J BIOCHEM MOLECULAR TOXICOLOGYVolume 16, Number 6, 2002
Biochemical Characteristics of Purified Beef LiverNADPHCytochrome P450 ReductaseEmel Arinc and Haydar LelikJoint Graduate Program in Biochemistry, Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey;E-mail: firstname.lastname@example.org
Received 20 June 2002; revised 12 September 2002; accepted 5 October 2002
ABSTRACT: NADPHcytochrome P450 reductase, anobligatory component of the cytochrome P450 depen-dent monooxygenase system, was purified to elec-trophoretic homogeneity from beef liver microsomes.The purification procedure involved the ion exchangechromatography of the detergent-solubilized micro-somes on first and second DEAE-cellulose columns,followed by 2,5-ADP Sepharose affinity chromatog-raphy. Further concentration of the enzyme and re-moval of Emulgen 913 and 2-AMP were accomplishedon the final hydroxylapatite column. The enzyme waspurified 239-fold and the yield was 13.5%. Monomermolecular weight of the enzyme was estimated to be76000 3000 (N = 5) by SDS-PAGE. The absolute ab-sorption spectrum of beef reductase showed two peaksat 455 and 378 nm, with a shoulder at 478 nm, charac-teristics of flavoproteins. The effects of cytochrome cconcentration, pH, and ionic strength on enzyme ac-tivity were studied. Reduction of cytochrome c withthe enzyme followed MichaelisMenten kinetics, andthe apparent Km of the purified enzyme was foundto be 47.7 M for cytochrome c when the enzyme ac-tivity was measured in 0.3 M potassium phosphatebuffer (pH 7.7). Stability of cytochrome c reductaseactivity was examined at 25 and 37C in the pres-ence and absence of 20% glycerol. The presence ofglycerol enhanced the stability of cytochrome c reduc-tase activity at both temperatures. Sheep lung micro-somal cytochrome P4502B and NADPHcytochromeP450 reductase were also purified by the already ex-isting methods developed in our laboratory. Both beefliver and sheep lung reductases were found to be ef-fective in supporting benzphetamine and cocaine N-demethylation reactions in the reconstituted systemscontaining purified sheep lung cytochrome P4502Band synthetic lipid, phosphatidylcholine dilauroyl.C 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol16:286297, 2002; Published online in Wiley InterScience(www.interscience.wiley.com). DOI 10.1002/jbt.10054
Correspondence to: Emel Arinc.Contract Grant Sponsor: Turkish Academy of Sciences (TUBA).
c 2002 Wiley Periodicals, Inc.
KEYWORDS: Cytochrome P450 Reductase; BeefLiver; Sheep Lung; Cytochrome P4502B; Kinetics;Benzphetamine; Cocaine
NADPHcytochrome P450 reductase (NADPH:ferrihemoprotein oxidoreductase, EC 126.96.36.199) is an inte-gral membrane flavoprotein that catalyzes the transferof electrons from NADPH to cytochrome P450 and isan essential component of the microsomal cytochromeP450 monooxygenase system. The monooxygenase sys-tem consists of NADPHcytochrome P450 reductaseand a family of heme proteins, cytochromes P450,and is involved in oxidative metabolism of both en-dogenous and exogenous compounds . Further-more, cytochrome P450 reductase is known to playimportant roles in the reductive metabolism of someanticancer drugs and antibiotics, as well as in lipidperoxidation and in the production of reactive oxygenspecies that can result in genotoxicity and cytotoxicity.
Functional and structural properties of cytochromeP450 reductase purified from microsomes of guinea pigliver , pig liver [10,11], pig kidney , sheep lungand liver [12,13], rat liver , rabbit liver , humanliver [16,17], and three fish species (scup liver , troutliver , and mullet liver ), and from a nonmam-malian species (house fly ) have been described indetail and some kinetic, structural, and functional dif-ferences are known. Cytochrome P450 reductase wasalso isolated from a plant species, Jerusalem artichoke,and the isolated reductase was found to be immuno-logically distinct from previously described reductases.
In recent years, there has been a great deal of in-terest in the study of metabolism of exogenous com-pounds such as veterinary drugs, chemical carcino-gens, antibiotics, growth factors by drug-metabolizingenzymes and formation of reactive oxygen species
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in veterinary animals, especially in beef, cattle, andsheep. However, very little information is availableon the role of cytochrome P450 dependent monooxy-genases in drug and xenobiotic metabolism by beefliver, although beef liver has a very high nutri-tional value and is extensively consumed by hu-mans. Studies with beef have been concentrated pri-marily on the steroid biosynthesis in adrenal cor-tex where cytochrome P450 and cytochrome b5 playa significant role in the synthesis of different typesof steroids. Therefore, elucidation of the characteris-tics and activity of the beef hepatic cytochrome P450system and NADPHcytochrome P450 reductase indrug and xenobiotic metabolism will make an im-portant contribution to a better understanding of thestructure and function of these enzymes, and theirrole in the metabolism of foreign compounds, in-cluding veterinary drugs. Different types of veteri-nary drugs and antibiotics are being used for ani-mal health in stockbreeding all over the world anda wide range of these drugs and antibiotics are me-tabolized by cytochrome P450 dependent monooxyge-nases and NADPHcytochrome P450 reductase alone.This study was undertaken to purify amphipathic cy-tochrome P450 reductase from beef liver and charac-terize its spectral, structural, biocatalytic, and kineticproperties.
MATERIALS AND METHODS
Glycerol, sodium dodecyl sulfate (SDS), ethylene-diaminetetraacetic acid (EDTA), and methanol werepurchased from E. Merck, Darmstadt, Germany.Cytochrome c, DL-dithiothreitol (DTT), phenyl-methylsulfonyl fluoride (PMSF), -aminocaproicacid (-ACA), -nicotinamide adenine dinucleotidephosphate (NADPH, reduced form), -nicotinamideadenine dinucleotide phosphate (NADP+), L--phosphatidylcholine, dilauroyl (DPLC) were theproducts of Sigma, St. Louis, MI, USA. 2,5-ADPSepharose 4B was obtained from Pharmacia FineChemicals, Inc., Uppsala, Sweden. Hydroxylapatite(Bio-Gel HTP) was purchased from Bio-Rad Laborato-ries, Richmond, CA, USA. Emulgen 913 was a gift fromKao-Atlas Co., Ltd., Tokyo, Japan. Diethylaminoethyl(DEAE)-cellulose (DE microgranular preswollen) waspurchased from Whatman Biochemicals Ltd., Kent,England. BenzphetamineHCl was kindly provided byDr. J. F. Stiver of UpJohn Co., USA. CocaineHCl waspurchased from Tarm ve Koy Isleri Bakanlg, ToprakMahsulleri Ofisi, Ankara-Turkiye. All other chemicalswere of the highest grade available commercially.
Preparation of Beef Liver and SheepLung Microsomes
Livers from well-bled healthy bovine (12 yearsold) and lungs from Akkaraman sheep (612 monthsold) were obtained from a local slaughterhouse(Msrdal Slaughterhouse Sincan, Ankara) immedi-ately after killing. Liver and lung microsomes wereprepared by differential centrifugation as describedpreviously  except that homogenization solutioncontained 2 mM EDTA, 0.25 mM -ACA, and 0.1 mMPMSF in 1.15% KCl. The washed microsomal pelletswere finally suspended in 25% glycerol containing 1mM EDTA. For each gram of liver and lung, 0.5 and0.3 mL suspensions were used, respectively. Micro-somes were gassed with nitrogen gas and stored inplastic bottles at 70C in deep freezer until used forpurification studies.
Purification of Beef Liver MicrosomalNADPHCytochrome P450 Reductase
NADPHcytochrome P450 reductase of beef livermicrosomes was purified to apparent homogeneityfrom the detergent-solubilized microsomes by the com-bination and the refinement of methods previouslyused in this laboratory [12,13,20,24]. The purificationprocess involving two successive DEAE-cellulose col-umn and then 2-5-ADP Sepharose 4B column, and a fi-nal hydroxylapatite column chromatographies was re-peated six times to purify the beef reductase, with thehighest yield and specific content. All purification stepswere carried out at 04C.
Solubilization of Beef Liver Microsomes
Beef liver NADPHcytochrome P450 reductasewas solubilized by the treatment of liver microsomeswith Emulgen 913 and sodium cholate. In a typicalpreparation, the microsomal suspension during thesolubilization contained 20% glycerol, 2 mM EDTA,0.1 mM DTT, 0.25 mM -ACA, 0.5 mM PMSF, 1% (v/v)Emulgen 913, 0.4% (w/v) cholate, and 6 mg/mL pro-tein in 10 mM potassium phosphate buffer (pH 7.85)(at 25C). A stock solution of 10% Emulgen 913 (v/v in20% glycerol) and then 10% cholate (w/v) were addeddropwise with stirring to give a final concentration of1% (v/v) Emulgen 913 and 0.4% (w/v) sodium cholateover 10 min. The mixture was stirred on a magneticstirrer in an ice bath for an additional 40 min and thencentrifuged at 87000 g in type 35 rotor in Beckman ul-tracentrifuge for 120 min. The clear yellow supernatantwas taken. The pellet was discarded.
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First DEAE-Cellulose ColumnChromatography Step
The clear yellow solubilized microsomal fractionwas applied to a DEAE-cellulose column (3.0 34 cm)which was equilibrated with 20 mM potassium phos-phate buffer (pH 7.85) containing 20% glycerol, 2 mMEDTA, 0.1 mM DTT, 0.25 mM PMSF, 0.25 mM -ACA, 0.5% Emulgen 913, and 0.2% cholate (buffer I),at a flow rate of 150 mL/h. Following sample ap-plication, the column was extensively washed with2650 mL (approximately 11 column volumes) bufferI until NADHcytochrome b5 reductase activity wascompletely eluted. During the washing of the column,cytochrome P450s and NADHcytochrome b5 reduc-tase were eluted from the column in given order. Afterthe washing step, a 0.08 M KCl in buffer I (430 mL)was applied to the column in order to separate thecytochrome b5 from NADPHcytochrome P450 reduc-tase. Cytochrome b5 was then eluted from the columnas a sharp peak. The major NADPHcytochrome P450peak containing minor amounts of cytochrome b5 andcytochrome P450 was then obtained by the elution ofthe column with 0.15 M KCl in buffer I (400 mL). Furtherincreasing the KCl concentration of buffer I (340 mL) to0.3 M eluted the remaining cytochrome P450 reductase,cytochrome P450, and cytochrome b5 from the column.
Second DEAE-Cellulose ColumnChromatography Step
The fractions eluted from the first DEAE-cellulosecolumn in buffer I containing 0.15 M KCl with a highamount of NADPHcytochrome P450 reductase activ-ity were pooled and diluted 6.25 times with buffer IIwithout potassium phosphate. Then, the diluted sam-ple was applied to a second DEAE-cellulose column(2.6 6 cm), previously equilibrated with buffer II(30 mM potassium phosphate buffer, pH 7.85, contain-ing 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.25 mMPMSF, 0.25 mM -ACA, and 0.5% Emulgen 913). Af-ter sample application and washing steps, cytochromeb5 and minor amounts of cytochrome P450 reductasewere eluted from the column with 0.08 M KCl in bufferII (130 mL). The major cytochrome P450 reductase peakwas then eluted from the column by increasing theKCl concentration of buffer II (60 mL) to 0.3 M. Atthis step, cytochrome P450 reductase was concentratedabout 5.5 times.
Affinity Chromatography on 2,5-ADPSepharose 4B Column
The pooled cytochrome P450 reductase fractionseluted from the second DEAE-cellulose column with
0.3 M KCl in buffer II was dialyzed overnight againstbuffer III (50 mM potassium phosphate buffer, pH 7.1,containing 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT,0.25 mM PMSF, 0.25 mM -ACA, and 0.1% Emulgen913) and then subjected to affinity chromatographyon 2,5-ADP Sepharose 4B column (0.7 6 cm), previ-ously equilibrated with buffer III. Sample was appliedto the column at a very slow rate of about 2 mL/h.Then the column was washed extensively with at least70 column volumes of 0.2 M potassium phosphate con-taining buffer III (160 mL). Almost all the impurities(cytochrome b5 and cytochrome P450) present in cy-tochrome P450 reductase fractions with minor amountsof cytochrome P450 reductase were eluted from the col-umn during sample application and washing steps. Af-ter that, seven column volumes (15.5 mL) of 20 mMbuffer III (2.5 times diluted buffer III, -ACA concentra-tion was kept constant at 0.25 mM) were passed throughthe same column. Then cytochrome P450 reductase waseluted from the column with approximately 60 mL of20 mM buffer III containing 2.5 mM 2-AMP.
Hydroxylapatite ColumnChromatography Step
The pooled samples obtained from the previouscolumn were chromatographed on hydroxylapatitecolumn (1.5 2 cm), preequilibrated with buffer IV(20 mM potassium phosphate, pH 7.1, containing 20%glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF,0.1 mM -ACA, and 0.25% cholate) in order to re-move the 2-AMP and Emulgen 913 from the sampleand concentrate the cytochrome P450 reductase. Fol-lowing sample application, the column was washedwith buffer IV (90 mL) until the absorbances of the col-umn effluents fell to zero at 280 nm which monitors thepresence of 2-AMP and Emulgen 913 in the fractions.Then, NADPHcytochrome P450 reductase was elutedfrom the column as a sharp peak (6.6 mL) by increasingthe potassium phosphate concentration of buffer IV to0.3 M.
Purification of Sheep Lung MicrosomalNADPHCytochrome P450 Reductase
NADPHcytochrome P450 reductase was purifiedfrom sheep lung microsomes by slight modificationsof the already existing methods developed in our lab-oratory [12,24]. The purification procedure includesanion exchange chromatography of the detergent-solubilized microsomes on two successive DEAE-cellulose columns, affinity chromatography of the par-tially purified reductase on 2,5-ADP Sepharose 4Bcolumn, and further concentration and purification
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of the reductase on a final hydroxylapatite column.During the sample application and washing stepsin first DEAE-cellulose column chromatography, frac-tions having the highest cytochrome P450 content werepooled and used for the subsequent purification of cy-tochrome P4502B.
Purification of Sheep Lung MicrosomalCytochrome P4502B
Sheep lung microsomal cytochrome P4502B waspurified according to the method of Adal and Arinc with slight modifications. The purification pro-cedure involved anion exchange chromatography ofdetergent-solubilized microsomes on an initial DEAE-cellulose column, and further purification of the par-tially purified cytochrome P4502B on first hydrox-ylapatite and second DEAE-cellulose columns. Fi-nally, cytochrome P4502B was further purified andconcentrated on a second hydroxylapatite columnchromatography.
Protein concentration was determined according tothe method of Lowry et al.  using crystalline BSAas standard.
Cytochrome P450 content was measured by the de-tection of a peak around 447452 nm when sampleswere reduced and gassed with CO on a Hitachi 220Adouble beam recording spectrophotometer using an ex-tinct...