Evidence for Requirement of NADPH-Cytochrome P450 Oxidoreductase in the Microsomal NADPH-Sterol Δ7-Reductase System

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    Rabbpart odoreduabilityIn addmicrostransfeD7-reding deoxidorubilizaity coucolumntainedcarboncific inD7-redevidendistincD7-redKCN mdose-donly ferric ion restored the reductase activity in theEDTA-treated microsomes. These results sugguestthat Niron-delipid bi

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    1 To wBiochem8638, N1hokudai

    0003-9861CopyrightAll rights

    Archives of Biochemistry and BiophysicsVol. 374, No. 2, February 15, pp. 293298, 2000doi:10.1006/abbi.1999.1602, available online at http://www.idealibrary.com onADPH-sterol D7-reductase is membrane-boundpendent protein embedded in the microsomallayer. 2000 Academic PressWords: NADPH-cytochrome P450 oxidoreduc-icrosomal membrane; NADPH-sterol D7-reduc-stem.

    rough endoplasmic reticulum, its activity was detectedin microsomes when the enzyme was assayed in thepresence of cytosolic fractions (8, 9). The microsomalD7-reductase of higher eukaryotes, including both an-imals and higher plants, is strongly inhibited by

    hom correspondence should be addressed at Department of

    2 Abbreviations used: fpT, trypsin-treated form of NADPH-cyto-chrome P450 oxidoreductase; OR, detergent solubilized NADPH-cytochrome P450 oxidoreductase; SCP2, sterol carrier protein 2;AY9944, trans-1,4-bis(2-dichlorobenzylaminomethyl) cyclohexanedihydrochloride; SLO, Smith-Lemli-Opitz. Systematic names of ste-nce for Requirement of NADPH-Cyoreductase in the Microsomal NADeductase System

    ki Nishino and Teruo Ishibashi1

    ent of Biochemistry, Hokkaido University School of Medicin

    August 19, 1999, and in revised form October 31, 1999

    it antibodies raised against the hydrophilicf microsomal NADPH-cytochrome P450 oxi-ctase (denoted fpT) demonstrated a markedto inhibit NADPH-sterol D7-reductase activity.ition, trypsin and proteinase K treatment ofomes removed almost all microsomal electronr constituents from the microsomes, but the

    uctase activity could be reconstituted by add-tergent-solubilized NADPH-cytochrome P450eductase (denoted OR). Furthermore, after sol-tion from microsomes, the D7-reductase activ-ld be reconstituted with OR in a DEAE-cellulose

    chromatography eluate fraction, which con-little OR activity. In the microsomal system,monoxide, ketoconazole, and miconazole, spe-

    hibitors of cytochrome P450, had no effect onuctase activity. These results provide the firstce of an essential requirement of OR, which ist from cytochrome P450, in the NADPH-steroluctase system. EDTA, o-phenanthroline and

    arkedly lowered D7-reductase activity in aependent manner. Among metal ions tested,


    Thoccuis chdineboun1.3.1equitheNADchroelect

    Stoutliveristry, Hokkaido University School of Medicine, Sapporo 060-5 W7, Japan. Fax: 81-11-706-5169. E-mail: t-ishiba@med.


    rols refeterol, 5a3b-ol.

    /00 $35.00 2000 by Academic Pressof reproduction in any form reserved.chrome P450-Sterol

    Sapporo 060-8638, Japan

    he enzymes required for the synthesis of choles-om squalene, the first sterol intermediate, areto the endoplasmic reticulum (1), which is iso-rom cell-free homogenates as microsomes. Thisde it difficult to isolate individual microsomal

    es from cholesterogenic tissues and organs. Theanes of the endoplasmic reticulum contain aroportion of enzymes associated with electronort and oxidative metabolism.reduction step of 7-dehydrocholesterol,2 whichlate in the biosynthetic pathway to cholesterol,acterized by the requirement for reduced pyri-ucleotide, NADPH (24), and a microsomalenzyme referred to as sterol D7-reductase (EC1). However, little is known about how reducinglents are transferred from pyridine nucleotide touctase. Furthermore, it is not clear whether-cytochrome P450 oxidoreductase and cyto-P450 participate in the known microsomal

    n transport system involving NADPH.ies on the sterol D7-reductase have been carriedseveral laboratories using microsomes of rat, 6) and plant (7). Although the mammalianrred to in the text by their abbreviated names are lathos--cholest-7-en-3b-ol; 7-dehydrocholesterol, cholesta-5,7-dien-


  • AY9944 (10). However, there has been no report onstudies of the mode of regulation of the microsomalD7-reductase both in vivo or in vitro.

    As chbranessteroidpectedtissue.synthesequnclesteroducedthe preplasmavelopciencyteristicBecausits suspis of gAvailaductasmakequiredwill hein this

    HereNADPHtant reprocess


    Chemicial sourol), NADtrypsin ifrom BocelluloseG-100 (fiicals wer

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    Tryptiwere digphosphastoppedbuffer, msuspend

    PreparNADPHuntreateand Takgel filtra


    was separated from the whole blood. The DEAE-cellulose fraction ofg-globulin (IgG) from both immune and preimmune serum was pre-pared by the procedure of Masters et al. (20). The titer of the antibodywas checked by Ouchterlony double immunodiffusion (21). The anti-


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    294 NISHINO AND ISHIBASHIolesterol is an integral component of cell mem-and serves as a substrate for the biosynthesis ofs, sex hormones, and bile acids, it is not unex-that abnormalities can affect virtually everyFor example, the importance of cholesterol bio-sis for morphogenesis is illustrated by the con-es of deficiency of 7-dehydrocholesterol to cho-l conversion (11). This results in markedly re-cholesterol concentrations with accumulation ofcursor, 7-dehydrocholesterol and derivatives, inand tissues, especially brain (11). Tissues de-

    abnormalities and function poorly. This defi-is thought to cause the malformations charac-of the Smith-Lemli-Opitz (SLO) syndrome (11).e of its role in drug-induced malformations andected deficiency in SLO syndrome, this enzyme

    reat pharmacological and medical significance.bility of the cDNA (12, 13) for the D7-sterol re-e will allow cloning of the reductase gene andit possible to identify additional proteins re-for the conversion of 7-dehydrocholesterol. Thislp to determine whether inherited abnormalitiesor other genes cause the disorder.we present, for the first time, evidence that-cytochrome P450 oxidoreductase is an impor-

    gulatory enzyme in this complex biosynthetic.


    cals. The following materials were obtained from commer-ces: cholesterol, 7-dehydrocholesterol (cholesta-5,7-dien-3b-H, NADPH, miconazole, trypsin (bovine pancreas), and

    nhibitor (soybean) from Sigma; cytochrome c (horse heart)ehringer; Triton X-100 and Tween 80 from Wako; DEAE-(DE52) from Whatman; 29,59-ADP-Sepharose and Sephadexne) from Pharmacia; ketoconazole from ICN. All other chem-e of reagent grade.ation of microsomes. Male Wistar rats weighing 100200arved for 2 days and then refed a fat-free diet for 1 day priorkilled by decapitation (14). Microsomes (10 mg protein/ml)pared from rat liver as described previously (15). Samplesred at 270C prior to use.n was determined by the method of Lowry et al. (16) witherum albumin as a standard.c digestion of microsomes. Microsomes (1.5 mg of protein)ested with pancreatic trypsin (1 mg) in 0.1 M potassiumte buffer (pH 7.5) at 30C for 15 min (17). The reaction waswith soybean trypsin inhibitor (2 mg). After dilution withicrosomes were isolated by centrifugation, washed, and

    ed in 0.1 M potassium phosphate buffer (pH 7.5).ation of fpT and its antibody. The hydrophilic portion of

    -cytochrome P450 oxidoreductase (fpT) was prepared fromd rat liver microsomes according to the method of Omuraesue (18) with trypsin digestion followed by Sephadex G-100tion and DEAE-cellulose column chromatography.male rabbits were immunized with fpT by the method of

    et al. (19). Blood was collected from ear veins and serum

    fpT a

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    Trsomandthelevebody yields 91% inhibition of OR activity.

    ation of detergent-solubilized NADPH-cytochrome P450 ox-tase (OR). OR was purified from rabbit liver microsomese of 29,59-ADP-Sepharose affinity chromatography (22). Thethe final preparation was confirmed by SDS-slab gel elec-

    sis (23).H-cytochrome P450 oxidoreductase assay. The assay wasut with a Hitachi 220A spectrophotometer (24). In order toe the reductase activity, microsomes were added to 0.1 M

    m phosphate buffer (pH 7.5), which contained 0.02 mmol ofme c and 0.1 mmol of NADPH in a final volume of 1.0 ml.pyridine dinucleotide was added to initiate the reaction.for NADPH-sterol D7-reductase. The incubation mixture

    of 1 mg/ml of microsomal membrane, 3.6 mM NADPH,7-dehydrocholesterol, 0.3% (w/v) Tween 80, and 0.1 M

    m phosphate buffer (pH 7.5) in a total volume of 0.5 ml. Thewas started by adding NADPH under gentle stirring at 37Ceriods of time indicated in an atmosphere of nitrogen.minate the enzyme reaction, 0.5 ml of 20% KOH in 50%lcohol was added to the incubation mixture. Following sa-ion (1 h at room temperature), unsaponifiable materialsracted with 2 ml of hexane three times. The organic upperas pooled and evaporated under nitrogen, and the residueolved in 40 ml of 0.75 % 2-propanol in n-hexane. An aliquotof the solution was subjected to reversed-phase HPLC on an

    column (4.6 3 150 mm) (TSK gel, Tosoh) using a mobilensisting of 0.75% 2-propanol in n-hexane. The flow rate wasin, and the eluate was monitored for 7-dehydrocholesterol

    uring the absorbance at 280 nm (15).cases, the specific activity of the enzyme was calculated perol added as an internal standard after saponification. All

    ere repeated twice each on two duplicate enzyme prepara-gave essentially identical results. Results were the average

    ate samples, and representative data from at least threeexperiments are shown.


    t of anti-fpT on D7-sterol reductase activity.quirement of the NADPH-cytochrome P450 oxi-ctase was measured by means of inhibitionfor sterol D7-reductase activity (Fig. 1). A sig-

    t decrease (6070%) of the enzyme activity wased upon addition of the anti-fpT IgG preparationabbit serum, while preimmune IgG had no in-y effect. These results can be explained in termsnvolvement of OR in the reductase reaction.anti-fpT IgG did not inhibit microsomal NADH-ome c reductase or NADH-ferricyanide reduc-tivity (data not shown). Thus, the inhibitoryof anti-fpT on the electron-transferring reac-

    f microsomes seem to be specific to those whichd the participation of OR.sin treatment of microsomes. When micro-are treated with trypsin, the sterol D7-reductase

    activities were diminished (Fig. 2). However,uctase activity recovered almost to the controlpon addition of purified OR. These results fur-

  • ther sution.

    Resoand ORby solulose coumn (1KCl (0buffer3). Sterfractionaddedwere pOR).

    acontsosedig.ofe uinfeconinhl D


    FIG. 1.sterol D7microsom

    FIG. 2.reductaswith ORactivitiemicrosom


    4.n o

    295MICROSOMAL STEROL D7-REDUCTASE SYSTEMpport an OR requirement of the reductase reac-

    lution and reconstitution of sterol D7-reductase. Sterol D7-reductase fractions were preparedbilization using Triton X-100 and DEAE-cellu-lumn chromatography as before (25). The col-.4 3 10 cm) was eluted with a linear gradient of0.35 M) in 0.4% Triton X-10025 mM Tris-HCl(pH 7.7) and 5-ml fractions were collected (Fig.ol D7-reductase activity was not detected in any, but the activity appeared in the presence of

    OR. Fractions positive for the reductase activityooled (designated Fa; completely separated from

    FrTritimenmicrD7-rin Ftionrangtion


    Effects of antibodies against fpT on microsomal NADPH--reductase activity. The control value was 769 pmol/min/mges. See the text for details.

    Effects of trypsin treatment of microsomes on sterol D7-e activity and reconstitution of trypsin-treated microsomes. The control values of NADPH-sterol D7-reductase and ORs were 769 pmol/min/mg microsomes and 0.17 mmol/min/mg

    es, respectively. See the text for details.


    FIG.tratiotion Fa was dialyzed extensively against 0.4%X-100 buffer and used for reconstitution exper-with OR. That is, OR purified from rabbit liver

    omes was tested for its ability to support steroluctase activity in the presence of Fa. As shown4, the purified OR was effective for reconstitu-the reductase activity. In the concentrationp to 0.5 unit of OR, the rate of D7-sterol reduc-

    creased linearly.t of carbon monoxide and molecular oxygen.monoxide, ketoconazole, and miconazole, spe-ibitors of cytochrome P450, had no effect on the7-reduction (Table I), which implied that cyto-P450 is not involved in the enzyme reaction.

    rmore, molecular oxygen was not required (26).nding may have biogenetic implications.

    DEAE-cellulose column chromatography of sterol D7-re-with or without OR. See the text for details.

    Dependence of sterol D7-reductase activity on the concen-f OR. See the text for details.

  • EffecD7-redagentsand by(Fig. 5)the prethat thded in



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    Effects of O2 and Cytochrome P450 Inhibitorson Sterol D7-Reductase


    a ThemM.

    FIG. 5.D7-reducthroline,reductastivity, retase andmmol/mi


    Effects of Metal Ions on Sterol D7-Reductase







    296 NISHINO AND ISHIBASHIt of chelating agents and metal ions. The steroluctase activity was inhibited by chelating, such as EDTA, Tiron and o-phenanthroline,KCN, though they had no effect on OR activity. The inhibition by EDTA was partly reversed insence of Fe31 (Table II). These results suggeste reductase is an iron-dependent protein embed-the microsomal membrane.


    r microsomes exhibit two principal electronr processes, both of which are needed for thesion of lanosterol to cholesterol. Although the

    -cytochrome P450 transport chain is quantita-more important in liver microsomes and is in-in oxidation of a wide variety of structurally

    ilar compounds, only one step of cholesterol syn-appears to be cytochrome P450-dependent, i.e.,

    the odemthe ois abb5 inpathto ydepe

    ThcelluidentasefromitedlogicpossNADdemspecconanot iobsetase

    Thing Omulsupptheredubranof 5segmenzycytoformbranembthe rmayfluid36).fusio

    Enzyme activity(pmol/min/mg microsomes) (%)

    659 100641 97.3653 99.1

    zolea 666 101nazolea 643 97.6

    concentrations of inhibitors for cytochrome P450 were 10

    Effects of metal chelating agents and cyanide on steroltase activity. (h, n ) EDTA, ({, }) Tiron, (, ) o-phenan-(E, F) KCN. Open and closed symbols show sterol D7-

    e activity and NADPH-cytochrome P450 oxidoreductase ac-spectively. The control values of NADPH-sterol D7-reduc-OR activities were 769 pmol/min/mg microsomes and 0.17

    n/mg microsomes, respectively. See the text for details.




    a Tdation of the C-32 methyl groups. This oxidativeylation is...


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