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Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

1997; Elsevier BV; Volume: 272; Issue: 31 Linguagem: Inglês

10.1074/jbc.272.31.19615

1083-351X

Bernd Clement, Rüdiger Lomb, Wenke Möller,

Biochemical Acid Research Studies

Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochromeb 5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83–92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxicN-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason whyN-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups. Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochromeb 5, its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83–92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxicN-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason whyN-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups. The metabolism of nitrogen-containing functional groups has become a topic of considerable interest since the early discovery thatN-hydroxylated intermediates are often responsible for the toxic and/or carcinogenic properties of aromatic amines, hydrazines, and amides (1Testa B. Testa B. Caldwell J. The Metabolism of Drugs and other Xenobiotics. Academic Press, Inc., San Diego, CA1995: 415-433Google Scholar). On the other hand, the more facileN-oxygenation of secondary and tertiary alkylamines to hydroxylamines and N-oxides was considered as a route for detoxication (2Bickel M.L.C. Pharmacol. Rev. 1969; 21: 325PubMed Google Scholar, 3Coutts R.T. Beckett A.H. Drug Metab. Rev. 1977; 6: 51-69Crossref Scopus (36) Google Scholar), and it was generally assumed that the strongly basic nitrogen compounds were metabolically stable. However, we have demonstrated that even the protonated hydrophilic amidines (4Clement B. Xenobiotica. 1983; 13: 467-473Crossref PubMed Scopus (32) Google Scholar, 5Clement B. Kämpchen T. Chem. Ber. 1985; 118: 3481-3491Crossref Scopus (27) Google Scholar, 6Clement B. Zimmermann M. Xenobiotica. 1987; 17: 659-667Crossref PubMed Scopus (18) Google Scholar), as well as diamidines such as pentamidine and diminazene and also guanidines and amidinohydrazones, are capable of undergoing metabolicN-oxygenation by liver microsomal cytochrome P450 monooxygenases (7Clement B. Jung F. Pfundner H. Mol. Pharmacol. 1993; 43: 335-342PubMed Google Scholar, 8Clement B. Jung F. Drug Metab. Dispos. 1994; 22: 486-500PubMed Google Scholar). The N-oxygenation of these functional groups produces more reactive metabolites, and the genotoxic properties of benzamidoxime are well known (9Clement B. Schmezer P. Schlehofer J.R. Schmitt S. Pool B.L. J. Cancer Res. Clin. Oncol. 1988; 114: 363-368Crossref PubMed Scopus (15) Google Scholar). During investigations of the metabolic fate of strongly basic N-hydroxylated xenobiotics, we observed that they were readily reduced both in vivo andin vitro by a microsomal system present in all mammalian species (rats, rabbits, pigs, and humans) tested to date (10Clement B. Schmitt S. Zimmermann M. Arch. Pharm. 1988; 321: 955-956Crossref PubMed Scopus (30) Google Scholar, 11Hauptmann J. Paintz M. Kaiser B. Richter M. Pharmazie. 1988; 43: 559-560PubMed Google Scholar, 12Clement B. Schulze-Mosgau M. Wohlers H. Biochem. Pharmacol. 1993; 46: 2249-2267Crossref PubMed Scopus (65) Google Scholar, 13Clement B. Immel M. Schmitt S. Steinmann U. Arch. Pharm. 1993; 326: 807-812Crossref PubMed Scopus (15) Google Scholar). Preliminary experiments indicated that this system (10Clement B. Schmitt S. Zimmermann M. Arch. Pharm. 1988; 321: 955-956Crossref PubMed Scopus (30) Google Scholar) had many of the characteristics of the microsomal O2-insensitive hydroxylamine reductase described by Kadlubar et al. (14Kadlubar F.F. McKee E.M. Ziegler D.M. Arch. Biochem. Biophys. 1973; 156: 46-57Crossref PubMed Scopus (86) Google Scholar,15Kadlubar F.F. Ziegler D.M. Arch. Biochem. Biophys. 1974; 162: 83-92Crossref PubMed Scopus (64) Google Scholar), which required NADH-cytochrome b 5reductase, cytochrome b 5, and a third unidentified protein component. In this report, we describe the isolation, purification, and characterization of this component from pig liver microsomes and show that the system catalyzing the O2-insensitive reduction of benzamidoxime requires NADH-cytochrome b 5 reductase, cytochromeb 5, a cytochrome P450, and phospholipid (Fig.1). Pig liver microsomes were prepared by fractional acid precipitation according to the procedure of Ziegler and Pettit (16Ziegler D.M. Pettit F.H. Biochem. 1966; 5: 2932-2938Crossref PubMed Scopus (87) Google Scholar) with slight modifications (7Clement B. Jung F. Pfundner H. Mol. Pharmacol. 1993; 43: 335-342PubMed Google Scholar). Cytochromeb 5, NADH-cytochrome b 5reductase, and NADPH-cytochrome P450 reductase were separated by modifications of the procedure described by Kling et al.(17Kling L. Legrum W. Netter K.J. Biochem. Pharmacol. 1985; 34: 85-91Crossref PubMed Scopus (22) Google Scholar). Thesit (Boehringer Mannheim) was used in place of Emulgen 913 to solubilize the microsomal proteins and in the elution buffers. All purification steps were performed at 4 °C. The solubilized pig liver microsomes were applied to an octyl-Sepharose CL 4B (Pharmacia, Freiburg, FRG) column (inner diameter, 3.8 cm; length, 38 cm), and enzymes were eluted by a stepwise increase in the detergent concentration and a decrease in the salt concentration in elution buffers (buffer A: 10 mm potassium phosphate, pH 7.4, 1 mm EDTA (Serva, Heidelberg, FRG), 1 mmdithiothreitol, 20% (w/v) glycerol, 0.5% (w/v) sodium cholate, and 0.5 m NaCl; buffer B: 10 mm potassium phosphate, pH 7.4, 1 mm EDTA, 1 mmdithiothreitol, 20% (w/v) glycerol, 0.44% (w/v) sodium cholate, 0.2% (w/v) Thesit, and 0.5 m NaCl; buffer C: 10 mmpotassium phosphate, pH 7.4, 1 mm EDTA, 1 mmdithiothreitol, 20% (w/v) glycerol, 0.2% (w/v) sodium cholate, and 2% (w/v) Thesit). The flow rate was 50 ml/h; the volume of the fractions collected was 8.5 ml; and the eluate was monitored at 280 nm (Fig. 2). The fractions containing NADPH-cytochrome P450 reductase activity (Fig. 2, peak 2) and those containing NADH-ferricyanide reductase activity (Fig. 2,peak 4) were collected. In addition, the fractions with the highest absorbance at 417 nm (cytochrome b 5) were combined (Fig. 2, peak 3). NADH-cytochromeb 5 reductase (Fig. 2, peak 4) was purified to homogeneity by affinity chromatography on 5′-AMP-Sepharose 4B (Pharmacia) similar to the procedure described for the purification of NADPH-P450 reductase (18Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar). The fractions containing the highest NADH-ferricyanide reductase activity were combined and concentrated, followed by gel filtration (NAP 10, Pharmacia). The specific activity of the purified reductase was 27 units/mg, and only one band was detectable on SDS-PAGE 1The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; DLPC,l-α-dilauroyl phosphatidylcholine. (data not shown). Cytochromeb 5, present in the highest concentration in peak 3 (Fig. 2), was purified on a DEAE-cellulose column (inner diameter, 2.5 cm; length, 30 cm) equilibrated with buffer D (10 mmpotassium phosphate, pH 7.4, 0.1 mm EDTA, 0.1 mm dithiothreitol, 20% (w/v) glycerol, 0.5% (w/v) sodium cholate). After equilibration, elution was performed with a linear gradient from 0 to 1 m KCl in buffer D, collecting 10-ml fractions, and those with the highest absorbance at 417 nm were combined (data not shown) and further processed by gel filtration on Sephadex G-100 (inner diameter, 1.5 cm; length, 45 cm), equilibrated in buffer D, at a flow rate of 15 ml/h. The fractions containing cytochrome b 5 were concentrated by ultrafiltration (Amicon, Witten, FRG; exclusion size, 10 kDa), and the buffer was changed to buffer D, lacking sodium cholate, by gel filtration on NAP 25 columns (Pharmacia). The final cytochromeb 5 fraction contained 10.2 nmol of cytochromeb 5/mg of protein and exhibited one band at 16 kDa after SDS-PAGE (data not shown). The NADPH-cytochrome P450 reductase (Fig. 2, peak 2) was further purified by affinity chromatography on 2′,5′-ADP-Sepharose 4B (Pharmacia) and subsequently by gel filtration chromatography on acrylamide-agarose (AcA 44 Ultrogel; Pharmacia), as described by Yasukochi and Masters (18Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar). The preparation also appeared homogenous on SDS-PAGE (data not shown). Preliminary measurements indicated that in addition to cytochromeb 5 and its reductase, further components present in fractions 88–101 (Fig. 2, peak 4) were also required for reconstitution of amidoxime reductase. The combined fractions 88–101 were concentrated by ultrafiltration (exclusion size, 30 kDa; Amicon), desalted by gel filtration (NAP10, Pharmacia), and chromatographed on an anion exchange column by preparative HPLC with a conventional HPLC system (L-6210 Intelligent Pump, 655 A-22 UV detector, D-2500 integrator; Merck/Hitachi, Darmstadt, FRG). 1 mg of protein was applied to a semipreparative Fractogel EMD TMAE 650(S) column (inner diameter, 10 mm; length, 150 mm; particle size, 25–40 μm; Merck), which had been equilibrated with buffer E (10 mm Tris acetate, pH 7.4, 20% (w/v) glycerol, 0.5% (w/v) Thesit, 0.1 mm EDTA, 0.1 mm dithiothreitol). After equilibration, fractions were eluted with a one-step increase in the salt concentration up to 500 mm in buffer E at a flow rate of 1 ml/min. The fractions with the highest absorbance at 280 nm (Fig.3) were pooled and concentrated by pressure dialysis, tested for NADH cytochrome b 5reductase activity, and tested for benzamidoxime reductase activity in the reconstituted system. TM1 showed only one band on SDS-PAGE (Fig.4).Figure 4SDS-PAGE of the prepurified enzymes after preparative HPLC separation on a Fractogel-TMAE(S) column. STD, protein standards. Lane 1, peak 4 (Fig. 2);lane 2, fraction TM2 (Fig. 3); lane 3, fraction TM1 (Fig. 3). For details, see "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fraction TM1 (Fig. 3, peak 1), which contained the third component required for benzamidoxime reductase activity in the reconstituted system and the UDP-glucuronyltransferase (both M r 50,000), was applied to a UDP-hexanolamine-Sepharose (Sigma) column (inner diameter, 1.2 cm; length, 4.5 cm) equilibrated with 10 mm Tris acetate buffer, pH 7.4, and washed with 50 ml of buffer F, consisting of 50 mm KCl and 40 μm phosphatidylcholine in 10 mm Tris acetate buffer, pH 7.4. Activity was recovered quantitatively in the effluent, which showed only one band (50 kDa) on SDS-PAGE (like TM1 in Fig. 4). This fraction is named the third protein or the third component. Detergents were removed from the purified enzymes by shaking the concentrated fractions for 4 h with Calbiosorb (Calbiochem, La Jolla, CA) at 4 °C. Benzamidoxime was synthesized by a published method (19Krüger P. Ber. Dtsch. Chem. Ges. 1885; 18: 1055-1060Crossref Google Scholar). Benzamidine was obtained from Aldrich-Chemie (Steinheim, FRG), dextromethorphan was from Sigma, and dextrophan-d-tartrate was from ICN Biochemicals Inc. NADPH and NADH were purchased from Merck. Spectrophotometric measurements were performed with a Kontron Uvicon 930 spectrophotometer. For HPLC analysis, a conventional system was used: L-5000 controller, 655 A-11 HPLC gradient pump, L-422 UV detector, AS-2000 autosampler, and D-2500 integrator (Merck/Hitachi). Amino acid sequence analyses on peptide fragments of the purified proteins were carried out by TopLab (Munich, FRG). All protein concentrations were measured using the method described by Smith et al. (20Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18507) Google Scholar) with bicinchoninic acid (BCA reagent kit; Pierce). The SDS-PAGE analyses were carried out by the method of Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar), with a 3% stacking gel. For cytochromeb 5 preparations a 12% separation gel was used, while all other preparations were separated on an 8% gel (1.5-mm thickness). Staining was performed with Coomassie Brilliant Blue R250 (Serva, Heidelberg, FRG). Standards and samples were pretreated with β-mercaptoethanol for 5 min at 90 °C. The following proteins were used as standards (high molecular weight calibration kit; Pharmacia): myosin (M r 205,000), β-galactosidase (M r 116,000), phosphorylase b(M r 94,000), albumin (M r67,000), ovalbumin (M r 45,000), and carbonic anhydrase (M r 29,000). Cytochromeb 5 concentration was determined by recording the reduced minus the oxidized spectrum (absorbance 185 mm−1 cm−1) as described by Estabrook and Werringloer (22Estabrook R.W. Werringloer J. Methods Enzymol. 1978; 52: 212-220Crossref PubMed Scopus (302) Google Scholar). Cytochrome P450 concentrations were determined by measuring the carbon monoxide difference spectra after reduction with dithionite as described by Omura und Sato (23Omura T. Sato R. J. Biol. Chem. 1964; 239: 2370-2378Abstract Full Text PDF PubMed Google Scholar). In the case of TM1 protein, cytochrome P450 was also quantified by CO difference spectra after reduction (50 μg of protein) with 3 units of NADPH-P450 reductase, 1.5 mm NADPH and 40 μml-α-dilauroyl phosphatidylcholine (DLPC) (Sigma) in a total volume of 1 ml of 100 mmpotassium phosphate, pH 7.4. After preincubation for 5 min at 25 °C, the cuvettes were saturated with CO, and the difference spectra were recorded. NADH-cytochromeb 5 was measured by its NADH-ferricyanide reductase activity (1 unit = 1 μmol of reduced ferricyanide/min) as described by Mihara and Sato (24Mihara K. Sato R. Methods Enzymol. 1978; 52: 102-108Crossref PubMed Scopus (93) Google Scholar). NADPH-cytochrome P450 reductase activity was measured by following the reduction of cytochrome c (type 4 horse heart; Sigma) as described (25Williams Jr., C.H. Kamin H. J. Biol. Chem. 1962; 237: 587-595Abstract Full Text PDF PubMed Google Scholar) with minor modifications (18Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar) (1 milliunit = 1 nmol of reduced cytochrome c/min). The incubation mixture for the reconstituted enzyme system consisted of 50 pmol of cytochrome b 5, 0.5 unit of NADH cytochromeb 5 reductase, 5 μg of fraction TM1 (Fig. 3,peak 1) or 5 μg of third protein, 7.5 μg of DLPC (final concentration, 40 μm; Sigma), and 150 nmol of benzamidoxime (final concentration, 0.5 mm) in a 100 mm potassium phosphate buffer, pH 6.3. After a 5-min preincubation period at 37 °C, the reaction was initiated by the addition of NADH (final concentration, 1 mm) to a total volume of 300 μl. After 20 min, the reaction was terminated by adding 300 μl of methanol on ice. Precipitated proteins were sedimented by centrifugation at 10,000 × g for 5 min, and the supernatant was analyzed by HPLC (26ät zu KielWohlers, H. (1994) Studien zur Biotransformation von Guanidinen und Amidinen mit Hepatozyten und Mikrosomen, Ph. D. thesis, ChristianAlbrecht-Universität zu Kiel.Google Scholar). Aliquots of 10 μl were injected into a LiChrospher RP-Select B column (125 × 5 mm, 5 μm; Merck) with an RP-Select B precolumn (4 × 4 mm; Merck). The separation was carried out at room temperature with 3 mm1-octanesulfonic acid, pH 2.5 (adjusted with 85% phosphoric acid)/acetonitrile (88:12, v/v) as the mobile phase, at a flow rate of 0.7 ml/min. The effluent was monitored at 229 nm. The retention time for benzamidine was 16.5 ± 0.4 min and 14.3 ± 0.2 min for benzamidoxime when injected as controls (Fig.5). The rate of product formation was linear for about 60 min, and the limit of detection of benzamidine was 10 pmol/injection. Standard curves with 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, and 50.0 μm benzamidine were constructed and found to be linear over this range with correlation coefficients >0.9993. The recovery of benzamidine from incubation mixtures was 98.5 ± 1.3% (n = 36) of that obtained using samples that contained the same amount of benzamidine dissolved in phosphate buffer. The reductase activities of pig and human liver microsomes were measured by following the reduction of benzamidoxime, as described above for the reconstituted system, in 300 μl of 100 mm phosphate, pH 6.3, containing 500 μm benzamidoxime, 1 mm NADH, and 0.05–0.1 mg of human liver microsomes or 0.2 mg of pig liver microsomes. Ten different samples of human liver microsomes obtained from Human Biologics, Inc. (Phoenix, AZ) were tested. Various enzymatic activities related to specific human P450 subfamilies had been determined by Human Biologics, Inc. TheO-demethylation of dextromethorphan was carried out essentially as described (27Jaruratanasirikul S. Hortiwakul R. J. Pharm. Pharmacol. 1994; 46: 933-935Crossref PubMed Scopus (11) Google Scholar) in 100 mm phosphate, pH 7.4, containing 0.3 units of NADPH-cytochrome-P450 reductase, 5 μg of third protein component or 5 μg of fraction TM1 (Fig. 3), 40 μm DLPC, and 3.3 mm MgCl2 in a final volume of 300 μl. The reconstituted assay for the reduction of benzamidoxime was carried out with 0.1 mm NADH (final concentration), and the oxidation of NADH during the incubation was monitored by absorbance at 340 nm. Further procedures were performed as described above. The data summarized in TableI demonstrate that reduction of benzamidoxime by NADH requires three proteins purified to apparent homogeneity from pig liver microsomes. The fastest rates were consistently observed with mixtures containing 0.5 units of NADH-cytochrome b 5 reductase, 50 pmol of cytochrome b 5, and 5 μg of the third protein isolated from peak 4 (Fig. 2) along with DLPC in phosphate buffer. DLPC was the least essential ingredient, and omitting this phospholipid decreased activity by only 30%. On the other hand, omitting either cytochrome b 5 or its reductase virtually abolished activity, and omission of the third protein component decreased activity by almost 90% compared with rates obtained with the complete system (Table I). Replacing NADH with NADPH at the same initial concentration (1 mm) reduced activity by almost 60%. The consumption of NADH was linear during the incubation with the complete system. The molar ratio of benzamidine production and depletion of NADH was 1. In the absence of the third protein component, the decrease of absorbance was the same as the blank (without substrate).Table IN-Reduction of benzamidoximeComposition of incubation mixturen 1-aNumber of determinations.Activity1-bActivity values are in nmol of benzamidine/min/mg of third protein ± S.D.Percentage1-cBased upon activity in the complete system.%Complete system1298.8 ± 7.8100Without NADH9ND1-dND, not detectable.0.0Without NADH/with NADPH (1 mm)937.2 ± 4.937.7 ± 5.0Without cytochromeb 592.1 ± 0.72.1 ± 0.7Without NADH cytochrome b 5reductase9ND 1-dND, not detectable.0.0Without third protein910.6 ± 0.910.7 ± 0.9Without DLPC970.3 ± 1.671.1 ± 1.6With superoxide dismutase (500 units/ml)9148.2 ± 2.5150 ± 2.5A complete incubation mixture for optimized conditions consisted of 50 pmol of cytochrome b 5, 0.5 unit of NADH cytochromeb 5 reductase, 5 μg of third protein, 0.5 mm benzamidoxime, 1 mm NADH, 7.5 μg of DLPC (final concentration, 40 μm), in 0.3 ml of 100 mm phosphate buffer, pH 5.5. Incubation, sample preparation, and HPLC were performed as described under "Experimental Procedures." Each incubation was measured in triplicate. At least two separate incubations were performed. Values are mean ± S.D.1-a Number of determinations.1-b Activity values are in nmol of benzamidine/min/mg of third protein ± S.D.1-c Based upon activity in the complete system.1-d ND, not detectable. Open table in a new tab A complete incubation mixture for optimized conditions consisted of 50 pmol of cytochrome b 5, 0.5 unit of NADH cytochromeb 5 reductase, 5 μg of third protein, 0.5 mm benzamidoxime, 1 mm NADH, 7.5 μg of DLPC (final concentration, 40 μm), in 0.3 ml of 100 mm phosphate buffer, pH 5.5. Incubation, sample preparation, and HPLC were performed as described under "Experimental Procedures." Each incubation was measured in triplicate. At least two separate incubations were performed. Values are mean ± S.D. Benzamidoxime reductase activity of the reconstituted system was insensitive to catalase and cyanide. A complete inhibition of the benzamidoxime reductase activity was observed upon the addition of 0.5 mm N-methylhydroxylamine (data not shown). Superoxide dismutase (500 units/ml) increased the rate of reduction with some preparations as much as 50%. We also investigated the addition of NADPH-cytochrome P450 reductase. The presence of NADPH-cytochrome P450 reductase in the reconstituted system decreased the benzamidoxime reductase activity by 35% (TableII). In addition to this, added NADPH (1 mm) resulted in a further 25% inhibition compared with the complete system. The inhibitory effect of NADPH-P450 reductase was reversed by the addition of superoxide dismutase. The replacement of NADH-cytochrome b 5 reductase by NADPH-P450 reductase in presence of superoxide dismutase diminished activity by about 80%; in the absence of superoxide dismutase, however, there was no detectable activity (Table II).Table IIInfluence of NADPH-cytochrome P450 reductase on the reconstituted systemComposition of incubation mixture2-afp1, NADH-cytochrome b 5 reductase; fp2, NADPH-cytochrome P450 reductase (0.5 units); SOD, superoxide dismutase (final concentration, 500 units/ml).n 2-bNumber of determinations.Activity2-cActivity values are in nmol of benzamidine/min/mg of third protein ± S.D.Percentage2-dBased upon activity in the complete system.%Complete system12147.9 ± 5.3100 ± 3.6With fp2895.4 ± 8.764.5 ± 5.9With fp2/with SOD4220.7 ± 3.7149.2 ± 2.5With fp2/with NADPH861.5 ± 14.541.6 ± 9.8Without fp1/with fp2/with SOD429.1 ± 1.820.1 ± 1.2Without fp1/with fp24ND2-eNot detectable.0.0The assays were performed as described in Table I. Values are mean ± S.D.2-a fp1, NADH-cytochrome b 5 reductase; fp2, NADPH-cytochrome P450 reductase (0.5 units); SOD, superoxide dismutase (final concentration, 500 units/ml).2-b Number of determinations.2-c Activity values are in nmol of benzamidine/min/mg of third protein ± S.D.2-d Based upon activity in the complete system.2-e Not detectable. Open table in a new tab The assays were performed as described in Table I. Values are mean ± S.D. The addition of dextromethorphan (final concentration, 0.5 mm), NADPH-P450 reductase (0.5 unit), and NADPH (final concentration, 1 mm) to the reconstituted benzamidoxime reductase system diminished the appearance of the benzamidine product by about 30% in presence of superoxide dismutase (500 units/ml) (data not shown). In addition to benzamidoxime, the reconstituted system also catalyzed the reduction of guanoxabenz and N-hydroxydebrisoquine (data not shown). Although the reduction of these N-hydroxylated substrates by NADH required the same components, the optimal pH for reduction of N-hydroxydebrisoquine (near pH 7) was higher than that for the reduction of guanoxabenz (pH 6) or benzamidoxime (pH 5.5). Spectra of this protein purified to apparent homogeneity from peak TM1 (Fig. 3) were similar to those of other cytochrome P450 isoenzymes and, like the latter, gave typical reduced minus oxidized difference spectra in the presence of CO (data not shown). The amino acid sequences of three peptides isolated from a proteolytic digest of the purified protein are shown in Table III. The sequence of peptide 2 is identical to that of a sequence in P450 2D described by Tsuneoka et al. (28Tsuneoka Y. Matso Y. Higuchi R. Ichikawa Y. Eur. J. Biochem. 1992; 208: 739-746Crossref PubMed Scopus (17) Google Scholar), and the sequences of peptides 1 and 3 are similar to sequences of P450 2D (peptide 1, 66.7%; peptide 3, 83.3%) and P450 2D6 (peptide 1, 73.3%; peptide 3, 75%) (28Tsuneoka Y. Matso Y. Higuchi R. Ichikawa Y. Eur. J. Biochem. 1992; 208: 739-746Crossref PubMed Scopus (17) Google Scholar, 29Gonzalez F.J. Skoda R.C. Kimura S. Umeno M. Zanger U.M. Nebert D.W. Gelboin H.V. Hardwick J.P. Meyer U.A. Nature. 1988; 331: 442-446Crossref PubMed Scopus (634) Google Scholar). The sequence of peptide 3 also shows similarity to P450 isoenzymes of the subfamilies CYP2A (≤57.8%), CYP1A (≤57.8%) and CYP2J (≤56.3%), while the sequence of peptide 2 shows a slight similarity to the subfamily CYP3A.Table IIIAmino acid sequences of peptides of third protein component compared to sequences of CYP 2D and CYP 2D6Peptide 1Met Leu Lys Leu Leu Asp Leu Val Leu Glu Gly Leu Lys Glu GluSequence of 2D3-aRef. 28. See "Experimental Procedures."Ile Ile –3-b–, same amino acid as in third protein component. – – –– Thr Glu Asp – – – – –Sequence of 2D63-cRef. 29. See "Experimental Procedures."Phe – Arg – – – – Thr Gln – – – – – –Peptide 2Met Ile Leu His Pro Asp Val Gln Arg Arg Val Gln Gln Glu Ile AspSequence of 2D3-aRef. 28. See "Experimental Procedures."– – – – – – – – – – – – – – – –Sequence of 2D63-cRef. 29. See "Experimental Procedures."– – – – – – – – – – – – – – – –Peptide 3Asp Glu Val Ile Gly His Val Arg Gln Pro Glu MetSequence of 2D3-aRef. 28. See "Experimental Procedures."– – – – – Gln – – Arg – – –Sequence of 2D63-cRef. 29. See "Experimental Procedures."– Asp – – – Gln – – Arg – – –3-a Ref. 28Tsuneoka Y. Matso Y. Higuchi R. Ichikawa Y. Eur. J. Biochem. 1992; 208: 739-746Crossref PubMed Scopus (17) Google Scholar. See "Experimental Procedures."3-b –, same amino acid as in third protein component.3-c Ref. 29Gonzalez F.J. Skoda R.C. Kimura S. Umeno M. Zanger U.M. Nebert D.W. Gelboin H.V. Hardwick J.P. Meyer U.A. Nature. 1988; 331: 442-446Crossref PubMed Scopus (634) Google Scholar. See "Experimental Procedures." Open table in a new tab The data summarized in Table IVdemonstrate that the third protein component can function as a monooxygenase in the system reconstituted with the NADPH-cytochrome P450 reductase. The results show that the protein purified to homogeneity from fraction TM1 also has the catalytic activity expected of this class of P450-dependent monooxygenases.Table IVDemethylation of dextromethorphanComposition of incubation mixturen 4-aNumber of determinations.Activity4-bActivity values are in nmol of dextrophan/min/mg of third protein.Complete system TM185 ± 0.2 Third protein810 ± 0.5Without NADPH8ND4-cND, not detectable.Without P450 reductase8NDWithout third protein8NDThe complete incubation mixture consisted of 5 μg of third protein (TM1 or UDP1), 0.3 units of NADPH cytochrome P450 reductase, 7.5 μg of DLPC (final concentration, 40 mm), 3.3 mmMgCl2, 200 μm NADPH, and 50 μmdextromethorphan in 0.3 ml of 100 mm phosphate buffer, pH 7.4, as described under "Experimental Procedures." Each incubation was performed in duplicate on two separate occasions, and the analysis of each incubation was carried out twice. Values are mean ± S.D.4-a Numb