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• W2034928061 abstract "The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties. The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties. 5-methoxycarbonylamino-N-acetyltryptamine quinone reductase 2 octylglucopyranoside dihydrobenzylnicotinamide dichoromethane O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate agomelatine N-methyl-{1-[2-(acetylamino)ethyl]naphthalen-7-yl}-carbamate N-[2-(7-amino-1-naphthyl)ethyl]acetamide Melatonin, a neurohormone produced at night in the pineal gland, is suspected to relay to the peripheral organs the circadian rhythm detected by the central nervous system. Several high affinity melatonin receptors have been identified to date, among which the mt1(1Reppert S.M. Weaver D.R. Ebisawa T. Neuron. 1994; 13: 1177-1185Abstract Full Text PDF PubMed Scopus (1019) Google Scholar) and MT2 (2Reppert S.M. Godson C. Mahle C.D. Weaver D.R. Slaugenhaupt S.A. Gusella J.F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8734-8738Crossref PubMed Scopus (813) Google Scholar) receptors have been cloned from human tissues. The pharmacology of these two receptors is well documented, and several compounds, including melatonin, are ligands with picomolar binding affinity (for review, see Ref. 3Dubocovich M.L. Trends Pharmacol. Sci. 1995; 16: 50-56Abstract Full Text PDF PubMed Scopus (405) Google Scholar). Another putative melatonin receptor was identified on pharmacological grounds, with lower melatonin affinity (nanomolar range), very rapid ligand association/dissociation kinetics, and an original pharmacological profile (4Duncan M.J. Takahashi J.S. Dubocovich M.L. Endocrinology. 1988; 122: 1825-1833Crossref PubMed Scopus (135) Google Scholar, 5Molinari E.J. North P.C. Dubocovich M.L. Eur. J. Pharmacol. 1996; 301: 159-168Crossref PubMed Scopus (95) Google Scholar, 6Paul P. Lahaye C. Delagrange P. Nicolas J.P. Canet E. Boutin J.A. J. Pharmacol. Exp. Ther. 1999; 290: 334-340PubMed Google Scholar). In line with mt1 and MT2receptors, this putative receptor was named MT3 , according to the nomenclature recommendations of the IUPHAR (7Dubocovich M.L. Cardinali D.P. Guardiola-Lemaı̂tre B. Hagan R.M. Krause D.N. Sugden B. Vanhoutte P.M. Yocca F.D. The IUPHAR Compendium of Receptor Characterization and Classification. IUPHAR Media, London1998: 187-193Google Scholar). So far, the known inhibitors of MT3 hardly reach the nanomolar range and encompass an unusually large structural diversity of highly hydrophobic cyclic or polycyclic compounds (Refs. 5Molinari E.J. North P.C. Dubocovich M.L. Eur. J. Pharmacol. 1996; 301: 159-168Crossref PubMed Scopus (95) Google Scholarand 6Paul P. Lahaye C. Delagrange P. Nicolas J.P. Canet E. Boutin J.A. J. Pharmacol. Exp. Ther. 1999; 290: 334-340PubMed Google Scholar, and for review, see Ref.3Dubocovich M.L. Trends Pharmacol. Sci. 1995; 16: 50-56Abstract Full Text PDF PubMed Scopus (405) Google Scholar). 1O. Nosjean, J. P. Nicolas, F. Klupsch, P. Delagrange, E. Canet, and J. A. Boutin, submitted for publication. 1O. Nosjean, J. P. Nicolas, F. Klupsch, P. Delagrange, E. Canet, and J. A. Boutin, submitted for publication. All pharmacological investigations on mt1, MT2, andMT3 were performed using the radioligand [125I]melatonin, a ligand with high affinity for mt1 and MT2 (K d = 10–200 pm) and with lower affinity for MT3 (K d = 3–9 nm). The hamster kidney, liver, and brain have been used as model tissues forMT3 pharmacological studies, and our recent data confirmed that among a wide range of mammals, this rodent was indeed the best source of MT3 .1 Hence, the binding specificity for [125I]melatonin competition studies on MT3 is achieved by preparing material from hamster tissues, and the fast dissociation kinetics is overcome by operating at 4 °C. In addition, iodination of the known very specific MT3 inhibitor, 5-methoxycarbonylamino-N-acetyltryptamine (MCA-NAT),2 paved the way to more accurate and reliable investigations on MT3 (5Molinari E.J. North P.C. Dubocovich M.L. Eur. J. Pharmacol. 1996; 301: 159-168Crossref PubMed Scopus (95) Google Scholar). This ligand, combined with recently improved operating conditions, made possible for us to perform specific MT3 pharmacological studies at room temperature.1 Confident in the interest of discovering novel melatoninergic pharmacological targets, we recently set up a biochemical approach to identify and characterize MT3 . The present report describes a specific purification procedure of MT3 from hamster kidney, which led to a homogeneous single protein of 26 kDa, identified by tandem mass spectrometry as a homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). This identification was confirmed by confronting MT3 pharmacological and QR2 enzymatic data obtained under different cellular and biochemical conditions corresponding to MT3 or QR2 typical conditions. The quinone reductase family comprises two isoforms, QR1 and QR2, which have been sequenced (8Jaiswal A.K. McBride O.W. Adesnik M. Nebert D.W. J. Biol. Chem. 1988; 263: 13572-13578Abstract Full Text PDF PubMed Google Scholar, 9Jaiswal A.K. Burnett P. Adesnik M. McBride O.W. Biochemistry. 1990; 29: 1899-1906Crossref PubMed Scopus (117) Google Scholar) and crystallized (10Li R. Bianchet M.A. Talalay P. Amzel L.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8846-8850Crossref PubMed Scopus (307) Google Scholar, 11Foster C.E. Bianchet M.A. Talalay P. Zhao Q. Amzel L.M. Biochemistry. 1999; 38: 9881-9886Crossref PubMed Scopus (96) Google Scholar). QR2 lacks a 47-amino acid C-terminal sequence present in QR1, resulting in a different substrate specificity. It is noteworthy that the literature on QR2 enzymology is rather scarce (12Liao S. Dulaney J.T. Williams-Ashman H.G. J. Biol. Chem. 1962; 237: 2981-2987Abstract Full Text PDF PubMed Google Scholar, 13Jaiswal A.K. J. Biol. Chem. 1994; 269: 14502-14508Abstract Full Text PDF PubMed Google Scholar, 14Zhao Q. Yang X.L. Holtzclaw W.D. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1669-1674Crossref PubMed Scopus (103) Google Scholar). Interestingly, QR2 was originally discovered in 1962 as a flavoenzyme (12Liao S. Dulaney J.T. Williams-Ashman H.G. J. Biol. Chem. 1962; 237: 2981-2987Abstract Full Text PDF PubMed Google Scholar), later re-discovered as the QR1-related enzyme (14Zhao Q. Yang X.L. Holtzclaw W.D. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1669-1674Crossref PubMed Scopus (103) Google Scholar), and was recently found again in porcine kidney as a puromycin aminonucleoside-binding protein (15Kodama T. Wakui H. Komatsuda A. Imai H. Miura A.B. Tashima Y. Nephrol. Dial. Transplant. 1997; 12: 1453-1460Crossref PubMed Scopus (8) Google Scholar). We now unveiled a new facet of QR2 as the melatonin-binding siteMT3, opening new perspectives in melatonin investigations as well as in quinone reduction studies. [125I]MCA-NAT (2200 Ci/mmol) was custom synthesized by Amersham Pharmacia Biotech (Orsay, France). 2-Iodomelatonin and MCA-NAT were purchased from Tocris (Bioblock, Illkirch, France), dihydrobenzylnicotinamide was obtained from Maybridge (Interchim, Montluçon, France), and all other reagents were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Frozen hamster kidneys were obtained from Charles River Breeding Laboratories (Saint Aubin les Elbeuf, France). The tissues were thawed, chopped, and added to 5 ml/g of homogenization buffer (50 mm Tris-HCl, pH 7.5, 0.2 m sucrose, 1 mm CaCl2, and CompleteTM mixture of protease inhibitors). The cells were gently disrupted using a Dounce homogenizer and unbroken material and nuclei were pelleted at 280 × g. The pellet (P1) was treated identically a second time and the two 280 × g supernatants were pooled (S1) and supplemented with 5 mm final β-octyl glucopyranoside (OG) prior to a 30-min incubation under agitation. Cytoplasm and loose membrane-associated material was recovered in the supernatant of a 100,000 × gcentrifugation (SOG) and the pellet (POG) was conserved for analysis. The SOG fraction was dialyzed against 20 mm Tris-HCl, pH 7.5, 1 mmCaCl2 and applied to a 6 × 5-cm DEAE Bio-Gel A column (Bio-Rad) pre-equilibrated with the same buffer. The elution of proteins was triggered by a stepwise gradient of 0–1 mNaCl in the application buffer and was monitored by absorbence at 280 nm. The fractions of interest were pooled and dialyzed against the application buffer, concentrated by laying the dialysis tubing onto 20,000 Da polyethylene glycol, and further dialyzed against the application buffer. All procedures were carried out at 4 °C. Most often, the OG solubilization sample SOG was flash frozen in liquid nitrogen and stored until application on the ion-exchange phase. For binding studies, this partially purified MT3 sample was dialyzed against 20 mm Tris-HCl, pH 7.5, 1 mm CaCl2 in order to remove the sucrose which inhibited MCA-NAT binding. The final DEAE sample was frozen identically before further purification or analysis. The ligandN-[2-(7-amino-1-naphthyl)ethyl]-acetamide (S27145, Fig. 1) was prepared in three steps from agomelatine (S20098 (16Depreux P. Lesieur D. Mansour H.A. Morgan P. Howell H.E. Renard P. Caignard D.H. Pfeiffer B. Delagrange P. Guardiola B. J. Med. Chem. 1994; 37: 3231-3239Crossref PubMed Scopus (149) Google Scholar)) and attached to the polymer resin over a spacer, in two further steps, as follows. A solution of N-[2-(7-methoxy-1-naphthyl)ethyl]acetamide (10 g, 41 mmol) in DCM (50 ml) was treated with BBr3 (25 ml) at −15 °C under nitrogen. The reaction mixture was kept at −15 °C for 1 h, then poured on, hydrolyzed with 1 nNaHCO3 and extracted with methylethylketone. The organic layer was dried over sodium sulfate and concentrated. The residue was taken up with DCM (100 ml) and the precipitate collected and dried to afford 8.77 g (93%) ofN-[2-(7-hydroxy-1-naphthyl)ethyl]acetamide (compound1). A solution 1 (8.7 g, 38 mmol) in DCM (400 ml) was treated with triethylamine (6.9 ml, 50 mmol) andN-phenyl-bistrifluoromethane sulfonimide (20 g, 53 mmol). The reaction mixture was refluxed for 12 h, then cooled, concentrated, taken up with diethyl ether, washed with 1 nNaHCO3, water, and then dried over magnesium sulfate. The crude product was purified on silica gel with DCM/ethyl acetate (95:5) to afford 11.5 g (84%) ofN-[2-(7-trifluoromethylsulfoxy-1-naphthyl)ethyl]acetamide (compound 2). A mixture of 2 (4.0 g, 11 mmol) with benzophenonimine (7 g, 38 mmol), bis(diphenylphosphino-1–1′-binaphthalene (310 mg, 0.5 mmol), palladium acetate (70 mg, 0.3 mmol), and cesium carbonate (5 g, 15 mmol) in dimethoxyethane (160 ml) was refluxed under nitrogen for 12 h. The resulting reaction mixture was hydrolyzed with water and extracted with diethyl ether. The organic layer was washed with 10% citric acid and water, then dried over sodium sulfate and concentrated. The resulting crude product was dissolved in tetrahydrofuran (150 ml), treated with 1n HCl (200 ml), and heated at 60 °C for 1 h. The reaction was cooled, extracted with diethyl ether, and the aqueous phase was brought to pH 11 with NaOH, then extracted with DCM. The organic layer was dried over sodium sulfate, concentrated, and purified on silica gel in DCM/methanol (97:3) to afford 1.44 g ofN-[2-(7-amino-1-naphthyl)ethyl]acetamide. The corresponding hydrochloride salt (1.61 g, 55%, compound 3) was obtained by treatment of the base in ethyl acetate with HCl in diethyl ether. Compound 3 (876 mg, 3.31 mmol) and 6-tert-butoxycarbonylamino hexanoic acid (1.15 g, 4.97 mmol) were dissolved in DCM (20 ml) and neat HATU (1.89 g, 4.97 mmol) was added in one single portion, followed by a slow addition of diisopropylethylamine (2.57 g, 14.90 mmol). After a 15-min reaction at room temperature, high performance liquid chromatography showed no remaining amine and one major new product. Ethyl acetate (100 ml) was then added and the solution washed 3 times with brine (3 × 25 ml), 1 m HCl (3 × 25 ml), 5% NaHCO3(3 × 25 ml), and brine (3 × 25 ml), dried over magnesium sulfate and evaporated under vacuum. The yield of {5-[7-(2-acetylamino-ethyl)-naphthalen-2-ylcarbamoyl]-pentyl}-carbamic acid tert-butyrate, compound 4 (1.4 g), was 95% and its high performance liquid chromatography purity was 99%. The Boc group was deprotected by treatment of 4 (1.40 g, 3.17 mmol) with trifluoroacetic acid (10 ml) in DCM (20 ml). After 15 min at room temperature, the solution was evaporated under vacuum and lyophilized in water/acetonitrile (9:1). The trifluoroacetic acid salt was then attached to the Novasyn TG carboxy resin (Novabiochem, 0.25 mmol/g) (12 g) on a semiautomatic synthesizer with axial shaking, using HATU (1.21 g, 3.17 mmol) in the presence of diisopropylethylamine (2.05 ml, 11.88 mmol) in a mixture of 200 ml of DCM/DMF (1:1) as solvent. The reaction mixture was shaken for 64 h at room temperature. After filtration, the usual washings were performed: 3 × with DMF and isopropyl alcohol, alternatively and 3 times with DCM. The resin was then capped by treatment with a large excess of glycine methyl ester hydrochloride (3.98 g, 31.7 mmol) using HATU (12.05 g, 31.7 mmol) in the presence of diisopropylethylamine (7.65 ml, 44.4 mmol) in 200 ml of a mixture of DCM/DMF (1:1). The same washings were performed after filtration. Estimation of the substitution level of the resin was carried out by microanalysis of the nitrogen percentage content: 0.11 mmol ligand/g of resin. The partially purified MT3 sample was subjected to affinity chromatography at 4 °C. The phase was synthesized as described above and used in a batch procedure. It was washed three times with 20 mm Tris-HCl, pH 7.5, 1 mm CaCl2 and mixed with the sample at a ratio of 50 μg of protein/mg of phase. The incubation was performed for 15 min under gentle agitation after which the sedimentation of the resin was left to occur. The unbound material was removed by pipetting over the supernatant and the phase was washed twice with the equilibration buffer. The bound proteins were eluted by incubating the phase for 15 min in the equilibration buffer supplemented with 50 μmMCA-NAT. The eluate was recovered by pipetting, dialyzed against water, and concentrated on polyethylene glycol as described above. The precipitate obtained in dry ice-cold acetone was dried under a flow of nitrogen and analyzed by Laemmli polyacrylamide gel electrophoresis (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The proteins were detected in the gel by Coomassie Blue. The protein spot in the Laemmli electrophoresis performed after affinity chromatography was excised from the Coomassie Blue-stained gel and washed with 50% acetonitrile. Gel pieces were dried in a vacuum centrifuge and reswollen in 20 μl of 25 mmNH4 HCO3 containing 0.5 μg of trypsin (Promega, sequencing grade). After 4 h incubation at 37 °C, the gel pieces were extracted with 5% formic acid and acetonitrile. The extracts were evaporated to dryness. The residues were dissolved in 0.1% formic acid and desalted using a Zip Tip (Millipore). Elution of the peptides was performed with 5–10 μl of 50% acetonitrile, 0.1% formic acid solution. The peptide solution was introduced onto a glass capillary (Protana) for nanoelectrospray ionization. Tandem mass spectrometry experiments were carried out on a Q-TOF hybrid mass spectrometer (Micromass, Altrincham, United Kingdom) in order to obtain sequence information. MS/MS sequence information was used for data base searching using the programs MS-Edman located at the University of California San Francisco and BLAST located at the NCBI. The QR2 coding sequence was isolated by reverse transcriptase-polymease chain reaction from human liver mRNA (CLONTECH, Palo Alto, CA) using the 5′ sense primer (5′-GAATTCTCCACCATGGCAGGTAAGAAAGTACTCATGTC-3′, nucleotides 176–202) and the 3′ antisense primer (5′-GCGGCCGCTCATTATTGCCCGAAGTGCCAGTGGGCTGTGC-3′, nucleotides 843–871) generated from the published sequence (Ref.9Jaiswal A.K. Burnett P. Adesnik M. McBride O.W. Biochemistry. 1990; 29: 1899-1906Crossref PubMed Scopus (117) Google Scholar; access number JO2888). Liver mRNA (200 ng) was reverse-transcribed with oligo(dT)12–18 in accordance with the first-strand cDNA synthesis protocol from Amersham Pharmacia Biotech. Polymerase chain reactions were performed in 100 μl containing 10 mm Tris-HCl, pH 8.3, 1.5 mmMgCl2, 0.2 mm dNTP, 2 μl of the single-stranded cDNA preparation, 0.3 μm of each primer, and 2 units of pfu native polymerase (Stratagene) with a 35 cycles program of 94 °C for 1 min, 65 °C for 2 min, and 72 °C for 2 min and a final extension at 72 °C for 8 min. The amplified cDNA was then subcloned in-frame into EcoRI andNotI site of the pcDNA3.1(+) vector (Invitrogen, San Diego, CA). CHO-K1 cells maintained in Ham's F12 medium supplemented with 10% fetal calf serum, 2 mm glutamine, 500 IU/ml penicillin, and 500 μg/ml streptomycin were transiently transfected by the pcDNA3.1(+)-QR2 plasmid using LipofectAMINE as described by the manufacturer (Life Technologies). Fourty-eight hours after the beginning of transfection, the adherent cells were washed by phosphate-buffered saline and harvested in 10 ml of homogenization buffer (see “Purification ofMT3 ,” above) and transferred from flask to flask. The cell supension obtained was adjusted to 0.2 msucrose and spun at 280 × g. The resulting nuclei-free supernatant was assayed for protein andMT3 /QR2 content. All assays were performed in triplicate and data presented herein are representative of two to six individual experiments. The protein concentration was determined by the method of Lowry (18Lowry O.H. Roseborough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. TheMT3 binding was performed according to our original procedure.1 Briefly, 100 μg (samples from animals) or 20 μg (samples from CHO) of proteins were incubated at 20–22 °C for 10–20 min with 200 pm [125I]MCA-NAT in the presence (nonspecific binding) or absence (total binding) of 10 μm MCA-NAT and, for competition studies, with varying concentrations of test compounds. The final volume was 150 μl in 20 mm Tris-HCl, pH 7.5, 1 mm CaCl2. Incubation was stopped by filtration through a 96-well filtration support disposed directly onto a Multiscreen filtering apparatus (Milllipore) connected to a vacuum pump, allowing rapid filtration after the samples were loaded using a 96-well pipetting device (Transtar, Costar). The filter-associated radioactivity was measured in a β-scintillation counter (TopCount NXT, Packard). Samples from CHO culture and from the purification of MT3 up to the ion exchange chromatography step were analyzed on glass fiber filters (GF/B Unifilter, Packard), while elution from the ion-exchange resin was followed using polyvinylidene difluoride filters (ImmobilonTM Multiscreen, Millipore) pre-soaked in methanol and rinsed three times by 200 μl of binding buffer. Alternatively, samples from the final purification steps, affinity chromatography, and ion exchange chromatography as an internal reference, were assayed forMT3 binding using 96-well format size-exclusion chromatography. Eighty μl of dry Sephadex G-25 fine (APB) were distributed into 96-well format polyvinylidene difluoride filters (DuraporeTM Multiscreen, Millipore) using a Multiscreen powder dispensing apparatus. The exclusion phase was hydrated with 250 μl of 20 mm Tris-HCl, pH 8.5, and spun at 550 × g for 1 min in a 96-well plate basket. The phase was further rinsed three times using 120 μl of the same buffer, left to equilibrate at 4 °C for 30 min, and spun before sample application. The samples were preincubated at 4 °C during 30 min with 200 pm [125I]MCA-NAT as described above, and 120 μl of the mixture were loaded onto the exclusion phase. The plates were immediately spun at 550 × g for 1 min. The free radioligand was excluded from the eluate by diffusion in the chromatographic medium, and 90 μl of the eluate were used for scintillation counting. For competition studies, the data presented are affinity constants (K i) calculated from specific binding values of logarithmic compound concentrations, according to the method of Cheng and Prusoff (19Cheng Y.C. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12132) Google Scholar). The measurements of QR1and QR2 quinone reductase activities were adapted from Jaiswal et al. (9Jaiswal A.K. Burnett P. Adesnik M. McBride O.W. Biochemistry. 1990; 29: 1899-1906Crossref PubMed Scopus (117) Google Scholar) and Zhao et al. (14Zhao Q. Yang X.L. Holtzclaw W.D. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1669-1674Crossref PubMed Scopus (103) Google Scholar). QR1 activity was measured using 100 μmmenadione as substrate and 100 μm NADH as co-substrate, while QR2 activity was measured using 100 μmmenadione as substrate, 100 μm dihydrobenzylnicotinamide (BNAH) as co-substrate, and 100 μm dicoumarol as QR1 inhibitor. In both cases, the activities were measured at 25 °C in 200 μl of 20 mm Tris-HCl, pH 7.5, 1 mm OG and the reactions were followed at 440 nm using the intrinsic fluorescence of the two co-substrates with excitation at 340 nm (PolarStar 96-well plate reader, BMG, Offenburg, Germany). Samples were diluted before use in the measurement buffer supplemented with 10% glycerol, in order to apply the desired amount of protein in 20 μl. The instrument was calibrated using a range of co-substrate concentrations. The IC50 values were calculated from inhibition curves using semi-logarithmic plots of the compound concentrations (8 points). This work primarily focused on the melatonin-binding site MT 3, which was studied and purified using [125I]MCA-NAT binding (5Molinari E.J. North P.C. Dubocovich M.L. Eur. J. Pharmacol. 1996; 301: 159-168Crossref PubMed Scopus (95) Google Scholar) as a specific assay, designated herein as the “MT 3assay.” Purification of MT 3 from hamster kidney provided “partially purified MT 3” and “MT 3 purified to homogeneity.” The cloning and expression of the human QR2 gene in CHO provided an identified source of this enzyme, which was assayed using the well described oxidoreduction mechanism involving a quinone (electron acceptor) and a nicotinic derivative (electron donor). For assaying the QR2 enzymatic activity, menadione was the substrate and a commercially available fluorescent NADH analogue, dihydrobenzylnicotinamide (BNAH, Powell et al. (20Powell M.F. Wong W.H. Bruise T.C. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4604-4608Crossref PubMed Scopus (19) Google Scholar)), was used for the first time as the co-substrate. Dicoumarol, a potent QR1 inhibitor and poor QR2 inhibitor (14Zhao Q. Yang X.L. Holtzclaw W.D. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1669-1674Crossref PubMed Scopus (103) Google Scholar) was added to the assay to ensure QR2 specificity. Control assays of QR1 activity were performed when necessary, using menadione as the substrate and NADH as the co-substrate, which provided good QR1 specificity since NADH is a very poor co-substrate for QR2 (14Zhao Q. Yang X.L. Holtzclaw W.D. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1669-1674Crossref PubMed Scopus (103) Google Scholar,20Powell M.F. Wong W.H. Bruise T.C. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4604-4608Crossref PubMed Scopus (19) Google Scholar). 3O. Nosjean and J. A. Boutin, unpublished observations. , Although, in the light of our results presented thereafter, MT 3 and QR2 seem to designate a unique protein, for convenience in the present report, we alternatively refer to hamsterMT 3 orMT 3/QR2 and human QR2, depending on the methodological approach involved. The purification of the melatonin-binding siteMT 3 was performed as described under “Experimental Procedures” and the intermediate fractions were assayed for [125I]MCA-NAT binding and QR2enzymatic activity. QR1 activity was also assayed in all samples as a control. We started the purification of theMT 3 melatonin-binding site by preparing a nuclei-free subcellular fraction, from which MT 3was recovered with high yield in the supernatant of a 100,000 ×g centrifugation after mild detergent treatment (5 mm octylglucoside). Dialysis removed most of the detergent molecules thanks to the high critical micellar concentration of OG (CMCOG = 25 mm (21Helenius A. McCaslin D.R. Fries E. Tanford C. Method. Enzymol. 1979; 56: 734-749Crossref PubMed Scopus (590) Google Scholar)). The dialysate was applied to a DEAE anion exchanger, from which MT 3 was eluted by a discontinuous gradient of NaCl. TheMT3 containing fractions were pooled and dialyzed in order to remove the NaCl. Finally, we purifiedMT 3 to homogeneity using an original affinity phase developed on the basis of the most specificMT 3 ligand known to date, MCA-NAT. The synthetic ligand (S27145, Fig. 1) bears an amine function in position 7 of naphthylethylacetamide, which was substituted by a 6-aminohexanoyl moiety in order to mimic the carbonylamide function of MCA-NAT. The free amino group of the aminohexanoic spacer was used for the attachment to the affinity matrix, a polyethylene glycol-derivatized polystyrene resin. The use of a naphthyl ring eliminated the photosensitivity associated with the indole ring. The affinity chromatographic step was performed at 4 °C in a batch procedure, and the proteins specifically adsorbed on the phase were eluted by 50 μm MCA-NAT. The eluate was analyzed by SDS-polyacrylamide gel electrophoresis, where it appeared as a single band of 26 kDa (Fig. 2). The band was recovered from the electrophoresis acrylamide gel as a trypsin digest and was analyzed by tandem mass spectrometry. The resulting five peptidic sequences were compared with protein data bases for alignment and showed 95% similarity with the human quinone reductase 2, QR2 (Fig. 3). TableI displays the yields of the successive purification steps as calculated from MT 3binding and QR2 enzymatic assay data. The [125I]MCA-NAT binding data are in good agreement with the QR2 enzymatic data, with a 2.5–3.5-fold enrichment ofMT 3/QR2 in the OG supernatant, and a 12.5–13.5-fold enrichment after DEAE chromatography, while QR1 was barely detectable in the ion exchange chromatography eluate. After the affinity chromatographic step, the purification of MT3 , as evaluated by [125I]MCA-NAT binding and QR2 enzymatic assay, reached a 10,000-fold factor of enrichment, confirming the identity of MT3 with QR2. Absorption and desorption of the protein preparation from the affinity medium gave a relatively low yield of recovery of MT3 and QR2 signals (about 20%), probably due to the rapid kinetics of ligand exchange of MT3 . Indeed, the dissociation constant at room temperature is about 0.3 s−1,1 and performing the affinity chromatographic step at 4 °C could not completely counterbalance the rapid dissociation kinetics.Figure 3Seq" @default.
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