Identification of the Missing Component in the Mitochondrial Benzamidoxime Prodrug-converting System as a Novel Molybdenum Enzyme
2006; Elsevier BV; Volume: 281; Issue: 46 Linguagem: Inglês
10.1074/jbc.m607697200
ISSN1083-351X
AutoresAntje Havemeyer, Florian Bittner, Silke Wollers, Ralf R. Mendel, Thomas Kunze, Bernd Clement,
Tópico(s)Biochemical Acid Research Studies
ResumoAmidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b5 and cytochrome b5 reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b5/NADH cytochrome b5 reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor. Amidoximes can be used as prodrugs for amidines and related functional groups to enhance their intestinal absorption. These prodrugs are reduced to their active amidines. Other N-hydroxylated structures are mutagenic or responsible for toxic effects of drugs and are detoxified by reduction. In this study, a N-reductive enzyme system of pig liver mitochondria using benzamidoxime as a model substrate was identified. A protein fraction free from cytochrome b5 and cytochrome b5 reductase was purified, enhancing 250-fold the minor benzamidoxime-reductase activity catalyzed by the membrane-bound cytochrome b5/NADH cytochrome b5 reductase system. This fraction contained a 35-kDa protein with homologies to the C-terminal domain of the human molybdenum cofactor sulfurase. Here it was demonstrated that this 35-kDa protein contains molybdenum cofactor and forms the hitherto ill defined third component of the N-reductive complex in the outer mitochondrial membrane. Thus, the 35-kDa protein represents a novel group of molybdenum proteins in eukaryotes as it forms the catalytic part of a three-component enzyme complex consisting of separate proteins. Supporting these findings, recombinant C-terminal domain of the human molybdenum cofactor sulfurase exhibited N-reductive activity in vitro, which was strictly dependent on molybdenum cofactor. Numerous drugs and drug candidates contain strongly basic functional groups, such as guanidines, amidinohydrazones, and amidines. These groups, however, impair drug absorption from the gastrointestinal tract, because they are protonated under physiological conditions. Therefore, the prodrug principle was developed to enhance oral bioavailability (1Ettmayer P. Amidon G.L. Clement B. Testa B. J. Med. Chem. 2004; 47: 2393-2404Crossref PubMed Scopus (328) Google Scholar). In the case of amidines, N-hydroxylation converts them to the corresponding N-hydroxyamidines that are less basic and therefore unprotonated under physiological conditions, thereby enhancing intestinal absorption by diffusion (2Clement B. Drug Metab. Rev. 2002; 34: 565-579Crossref PubMed Scopus (115) Google Scholar). This prodrug principle was applied to a wide range of mainly antiprotozoal and antithrombotic drugs (2Clement B. Drug Metab. Rev. 2002; 34: 565-579Crossref PubMed Scopus (115) Google Scholar). The wider application of amidoxime prodrugs requires the identification of the cellular N-reducing enzyme system. Previous studies described the ability of microsomes and mitochondria to reduce N-hydroxylated amidines (3Andersson S. Hofmann Y. Nordling A. Li X.Q. Nivelius S. Andersson T.B. Ingelman-Sundberg M. Johansson I. Drug Metab. Dispos. 2005; 33: 570-578Crossref PubMed Scopus (34) Google Scholar, 4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar, 5Hauptmann J. Paintz M. Kaiser B. Richter M. Pharmazie. 1988; 43: 559-560PubMed Google Scholar, 6Clement B. Schmitt S. Zimmermann M. Arch. Pharm. (Weinheim). 1988; 321: 955-956Crossref PubMed Scopus (31) Google Scholar), with the highest activities in the outer mitochondrial membrane (3Andersson S. Hofmann Y. Nordling A. Li X.Q. Nivelius S. Andersson T.B. Ingelman-Sundberg M. Johansson I. Drug Metab. Dispos. 2005; 33: 570-578Crossref PubMed Scopus (34) Google Scholar, 4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar). For mammals, it was demonstrated that cytochrome (cyt) 2The abbreviations used are: cyt, cytochrome; ABA3, A. thaliana molybdenum cofactor sulfurase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CT, C terminus; HPLC, high performance liquid chromatography; MALDI, matrix-assisted laser desorption ionization; Moco, molybdenum cofactor; MS, mass spectrometry; MS/MS, tandem mass spectrometry; OMV, outer membrane vesicle; TOF, time-of-flight; HMCS, molybdenum cofactor sulfurase. b5 and its FAD-containing reductase are involved in the reduction of hydroxylamines and N-hydroxylated amidines (7Kadlubar F.F. Ziegler D.M. Arch. Biochem. Biophys. 1974; 162: 83-92Crossref PubMed Scopus (65) Google Scholar, 8Clement B. Lomb R. Moller W. J. Biol. Chem. 1997; 272: 19615-19620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Both enzymes are present in a soluble and a membrane-bound form. The latter are components of an electron transport system associated with the mitochondrial outer membrane and the endoplasmatic reticulum in somatic cells (9Sottocasa G.L. Kuylenstierna B. Ernster L. Bergstrand A. J. Cell Biol. 1967; 32: 415-438Crossref PubMed Scopus (1820) Google Scholar), where they mediate electron transfer from NADH to a variety of final acceptors involved in lipid metabolism, such as fatty acid desaturase (10Strittmatter P. Spatz L. Corcoran D. Rogers M.J. Setlow B. Redline R. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4565-4569Crossref PubMed Scopus (454) Google Scholar), sterol oxidases (11Fukushima H. Grinstead G.F. Gaylor J.L. J. Biol. Chem. 1981; 256: 4822-4826Abstract Full Text PDF PubMed Google Scholar), and certain P450 enzymes (12Hildebrandt A. Estabrook R.W. Arch. Biochem. Biophys. 1971; 143: 66-79Crossref PubMed Scopus (455) Google Scholar). According to the physical electron transfer chain, an additional enzyme component is also postulated for drug metabolism. Kadlubar and Ziegler (7Kadlubar F.F. Ziegler D.M. Arch. Biochem. Biophys. 1974; 162: 83-92Crossref PubMed Scopus (65) Google Scholar) described the oxygen-independent microsomal hydroxylamine reductase as a multicomponent enzyme system consisting of cyt b5, its reductase, and a third unidentified protein fraction that catalyzes the reduction of hydroxylamine and a number of its mono- and disubstituted derivatives. Later a similar microsomal enzyme system was described: a microsomal N-hydroxy reductase (8Clement B. Lomb R. Moller W. J. Biol. Chem. 1997; 272: 19615-19620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), consisting of the membrane-bound electron transfer chain described above and a postulated microsomal P450 isoenzyme (Fig. 1) as N-reductive protein that should be able to reduce N-hydroxylated derivatives of amidines, sulfonamides, and numerous other N-hydroxylated functional groups (8Clement B. Lomb R. Moller W. J. Biol. Chem. 1997; 272: 19615-19620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Clement B. Behrens D. Amschler J. Matschke K. Wolf S. Havemeyer A. Life Sci. 2005; 77: 205-219Crossref PubMed Scopus (14) Google Scholar, 14Clement B. Lopian K. Drug Metab. Dispos. 2003; 31: 645-651Crossref PubMed Scopus (76) Google Scholar). The necessity of an additional N-reductive enzyme of the membrane-bound microsomal drug metabolism system was confirmed by Andersson et al. (3Andersson S. Hofmann Y. Nordling A. Li X.Q. Nivelius S. Andersson T.B. Ingelman-Sundberg M. Johansson I. Drug Metab. Dispos. 2005; 33: 570-578Crossref PubMed Scopus (34) Google Scholar), although the identity of the third protein remained open. Recently, it was found that also the outer mitochondrial membrane harbored a N-reductive system (4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar) similar to the one of microsomes, and a third protein component was postulated to be necessary for N-reduction. Here the identification of this third N-reductive component in the outer mitochondrial membrane of pig liver is described. It turned out to be a novel molybdenum-containing protein hitherto not described in eukaryotes or bacteria sharing significant homology with the C-terminal domain of the molybdenum cofactor sulfurase (HMCS-CT). Purification of the N-Reductive Mitochondrial Component—Pig liver mitochondria were obtained by differential centrifugation and an isotonic Percoll gradient (15Hovius R. Lambrechts H. Nicolay K. de Kruijff B. Biochim. Biophys. Acta. 1990; 1021: 217-226Crossref PubMed Scopus (311) Google Scholar) with modifications (4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar). The outer membrane vesicle (OMV) fraction was purified using the swell disruption method followed by two steps of sucrose density gradient centrifugation (16de Kroon A.I. Dolis D. Mayer A. Lill R. de Kruijff B. Biochim. Biophys. Acta. 1997; 1325: 108-116Crossref PubMed Scopus (195) Google Scholar) with modifications (4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar). Frozen OMV fraction was thawed, pooled, and adjusted to 20 mm Tris-base, 0.1 mm EDTA, 0.1 mm dithiothreitol, 20% (m/v) glycerol, 0.9 mm Zwittergent® 3-14, pH 7.4. The detergent/protein ratio was 0.5. This solubilization mixture was stirred for 60 min on ice. The solubilized membrane fraction (∼13 mg of protein) was applied to a DEAE-52 column (2.5 × 10 cm) (DEAE 52-Cellulose Servacel®; Serva Electrophoresis, Heidelberg, Germany) and equilibrated with buffer (20 mm Tris-base, 0.1 mm EDTA, 0.1 mm dithiothreitol, 20% (m/v) glycerol, 0.9 mm Zwittergent® 3-14, pH 7.4). After sample application, the column was washed at a flow rate of 0.8 ml/min with two column volumes of the equilibrating buffer and was developed with a linear concentration gradient (250 ml) of 0–1.0 sodium chloride in equilibrating buffer. Fractions of 4 ml were collected and assayed. Active fractions were pooled and stored at –80 °C. All operations were performed at 0–4 °C. Protein was assayed using bicinchonic acid (17Smith 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 (18792) Google Scholar), according to the manufacturer's directions (BCA protein assay kit; Pierce), using bovine serum albumin as a standard. Purification of NADH Cyt b5 Reductase—NADH Cyt b5 reductase was purified from pig liver microsomes by affinity chromatography on 5′-AMP-Sepharose 4 B (Amersham Biosciences), similar to the procedure described for the purification of NADH-P450 reductase (18Yasukochi Y. Masters B.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar) with modifications (8Clement B. Lomb R. Moller W. J. Biol. Chem. 1997; 272: 19615-19620Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Recombinant purified human NADH cyt b5 reductase was obtained from Abnova Corp. (Taipei City, Taiwan). SDS-PAGE—SDS-PAGE analyses were carried out by the method of Laemmli (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar), with a 5% stacking gel. Staining was performed with Coomassie Brilliant Blue R250 (Serva, Heidelberg, Germany). Standards and samples were pretreated with β-mercaptoethanol for 5 min at 90 °C. Mass Spectrometry—Proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass finger printing or nanocapillary liquid chromatography electrospray ionization tandem mass spectrometry (MS/MS). The analysis were performed by Planton GmbH (Kiel, Germany) and Richard Jones (National Center for Toxicological Research). Immunoblot Analysis—Immunoblot analysis was performed by gel-blotting protein-fraction A and B subjected to 12% SDS-PAGE using a primary polyclonal antibody raised against recombinantly expressed C terminus of Arabidopsis thaliana molybdenum cofactor sulfurase (ABA3-CT) (1:7000 dilution). The secondary horseradish peroxidase-conjugated anti-rabbit Ig (Sigma) was used in a 1:10,000 dilution, and chemiluminescence was detected using the ECL system (Amersham Biosciences). nit-1 Reconstitution—Neurospora crassa nit-1 extract was prepared as described previously (20Nason A. Lee K.Y. Pan S.S. Ketchum P.A. Lamberti A. DeVries J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 3242-3246Crossref PubMed Scopus (154) Google Scholar) and stored in aliquots at –70 °C. All reconstitutions were performed in nit-1 buffer (50 mm sodium phosphate, 200 mm NaCl, and 5 mm EDTA, pH 7.2) containing 2 mm reduced glutathione either in the absence or in the presence of 5 mm sodium molybdate. The reconstitution assay was performed in a 40-μl reaction volume containing 20 μl of gel-filtrated nit-1 extract. Complementation was carried out anaerobically for 2 h at room temperature. After the addition of 20 mm NADPH and incubation for 10 min in the dark, reconstituted NADPH-nitrate reductase activity was determined as described (20Nason A. Lee K.Y. Pan S.S. Ketchum P.A. Lamberti A. DeVries J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 3242-3246Crossref PubMed Scopus (154) Google Scholar). Importantly, the addition of sodium molybdate to the reaction mix did not significantly enhance reconstitution of NADPH-nitrate reductase activity, indicating that all fractions tested contained molybdenum cofactor (Moco) rather than molybdenum-free molybdopterin. Cloning of hmcs-CT—Polymerase chain reaction was performed with a human liver cDNA library (λ-Uni-ZAP XR, Stratagene) as template and by using primers HMCS-1429+ (5′-GGA TAC ATG TCG ACG CTG GAT GAT-3′) and HMCS-stop (5′-TTA GGA GGT AAC ATC CTG GTG TTT CTC-3′) derived from GenBank™ entry AK000740, which represents a full-length hmcs-cDNA. By this procedure, a partial hmcs cDNA fragment of ∼1.3 kb was obtained, which contained the 3′-region of the hmcs open reading frame and whose correctness was confirmed by sequencing. Restriction sites for BamHI were introduced by polymerase chain reaction using primers HMCS-CT-start/BamHI (5′-ACC CAG GGA TCC ATG TCA GAG AAA GCT GCA GGA GTC CTG-3′) and HMCS-CT-stop/BamHI (5′-ACG GTG GAT CCT TAG GAG GTA ACA TCC TGG TGT TTC TC-3′), which amplified the last 966 bp of the open reading frame encoding for the putative C-terminal domain of HMCS. Simultaneous introduction of an ATG codon at the N-terminal end in frame with the vector-encoded His6 tag enabled cloning into the pQE80-plasmid (Qiagen, Hilden, Germany), thereby allowing expression of a His6-HMCS-CT fusion protein with an estimated molecular mass of 37.4 kDa. Cloning and expression of ABA3-CT for generation of anti-ABA3-CT polyclonal antibodies was performed as described earlier by Heidenreich et al. (21Heidenreich T. Wollers S. Mendel R.R. Bittner F. J. Biol. Chem. 2005; 280: 4213-4218Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Expression and Purification of HMCS-CT—Routine protein expression was performed in freshly transformed Escherichia coli TP1000 cells (22Palmer T. Santini C.L. Iobbi-Nivol C. Eaves D.J. Boxer D.H. Giordano G. Mol. Microbiol. 1996; 20: 875-884Crossref PubMed Scopus (147) Google Scholar). Cells were grown aerobically in Luria broth medium in the presence of 100 μg/ml ampicillin at 22 °C to a A600 = 0.1 before induction with 15 μm isopropyl-β-d-thiogalactopyranoside and the addition of 1 mm sodium molybdate. After induction, cells were grown for a further 20 h at 22 °C. Expression in E. coli strains RK5206 and RK5204 (23Stewart V. MacGregor C.H. J. Bacteriol. 1982; 151: 788-799Crossref PubMed Google Scholar) was carried out likewise without the addition of sodium molybdate. Cells were harvested by centrifugation and stored at –70 °C until use. Cell lysis was achieved by several passages through a French pressure cell followed by sonication for 5 min. After centrifugation, His6-tagged protein was purified on a nickel-nitrilotriacetic acid superflow matrix (Qiagen, Hilden, Germany) under native conditions at 4 °C according to the manufacturer's manual. Eluted fractions were analyzed by SDS-PAGE. Molybdenum binding pterin bound to the purified proteins was detected and quantified by converting it to the stable oxidation product FormA-dephospho, according to Johnson et al. (24Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (275) Google Scholar). Oxidation, dephosphorylation, QAE chromatography, and high performance liquid chromatography (HPLC) analysis were performed as described previously (25Schwarz G. Boxer D.H. Mendel R.R. J. Biol. Chem. 1997; 272: 26811-26814Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). FormA-dephospho was quantified by comparison with a standard isolated from xanthine oxidase for which the absorptivity was ϵ380 = 13,200 m–1 cm–1 (24Johnson J.L. Rajagopalan K.V. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6856-6860Crossref PubMed Scopus (275) Google Scholar). Thereby, average saturation of HMCS-CT with MPT/Moco, determined as FormA-dephospho, was found to be about 41% (412 ± 96 pmol of FormA/nmol of protein; n = 5). NADH Cyt b5 Reductase Activity—Enzyme activity was determined by modification of the ferricyanide reduction assay (26Mihara K. Sato R. Methods Enzymol. 1978; 52: 102-108Crossref PubMed Scopus (93) Google Scholar). Heme Content—Heme content was estimated by recording the sodium dithionite-reduced minus the oxidized spectrum (27Estabrook R.W. Werringloer J. Methods Enzymol. 1978; 52: 212-220Crossref PubMed Scopus (303) Google Scholar). Assay for the Reduction of Benzamidoxime—For determination of benzamidoxime reduction, incubations were carried out under aerobic conditions at 37 °C in a shaking water bath. Incubation mixtures of the subcellular fractions contained 56 μg (mitochondria) or 6 μg of protein (OMV fraction), 0.5 mm benzamidoxime (synthesized from benzonitrile and hydroxylamine as described in Ref. 28Krüger P. Ber. Dtsch. Chem. Ges. 1885; 18: 1055-1060Crossref Google Scholar), and 1.0 mm NADH (or 0.4 mm NADH in the case of the OMV fraction) in a total volume of 150 μl of 100 mm potassium phosphate buffer, pH 6.0. After preincubation for 3 min at 37 °C, the reaction was initiated by the addition of NADH and terminated after 15 min (OMV fraction) or 20 min (mitochondria) by adding aliquots of methanol. If not otherwise stated, standard incubation mixtures of the reconstituted system contained 240 ng of purified mitochondrial enzyme fraction, 0.3 units of cyt b5 reductase, 100 pmol of recombinant purified cyt b5 (MoBiTec GmbH, Göttingen, Germany), 0.5 mm benzamidoxime, and 1.0 mm NADH in a total volume of 150 μl of 100 mm potassium phosphate buffer, pH 6.0. After preincubation for 3 min at 37 °C, the reaction was started by NADH and terminated after 15 or 30 min by adding aliquots of methanol. If not otherwise stated, incubations with HMCS-CT contained 135 μg of recombinant protein, 0.5 mm benzamidoxime, and 1.0 mm NADH in a total volume of 150 μl of 100 mm potassium phosphate buffer, pH 6.0. After preincubation for 3 min at 37 °C, the reaction was started by benzamidoxime and terminated after 10 min by adding aliquots of methanol. The precipitated proteins were sedimented by centrifugation, and the supernatant was analyzed by HPLC. Inhibition Studies—Incubations with the OMV fraction were performed as described above with slight modifications. After 5 min of preincubation of protein, inhibitor, and NADH, the reaction was started by the addition of benzamidoxime. Studies were performed with 0–100 μm sodium vanadate. HPLC Method for Benzamidine Quantification—The separation was carried out isocratically by 10 mm 1-octylsulfonate sodium salt and 17% acetonitrile (v/v) (pH not adjusted) by a LiChroCART® 250-4 HPLC-Cartridge with LiChrospher® 60 RP-select B (5 μm) and a LiChroCART® 4-4 guard column (Merck) at a flow rate of 1.0 ml/min. The effluent was monitored at 229 nm. For the determination of the recovery rate, reaction mixtures with defined concentrations of synthetic reference substance (1–300 μm) were incubated and worked up under the same conditions as the experimental samples but without adding cofactor. The standard curves were linear over this range with correlation coefficients of 0.999 (n = 48). The signals (peak areas) obtained were compared with those of the same amount of benzamidine dissolved in the mobile phase. The recovery rate from pig liver mitochondria amounted to 105% (r2 = 0.9993). Similar values were obtained from the OMV fraction. The retention times were 7.9 ± 0.1 min (benzamidoxime) and 26.7 ± 0.1 min (benzamidine). Mass Spectrometry—The formation of the reductive metabolite benzamidine was examined by mass spectrometry according to the method described by Froehlich et al. (29Frohlich A.K. Girreser U. Clement B. Xenobiotica. 2005; 35: 17-25Crossref PubMed Scopus (13) Google Scholar). Purification of a N-Reductive Protein Fraction—Identification of the third N-reductive component in the outer mitochondrial membrane remained elusive in previous studies. Therefore, a rigorously improved purification protocol was elaborated (for details, see "Experimental Procedures") consisting of three main steps. (i) The OMV fraction was prepared from pig liver mitochondria (4Clement B. Mau S. Deters S. Havemeyer A. Drug Metab. Dispos. 2005; 33: 1740-1747Crossref PubMed Scopus (39) Google Scholar). (ii) The membrane-bound N-reductive enzyme complex was solubilized in a stable and highly active form. Here, Zwittergent® 3-14 turned out to be superior to all other detergents (CHAPS, cholate, digitonin, thesit, and Triton X-100) tested, and full N-reductive activity was retained up to a detergent/protein ratio of 5. The activity was stable over a period of 6 days at 4 °C. Vanadate as inhibitor of benzamidoxime reductase almost abolished the N-reductive activity of the outer mitochondrial membrane, exerting an IC50 value of 8 μm. (iii) ion exchange chromatography of detergent-solubilized OMV fractions gave two pools (designated as fractions A and B) with benzamidoxime reductase activity (Fig. 2). Fraction A was devoid of any heme content (as tested by dithionite-reduced difference spectra) and of cyt b5 reductase activity, whereas fraction B contained minor contents of cyt b5 and its reductase. Therefore, fraction A was used for further analysis. Purification parameters are given in Table 1, and the results of SDS-PAGE analysis are shown in Fig. 3. The purified enzyme fraction A was stored at –80 °C, and the same activity in the reconstituted system was observed at least after one freezethaw cycle (data not shown).TABLE 1Purification of mitochondrial N-reductive componentsStageTotal activityTotal proteinSpecific activityPurificationYieldμmol/minmgμmol/min/mg-fold%Swell disruption homogenate36.520270.02aSpecific benzamidoxime reductase activity of membrane preparations (μmol/min/mg of total protein).1100Solubilized OMV fraction30.3130.23aSpecific benzamidoxime reductase activity of membrane preparations (μmol/min/mg of total protein).138Fraction A1.90.1910bSpecific benzamidoxime reductase activity of the reconstituted system (μmol/min/mg of protein of fraction A).556cCalculated to specific activity of the reconstituted system.5cCalculated to specific activity of the reconstituted system.a Specific benzamidoxime reductase activity of membrane preparations (μmol/min/mg of total protein).b Specific benzamidoxime reductase activity of the reconstituted system (μmol/min/mg of protein of fraction A).c Calculated to specific activity of the reconstituted system. Open table in a new tab FIGURE 3SDS-PAGE and Coomassie stain of mitochondrial preparations. A, pig liver mitochondria (Mt; 20 μg of protein); OMV fraction (OMV; 20 μgof protein). M, molecular mass marker (masses are indicated in kDa to the left of each panel). B, mitochondrial fraction A (A; 0.5 μg of protein).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Enzymatic Characterization of Fraction A—The reduction of benzamidoxime by NADH requires cyt b5, its reductase, and an additional N-reductive protein contained in the purified mitochondrial protein fraction A. Fraction A alone was not able to catalyze the reduction of benzamidoxime by NADH (Table 2). The low rate of benzamidoxime reduction obtained with the mitochondrial fraction A and purified NADH cyt b5 reductase (Table 2) is caused by the contamination of the reductase with small amounts of cyt b5. Similar results were obtained when replacing purified microsomal NADH cyt b5 reductase by purified recombinant NADH cyt b5 reductase (not shown). The N-reductive activity of the standard incubation mixture followed Michaelis-Menten kinetics (apparent Km 0.18 mm, Vmax was 12.25 μmol of benzamidine/min/mg of fraction A).TABLE 2N-Reduction of benzamidoxime by the reconstituted systemComposition of incubation mixtureSpecific activityFraction ATotal proteinμmol/min/mgnmol/min/mgComplete incubation mixture10.0 ± 0.14517 ± 7Complete incubation mixture with heat-denaturated fraction ANDaND, not detectable (below the limit of detection); limit of detection = 17 nmol/min/mg of fraction A.NDbND, not detectable (below the limit of detection); limit of detection = 1 nmol/min/mg of total protein.Complete incubation mixture without NADHNDaND, not detectable (below the limit of detection); limit of detection = 17 nmol/min/mg of fraction A.NDbND, not detectable (below the limit of detection); limit of detection = 1 nmol/min/mg of total protein.Complete incubation mixture without cyt b50.5 ± 0.0240 ± 2Complete incubation mixture without cyt b5 reductaseNDaND, not detectable (below the limit of detection); limit of detection = 17 nmol/min/mg of fraction A.NDcND, not detectable (below the limit of detection); limit of detection = 1 nmol/min/mg protein of fraction A and cyt b5.Complete incubation mixture without fraction A2.1 ± 0.4Only fraction ANDaND, not detectable (below the limit of detection); limit of detection = 17 nmol/min/mg of fraction A.a ND, not detectable (below the limit of detection); limit of detection = 17 nmol/min/mg of fraction A.b ND, not detectable (below the limit of detection); limit of detection = 1 nmol/min/mg of total protein.c ND, not detectable (below the limit of detection); limit of detection = 1 nmol/min/mg protein of fraction A and cyt b5. Open table in a new tab The specific activity of fraction B in the reconstituted system was slightly lower than the specific activity of fraction A (7.10 ± 0.08 μmol of benzamidine/min/mg (n = 4)). However, in contrast to fraction A, fraction B also reduced benzamidoxime without adding cyt b5 and its reductase with a specific activity of 30 ± 1 nmol of benzamidine/min/mg (n = 4), demonstrating that it is contaminated with cyt b5 and its reductase. Composition of Fraction A—SDS-PAGE analysis of fraction A showed an almost electrophoretically pure protein with two dominant spots (35 and 66 kDa) in a Coomassie-stained SDS gel (Fig. 3B). Whereas the 66-kDa protein was identified as monoamine oxidase-B by MALDI-TOF, tryptic digestion and electrospray ionization-MS/MS sequencing of the 35-kDa spot led to identification of four peptides, LWIYPVK, TEAYR, CILTTVDPDTGVIDRK, and VGDPVYR, which all displayed 100% identity to a putative protein from Homo sapiens hitherto referred to as "Moco sulfurase C-terminal domain-containing protein 2" (protein accession entry NP_060368). This result was confirmed by identifying the peptides LWIYPVKSCK, CILTTVDPDTGVIDRK, and VGDPVYRMV in a second independent experiment. Sequence comparisons showed that the protein identified shares 48% homology with the C-terminal domain of HMCS, which catalyzes the post-translational activation of the Moco-containing enzymes xanthine dehydrogenase and aldehyde oxidase (30Ichida K. Matsumura T. Sakuma R. Hosoya T. Nishino T. Biochem. Biophys. Res. Commun. 2001; 282: 1194-1200Crossref PubMed Scopus (118) Google Scholar). Antibodies raised against the heterologously expressed C-terminal domain of the Moco sulfurase ABA3 from A. thaliana, the plant counterpart of HMCS (31Bittner F. Oreb M. Mendel R.R. J. Biol. Chem. 2001; 276: 40381-40384Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), showed specific cross-reaction with the C-terminal domain of HMCS (HMCS-CT) (Fig. 4A). Also in fractions A and B from the outer membrane of pig liver mitochondria, the antibody specifically recognized the 35-kDa protein (Fig. 4B), which gives evidence for structural relation of the 35-kDa protein and the C termini of HMCS and ABA3. The upper band in Fig. 4B may derive from the 35-kDa protein with post-translational modifications, whic
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