concentration of free NADP+ in the target organ is dif-
`ficult to measure. However, a reasonable estimation can
`be made based on several previous reports. Total con-
`centration of NADP+ and NADPH in rat liver is
`approximately 100 lmol/L, and the NADP+/NADPH
`
`ratio is about 0.005. A significant portion of the NADP+
`and NADPH is bound to protein; in the case of NADH,
`over 80% of NADH is protein bound. Therefore, the
`concentration of free NADP+ would be much low-
`er.29–31 The estimated free NADP+ concentration in a
`target organ is approximately 0.1 lmol/L, which is more
`than 1000-fold lower than the concentration of NADP+
`used in the in vitro experiment. The high concentration
`of NADP+ in the in vitro experiment is necessary to ob-
`tain a reasonable signal size for measurement. There-
`fore, EC50 values of catechin gallates could be much
`lower than 0.3 lmol/L. In addition, it was recently re-
`ported that multiple treatments with catechins showed
`synergistic effects.32 Thus, multiple treatments will fur-
`ther decrease their EC50 values and our results may, in
`fact, be physiologically relevant to known in vivo activ-
`ities of green tea catechins in humans.
`
`3. Conclusion
`
`In this study, utilizing both in silico and conventional
`screening approaches, we identified that gallated cate-
`
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`E. S. Shin et al. / Bioorg. Med. Chem. 16 (2008) 3580–3586
`
`Figure 3. Effects of catechins and DHEA on NADPH production. The production of NADPH by 3T3-L1 cell lysates was measured in the presence
`of various concentrations of catechins and DHEA. (A) EGCG (d), ECG (h), GCG (m) and CG (·). (B) EGC (d), EC (h), GC (m) and C (·).
`(C) EGCG (d) and DHEA (s). (D) The production of NADPH by 6PGD in 3T3-L1 cell lysates was measured in the presence of various
`concentrations of EGCG (d) and DHEA (s). Results are represented as means ± SD of three-independent experiments.
`
`chins, but not ungallated catechins, were NADP+-com-
`petitive inhibitors of G6PD and other enzymes that em-
`ploy NADP+ as a coenzyme. Although the extent of the
`effects that are directly attributable to the inhibition of
`each enzyme remains unclear, these results along with
`previous reports concerning the inhibition effects of
`green tea catechins on FAS, PLA2 and GDH explain
`how green tea catechins can display such broad in vivo
`activities against obesity and oxidative stress-related dis-
`orders. Catechin gallates showed somewhat high IC50
`values in the NADPH production assay using cell ly-
`sates of differentiated 3T3-L1 adipocytes. However,
`these activities may still be physiologically relevant to
`known in vivo activities of green tea catechins in hu-
`mans, due to NADP+-competitive inhibition of catechin
`gallates and low in vivo concentrations of free NADP+.
`
`4. Experimental
`
`4.1. Materials
`
`Glucose-6-phosphate, sodium chloride, magnesium chlo-
`
`ride, b-NADP, ()-epigallocatechin gallate (EGCG),
`()-epigallocatechin (EGC),
`()-epicatechin gallate
`(ECG), ()-epicatechin (EC), ()-gallocatechin gallate
`()-gallocatechin (GC),
`()-catechin gallate
`(CG), ()-catechin (C), Glucose-6-phosphate dehydro-
`
`(GCG),
`
`genase (EC 1.1.1.49) from bakers yeast, 6-phosphoglu-
`conic dehydrogenase from Saccharomyces cerevisiae
`(EC 1.1.1.44), and isocitric dehydrogenase (NADP)
`
`(EC 1.1.1.42) from porcine heart were purchased from
`Sigma–Aldrich.
`
`4.2. Modeling of the binding site of G6PD
`
`Two binary complex structures of the G6PD human
`deletion mutant of Kotaka et al. with glucose-6-phos-
`phate (G6P, PDB code: 2bhl), and NADP+ (PDB code:
`2bh9) were selected for virtual screening.33 The superim-
`posed structure of the two binary complexes is shown in
`Fig. S-1. For convenience in structure-based modeling, a
`combined structure was constructed by copying G6P
`from the PDB structure 2bhl into the PDB structure
`2bh9,
`in a manner similar to that used by Kotaka
`et al.33 (Figure S-2). The software program IDPharmo
`version 2.0 (Equispharm Inc., Seoul, Korea)34 was used
`for virtual screening to search approximately three mil-
`lion commercially available library compounds in a per-
`iod of eight hours, using a 3GHz, four-CPU Linux PC.
`IDPharmo is fingerprint-based virtual screening soft-
`ware, and three essential physicochemical features com-
`prise its construction of
`reliable fingerprints:
`the
`hydrogen bond donor, the hydrogen bond acceptor,
`and the hydrophobic core. After several cycles of phar-
`macophore generation and refinement, 9 protein–ligand-
`binding features for the substrate- and coenzyme-bind-
`ing sites of G6PD were obtained, as
`shown in
`Figure S-2. Based on the combination of these 9 li-
`gand-binding features, 12 different pharmacophore
`models, termed PharmoMaps, were selected. Based on
`these 12 PharmoMaps, approximately 2000 ‘virtual hit’
`
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`E. S. Shin et al. / Bioorg. Med. Chem. 16 (2008) 3580–3586
`
`3585
`
`molecules were obtained from the three million-com-
`pound library in which each compound is pre-compiled
`in a maximum of 150 different conformations. The scor-
`ing methods utilized the combined root mean square
`deviation (RMSD) scores between the PharmoMaps
`and docked-molecules, and bump-penalties. In order
`to reduce the number of virtual hits carried forward fur-
`ther for testing, the 2000 hits were visually inspected for
`drug-likeness by assessing hydrophobicity, molecule
`size, and diversity of molecular structure. A total of
`250 compounds that could be purchased from different
`chemical
`library distribution companies were finally
`selected.
`
`4.3. Assay of enzyme activity
`
`Enzyme activity was determined using a Spectra MAX
`190 (Molecular Devices) by measuring absorbance at
`340 nm, at a temperature of 30 °C. The reaction mixture
`contained 150 mmol/L sodium chloride, 6 mmol/L mag-
`nesium chloride,
`0.5 mmol/L glucose-6-phosphate,
`0.25 mmol/L NADP+, and 0.1 mol/L Tris buffer (pH
`7.5) in a total volume of 0.2 mL. An amount of 0.0002
`enzyme units was used in each reaction. Glucose-6-phos-
`phate was replaced by 0.5 mmol/L 6-phosphogluconate
`for the 6PGD assay, or by 0.5 mmol/L isocitric acid
`for the IDH assay. The reaction mixture was prepared
`immediately prior to use. The reaction catalyzed the
`reduction of NADP+ to NADPH, and the rate of the
`reaction was calculated from the increase in the absor-
`bance at 340 nm. The extinction coefficient of NADPH
`1 cm
`1. The inhibition effect of the
`is 6.02 · 103 M
`inhibitors was examined by adding each inhibitor to
`the reaction mixture prior to the initiation of the reac-
`tion. The level of inhibition in the presence of the inhib-
`itor was measured by reference to the half-inhibition
`concentration (IC50). The IC50 values of the green tea
`catechins were determined graphically after
`linear
`regression of the inhibitory percentages expressed with
`the logarithmic concentration of the inhibitors.
`
`4.4. Determination of inhibition mechanism
`
`The mechanism of inhibition by catechin gallates was
`determined by constructing reciprocal plots of 1/V ver-
`sus 1/[S] for reactions with 1 mmol/L G6P and varying
`concentrations of NADP+, and with 0.5 mmol/L of
`NADP+ and varying concentrations of G6P. The plots
`were assessed by a weighted least-square analysis. The
`slopes of these reciprocal plots were then plotted against
`the concentration of the inhibitors (range: 0–2.0 lmol/L
`for EGCG, 0–10.0 lmol/L for CG)
`in a weighted
`analysis.
`
`4.5. Endogenous dehydrogenase enzyme assay
`
`G6PD and 6PGD enzyme activities were determined by
`measuring the rate of NADPH production as previously
`described with slight modifications.11 Because 6PGD
`also catalyzes a reaction in which NADPH is produced,
`NADPH production by 6PGD and NADPH produced
`by total dehydrogenases (G6PD + 6PGD) was measured
`separately. Fully differentiated 3T3-L1 adipocytes were
`
`lysed with NETN buffer (50 mmol/L Tris pH 7.8,
`100 mmol/L NaCl, 0.1% NP-40, 1 mmol/L EDTA) and
`centrifuged at 12,000 rpm at 4 °C for 15 min. Superna-
`tants were used immediately in assays measuring
`dehydrogenase activity. Each enzyme activity was
`measured after
`incubation of
`the supernatants
`in
`reaction buffer with indicated polyphenols and DHEA
`at 30 °C. For NADPH production assay, the reac-
`tion buffer contained 150 mmol/L sodium chloride,
`6 mmol/L magnesium chloride, 0.5 mmol/L glucose-6-
`phosphate,
`0.5 mmol/L
`6-phosphogluconate,
`0.25 mmol/L NADP+, and 0.1 mol/L (pH 7.5) Tris
`buffer. For 6PGD activity assay, the reaction buffer
`contained 150 mmol/L sodium chloride, 6 mmol/L mag-
`nesium chloride, 0.25 mmol/L 6-phosphogluconate,
`0.125 mmol/L NADP+, and 0.1 mol/L (pH 7.5) Tris buf-
`fer. In each assay, 1.0 lg of protein was added to the
`reaction buffer. Protein concentrations were determined
`using the Bradford method (Bio-Rad), and used to nor-
`malize enzyme activity. Inhibitors were dissolved in
`dimethylsulfoxide and then added to the reaction mix-
`ture. The volume of dimethylsulfoxide was less than
`0.2% (v/v) of the total reaction volume in order to avoid
`interference with the enzyme activity. In inhibitor-free
`control reactions, the same amount of dimethylsulfoxide
`was added to the reaction mixture.
`
`Acknowledgments
`
`This work was partly supported by the Korea Science
`and Engineering Foundation (KOSEF) through the Na-
`tional Research Laboratory Program of Korea Institute
`of Science (No. M10400000359-06J0000-35910),
`the
`Molecular and Cellular BioDiscovery Research Pro-
`gram (No. M10748000258-07N4800-25810), and the
`Technology Evaluation and Planning and Stem Cell Re-
`search Center of the 21st Century Frontier Research
`Program (No. R11-2005-009-01002-0). M. Ham, J.
`Park, and J. B. Kim are supported by the BK21 Re-
`search Fellowship from the Ministry of Education and
`Human Resource Development.
`
`Supplementary data
`
`Supplementary data associated with this article can be
`found,
`in the online version, at doi:10.1016/j.bmc.
`2008.02.030.
`
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