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Archives of Biochemistry and BiophysicsVol. 374, No. 2, February 15, pp. 293–298, 2000doi:10.1006/abbi.1999.1602, available online at http://www.idealibrary.com on
Evidence for Requirement of NADPH-Cytochrome P450Oxidoreductase in the Microsomal NADPH-SterolD7-Reductase System
Hideaki Nishino and Teruo Ishibashi1
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
Received August 19, 1999, and in revised form October 31, 1999
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Rabbit antibodies raised against the hydrophilicpart of microsomal NADPH-cytochrome P450 oxi-doreductase (denoted fpT) demonstrated a markedability to inhibit NADPH-sterol D7-reductase activity.In addition, trypsin and proteinase K treatment ofmicrosomes removed almost all microsomal electrontransfer constituents from the microsomes, but theD7-reductase activity could be reconstituted by add-ing detergent-solubilized NADPH-cytochrome P450oxidoreductase (denoted OR). Furthermore, after sol-ubilization from microsomes, the D7-reductase activ-ity could be reconstituted with OR in a DEAE-cellulosecolumn chromatography eluate fraction, which con-tained little OR activity. In the microsomal system,carbon monoxide, ketoconazole, and miconazole, spe-cific inhibitors of cytochrome P450, had no effect onD7-reductase activity. These results provide the firstevidence of an essential requirement of OR, which isdistinct from cytochrome P450, in the NADPH-sterolD7-reductase system. EDTA, o-phenanthroline andKCN markedly lowered D7-reductase activity in adose-dependent manner. Among metal ions tested,only ferric ion restored the reductase activity in theEDTA-treated microsomes. These results sugguestthat NADPH-sterol D7-reductase is membrane-boundiron-dependent protein embedded in the microsomallipid bilayer. © 2000 Academic Press
Key Words: NADPH-cytochrome P450 oxidoreduc-tase; microsomal membrane; NADPH-sterol D7-reduc-tase system.
1 To whom correspondence should be addressed at Department ofBiochemistry, Hokkaido University School of Medicine, Sapporo 060-
8638, N15 W7, Japan. Fax: 81-11-706-5169. E-mail: [email protected].t3
0003-9861/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.
All the enzymes required for the synthesis of choles-terol from squalene, the first sterol intermediate, arebound to the endoplasmic reticulum (1), which is iso-lated from cell-free homogenates as microsomes. Thishas made it difficult to isolate individual microsomalenzymes from cholesterogenic tissues and organs. Themembranes of the endoplasmic reticulum contain ahigh proportion of enzymes associated with electrontransport and oxidative metabolism.
The reduction step of 7-dehydrocholesterol,2 whichccurs late in the biosynthetic pathway to cholesterol,s characterized by the requirement for reduced pyri-ine nucleotide, NADPH (2–4), and a microsomalound enzyme referred to as sterol D7-reductase (EC
1.3.1.21). However, little is known about how reducingequivalents are transferred from pyridine nucleotide tothe reductase. Furthermore, it is not clear whetherNADPH-cytochrome P450 oxidoreductase and cyto-chrome P450 participate in the known microsomalelectron transport system involving NADPH.
Studies on the sterol D7-reductase have been carriedout by several laboratories using microsomes of ratliver (5, 6) and plant (7). Although the mammalianD7-reductase activity is localized in both smooth andrough 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
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) cyclohexane
ihydrochloride; SLO, Smith-Lemli-Opitz. Systematic names of ste-ols referred to in the text by their abbreviated names are lathos-
erol, 5a-cholest-7-en-3b-ol; 7-dehydrocholesterol, cholesta-5,7-dien-b-ol.293
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294 NISHINO AND ISHIBASHI
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 cholesterol is an integral component of cell mem-branes and serves as a substrate for the biosynthesis ofsteroids, sex hormones, and bile acids, it is not unex-pected that abnormalities can affect virtually everytissue. For example, the importance of cholesterol bio-synthesis for morphogenesis is illustrated by the con-sequnces of deficiency of 7-dehydrocholesterol to cho-lesterol conversion (11). This results in markedly re-duced cholesterol concentrations with accumulation ofthe precursor, 7-dehydrocholesterol and derivatives, inplasma and tissues, especially brain (11). Tissues de-velop abnormalities and function poorly. This defi-ciency is thought to cause the malformations charac-teristic of the Smith-Lemli-Opitz (SLO) syndrome (11).Because of its role in drug-induced malformations andits suspected deficiency in SLO syndrome, this enzymeis of great pharmacological and medical significance.Availability of the cDNA (12, 13) for the D7-sterol re-ductase will allow cloning of the reductase gene andmake it possible to identify additional proteins re-quired for the conversion of 7-dehydrocholesterol. Thiswill help to determine whether inherited abnormalitiesin this or other genes cause the disorder.
Here we present, for the first time, evidence thatNADPH-cytochrome P450 oxidoreductase is an impor-tant regulatory enzyme in this complex biosyntheticprocess.
EXPERIMENTAL PROCEDURES
Chemicals. The following materials were obtained from commer-ial sources: cholesterol, 7-dehydrocholesterol (cholesta-5,7-dien-3b-l), NADH, NADPH, miconazole, trypsin (bovine pancreas), andrypsin inhibitor (soybean) from Sigma; cytochrome c (horse heart)rom Boehringer; Triton X-100 and Tween 80 from Wako; DEAE-ellulose (DE52) from Whatman; 29,59-ADP-Sepharose and Sephadex-100 (fine) from Pharmacia; ketoconazole from ICN. All other chem-
cals were of reagent grade.Preparation of microsomes. Male Wistar rats weighing 100–200were starved for 2 days and then refed a fat-free diet for 1 day prior
o being killed by decapitation (14). Microsomes (10 mg protein/ml)ere prepared from rat liver as described previously (15). Samplesere stored at 270°C prior to use.Protein was determined by the method of Lowry et al. (16) with
ovine serum albumin as a standard.Tryptic digestion of microsomes. Microsomes (1.5 mg of protein)ere digested with pancreatic trypsin (1 mg) in 0.1 M potassiumhosphate buffer (pH 7.5) at 30°C for 15 min (17). The reaction wastopped with soybean trypsin inhibitor (2 mg). After dilution withuffer, microsomes were isolated by centrifugation, washed, anduspended in 0.1 M potassium phosphate buffer (pH 7.5).Preparation of fpT and its antibody. The hydrophilic portion ofADPH-cytochrome P450 oxidoreductase (fpT) was prepared fromntreated rat liver microsomes according to the method of Omurand Takesue (18) with trypsin digestion followed by Sephadex G-100el filtration and DEAE-cellulose column chromatography.
Adult male rabbits were immunized with fpT by the method ofederson et al. (19). Blood was collected from ear veins and serum
as 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-fpT antibody yields 91% inhibition of OR activity.
Preparation of detergent-solubilized NADPH-cytochrome P450 ox-idoreductase (OR). OR was purified from rabbit liver microsomesby the use of 29,59-ADP-Sepharose affinity chromatography (22). Thepurity of the final preparation was confirmed by SDS-slab gel elec-trophoresis (23).
NADPH-cytochrome P450 oxidoreductase assay. The assay wascarried out with a Hitachi 220A spectrophotometer (24). In order todetermine the reductase activity, microsomes were added to 0.1 Mpotassium phosphate buffer (pH 7.5), which contained 0.02 mmol ofcytochrome c and 0.1 mmol of NADPH in a final volume of 1.0 ml.
educed pyridine dinucleotide was added to initiate the reaction.Assay for NADPH-sterol D7-reductase. The incubation mixture
onsisted of 1 mg/ml of microsomal membrane, 3.6 mM NADPH,–50 mM 7-dehydrocholesterol, 0.3% (w/v) Tween 80, and 0.1 M
potassium phosphate buffer (pH 7.5) in a total volume of 0.5 ml. Thereaction was started by adding NADPH under gentle stirring at 37°Cfor the periods of time indicated in an atmosphere of nitrogen.
To terminate the enzyme reaction, 0.5 ml of 20% KOH in 50%methyl alcohol was added to the incubation mixture. Following sa-ponification (1 h at room temperature), unsaponifiable materialswere extracted with 2 ml of hexane three times. The organic upperphase was pooled and evaporated under nitrogen, and the residuewas dissolved in 40 ml of 0.75 % 2-propanol in n-hexane. An aliquotof 20 ml of the solution was subjected to reversed-phase HPLC on anODS-120 column (4.6 3 150 mm) (TSK gel, Tosoh) using a mobilephase consisting of 0.75% 2-propanol in n-hexane. The flow rate was0.4 ml/min, and the eluate was monitored for 7-dehydrocholesterolby measuring the absorbance at 280 nm (15).
In all cases, the specific activity of the enzyme was calculated perergosterol added as an internal standard after saponification. Allstudies were repeated twice each on two duplicate enzyme prepara-tions and gave essentially identical results. Results were the averageof duplicate samples, and representative data from at least threeseparate experiments are shown.
RESULTS
Effect of anti-fpT on D7-sterol reductase activity.The requirement of the NADPH-cytochrome P450 oxi-doreductase was measured by means of inhibitionstudies for sterol D7-reductase activity (Fig. 1). A sig-nificant decrease (60–70%) of the enzyme activity wasobserved upon addition of the anti-fpT IgG preparationfrom rabbit serum, while preimmune IgG had no in-hibitory effect. These results can be explained in termsof the involvement of OR in the reductase reaction.
The anti-fpT IgG did not inhibit microsomal NADH-cytochrome c reductase or NADH-ferricyanide reduc-tase activity (data not shown). Thus, the inhibitoryeffects of anti-fpT on the electron-transferring reac-tions of microsomes seem to be specific to those whichrequired the participation of OR.
Trypsin treatment of microsomes. When micro-somes are treated with trypsin, the sterol D7-reductaseand OR activities were diminished (Fig. 2). However,the reductase activity recovered almost to the control
level upon addition of purified OR. These results fur-
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295MICROSOMAL STEROL D7-REDUCTASE SYSTEM
ther support an OR requirement of the reductase reac-tion.
Resolution and reconstitution of sterol D7-reductaseand OR. Sterol D7-reductase fractions were preparedby solubilization using Triton X-100 and DEAE-cellu-lose column chromatography as before (25). The col-umn (1.4 3 10 cm) was eluted with a linear gradient ofKCl (0–0.35 M) in 0.4% Triton X-100–25 mM Tris-HClbuffer (pH 7.7) and 5-ml fractions were collected (Fig.3). Sterol D7-reductase activity was not detected in anyfraction, but the activity appeared in the presence ofadded OR. Fractions positive for the reductase activitywere pooled (designated Fa; completely separated fromOR).
FIG. 1. Effects of antibodies against fpT on microsomal NADPH-sterol D7-reductase activity. The control value was 769 pmol/min/mgmicrosomes. See the text for details.
FIG. 2. Effects of trypsin treatment of microsomes on sterol D7-reductase activity and reconstitution of trypsin-treated microsomeswith OR. The control values of NADPH-sterol D7-reductase and OR
activities were 769 pmol/min/mg microsomes and 0.17 mmol/min/mgicrosomes, respectively. See the text for details.
Fraction Fa was dialyzed extensively against 0.4%Triton X-100 buffer and used for reconstitution exper-iments with OR. That is, OR purified from rabbit livermicrosomes was tested for its ability to support sterolD7-reductase activity in the presence of Fa. As shownin Fig. 4, the purified OR was effective for reconstitu-tion of the reductase activity. In the concentrationrange up to 0.5 unit of OR, the rate of D7-sterol reduc-tion increased linearly.
Effect of carbon monoxide and molecular oxygen.Carbon monoxide, ketoconazole, and miconazole, spe-cific inhibitors of cytochrome P450, had no effect on thesterol D7-reduction (Table I), which implied that cyto-chrome P450 is not involved in the enzyme reaction.Furthermore, molecular oxygen was not required (26).This finding may have biogenetic implications.
FIG. 3. DEAE-cellulose column chromatography of sterol D7-re-ductase with or without OR. See the text for details.
FIG. 4. Dependence of sterol D7-reductase activity on the concen-tration of OR. See the text for details.
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296 NISHINO AND ISHIBASHI
Effect of chelating agents and metal ions. The sterolD7-reductase activity was inhibited by chelatingagents, such as EDTA, Tiron and o-phenanthroline,and by KCN, though they had no effect on OR activity(Fig. 5). The inhibition by EDTA was partly reversed inthe presence of Fe31 (Table II). These results suggestthat the reductase is an iron-dependent protein embed-ded in the microsomal membrane.
DISCUSSION
Liver microsomes exhibit two principal electrontransfer processes, both of which are needed for theconversion of lanosterol to cholesterol. Although theNADPH-cytochrome P450 transport chain is quantita-tively more important in liver microsomes and is in-volved in oxidation of a wide variety of structurallydissimilar compounds, only one step of cholesterol syn-thesis appears to be cytochrome P450-dependent, i.e.,
TABLE I
Effects of O2 and Cytochrome P450 Inhibitorson Sterol D7-Reductase
Enzyme activity(pmol/min/mg microsomes) (%)
ontrol 659 100O2 641 97.3
1CO 653 99.11Miconazolea 666 1011Ketoconazolea 643 97.6
a The concentrations of inhibitors for cytochrome P450 were 10mM.
FIG. 5. Effects of metal chelating agents and cyanide on sterol7-reductase activity. (h, ■) EDTA, ({, }) Tiron, (‚, Œ) o-phenan-
throline, (E, F) KCN. Open and closed symbols show sterol D7-reductase activity and NADPH-cytochrome P450 oxidoreductase ac-tivity, respectively. The control values of NADPH-sterol D7-reduc-
tase and OR activities were 769 pmol/min/mg microsomes and 0.17mmol/min/mg microsomes, respectively. See the text for details.the oxidation of the C-32 methyl groups. This oxidativedemethylation is sensitive to inhibition by CO (27). Onthe other hand, an attack of the C-4 methyl group (28)is absolutely dependent upon microsomal cytochromeb5 in yeast (29) and liver (30). The final oxidation in thepathway introducing the 5-double bond into lathosterolto yield 7-dehydrocholesterol is also cytochrome b5-dependent (25, 31).
The data in Fig. 3 indicate that Fb, one of two DEAE-cellulose fractions required for sterol D7-reductase, isidentical with NADPH-cytochrome P450 oxidoreduc-tase. Furthermore, antibodies against the purified ORfrom trypsin-digested microsomes significantly inhib-ited sterol D7-reductase activity (Fig. 1). The physio-logical role of OR is not exactly clear (32), but it ispossible that one role is to transfer electrons fromNADPH to terminal reductase. Although we could notdemonstrate the complete absence of cytochrome P450species in Fa, carbon monoxide, ketoconazole, and mi-conazole, specific inhibitors of cytochrome P450, didnot inhibit sterol D7-reductase activity (Table I). Theseobservations strongly suggest that the sterol D7-reduc-tase system is distinct from cytochrome P450 species.
The microsomal sterol D7-reductase system includ-ing OR and terminal reductase may be organized intomultienzyme systems in the membrane. This idea issupported by the strikingly high overall efficiency ofthe process. Molecular cloning of the cDNA of the D7-reductase revealed that the human enzyme is a mem-brane-bound protein with a predicted molecular massof 55 kDa and six to nine putative transmembranesegments (12). Alternatively, clustering of the complexenzyme system is possible, as has been proposed forcytochrome b5 and its reductase (33). However, no in-formation is available about clustering of these mem-brane components. The components of the system areembedded in the microsomal membrane and, therefore,the reaction rate of this putative multienzyme complexmay be significantly affected by changes in membranefluidity caused by changes in lipid composition (34–36). Increased fluidity can result in higher lateral dif-
TABLE II
Effects of Metal Ions on Sterol D7-Reductase
Enzyme activity(pmol/min/mg microsomes) (%)
Untreated Ms 878 100EDTA-washed Ms 136 15.41Fe31a 582 66.31Cu21 127 14.51Mn21 82 9.31Mg21 13 1.4
a The concentrations of metal ions were 1 mM.
fusion of 7-dehydrocholesterol, thus increasing the
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297MICROSOMAL STEROL D7-REDUCTASE SYSTEM
chance of its presentation to, and interaction with,membrane-bound D7-reductase. However, the correla-tion between membrane fluidity and the diffusion con-stant remains to be established. In our present system,several possibilities exist for the mode of interactionbetween the sterol D7-reductase and the NADPH-de-pendent electron transfer system in the microsomalmembrane (37–39). The terminal reductase moleculecould interact with each electron transfer componentby translational motion and collision within the micro-somal membrane (15). Studies on similar enzymes,e.g., acyl-CoA; acyltransferase (40), and squalene ep-oxidase (41), indicate that sterol processing enzymesare located on the cytoplasmic surface, but are buriedin the membrane. Because the enzymes are bound tothe same membrane, regulation of the various stepsmay be coordinated through common mechanisms.Therefore, the activities of all the membrane-boundenzymes may be regulated in concert (42). As the or-ganization of the enzymes in the membrane is betterunderstood, both the common mechanisms and thepossible synchrony of activity regulation should be-come apparent.
Scallen and his associates have demonstrated thatthe reduction of 7-dehydrocholesterol to cholesterol re-quires liver cytosol containing sterol carrier protein 2(SCP2) in addition to the microsomal enzyme (8, 9).Preliminary enzymatic studies by Shefer et al. demon-strated a marked deficiency of sterol D7-reductase inpatients with SLO syndrome (43). However, very littleis known about the structure or regulation of sterolD7-reductase and the role of associated proteins, suchas SCP2 or OR (44). Several laboratories have lookedor a deficiency of SCP2 by immunological means, but
all have found normal antigen levels (44).A key test of functionality of enzymes isolated from
membranes is to reconstitute the purified enzymes inartificial membranes (45). Such studies with thepresent microsomal biosynthetic enzymes are planned.Investigations of membrane enzymology should yield abetter understanding of membrane structure and ofthe turnover, function, and regulatory mechanisms ofmembrane components.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific Research(No. 09780558) and a Special Grant-in-Aid for Promotion of Educa-tion and Science in Hokkaido University provided by the Ministry ofEducation, Science, Sports, and Culture of Japan.
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