Vol. 173, No. 3
JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1035-1040 0021-9193/91/031035-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Induction and Substrate Specificity of the Lanosterol 14OxDemethylase from Saccharomyces cerevisiae Y222 GERARD D. WRIGHT AND JOHN F. HONEK*
Guelph-Waterloo Center for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3GJ Received
5
July 1990/Accepted 26 November 1990
The potential inducibility of the lanosterol 14at-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37°C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[alpyrene.
The lanosterol 14a-demethylase (P-45014DM) in yeasts and fungi is the target for the clinically and agriculturally important imidazole and triazole antifungal agents (5, 43, 55). Inhibition of this membrane-bound cytochrome P-450 monooxygenase (P-450) results in decreased biosynthesis of the major fungal sterol ergosterol and accumulation of C-14methylated sterol precursors. Ergosterol depletion results in altered membrane permeability and decreased activity of membrane enzymes such as chitin synthetase (42), which may contribute to inhibition of fungal growth. P-45014DM from the yeasts Saccharomyces cerevisiae and Candida albicans has been purified (15, 56) and cloned (18, 25). Many mammalian P-450s can be induced by a variety of compounds. In particular, these chemicals induce a specific enzyme, or group of enzymes, which catalyze distinct reactions (47, 53). There are several different classes of compounds which are known to induce mammalian P-450s; these include the phenobarbital class of compounds (12), polyaromatic hydrocarbons (e.g., 3-methylcholanthrene) (28), steroids (e.g., dexamethasone) (35), pituitary hormones (44), and ethanol (37), among others. P-450s in S. cerevisiae have been shown to catalyze a number of reactions, including lanosterol demethylation (2), A22-sterol desaturation (13), hydroxylation of benzo[a]pyrene (52), deethylation of 7-ethoxycoumarin (8), and activation of promutagens such as dimethylnitrosamine (6). Little is known about the induction of P-450 in yeasts. Ishidate and coworkers (17) and others (50) have shown that P-450 could be detected in S. cerevisiae cultures grown only at a low oxygen concentration (semianaerobic growth) and high glucose content (1 to 20%). Wiseman and Lim (49) indicated that S. cerevisiae P-450 was induced by phenobarbital, while in studies by Karenlampi et al. (20), P-450 in various yeast strains was not induced by a number of chemicals, including phenobarbital and 3-methylcholanthrene. King et al. (22) have shown that a benzo[a]pyrene hydroxylase can be induced in a particular strain of S. cerevisiae. Previous studies with this strain demonstrated a negative correlation between cellular cyclic AMP (cAMP) content and P-450 concentration (51), which is unlike the hormone-inducible mammalian P-450s involved in mammalian steroidogenesis in which cAMP is thought to be the second messenger (47). *
We have previously noted a relationship between iron and P-450 content in S. cerevisiae Y222 (54). While it seems probable that the various reactions catalyzed by P-450s are performed by distinct enzymes, only one P-450 locus has yet been identified in S. cerevisiae (18). Because of the small quantities of P-450 in yeast and fungi compared with those in mammalian sources, extensive studies on these enzymes are hampered by the availability of P-450. In this study we have investigated the induction of P-45014DM in S. cerevisiae Y222. It is shown that this enzyme is the only P-450 produced by this organism and that it does not metabolize a number of mammalian P-450 substrates.
MATERIALS AND METHODS Materials. trans-1,4-Bis(2-chlorobenzylaminomethylcyclohexane dihydrochloride) (AY-9944) was a gift from WyethAyerst Research (Princeton, N.J.). Benzo[a]pyrene, 3-methylcholanthrene, dilaurylphosphatidylcholine, Tergitol 15S-12, and lanosterol were from Sigma (St. Louis, Mo.). ,B-Naphthoflavone and N6,0-2'-dibutyryl (db)-cAMP were purchased from Aldrich (Milwaukee, Wis.). Dexamethasone was obtained from Fluka (Ronkonkoma, N.Y.). Phenobarbital was from Fisher (Toronto, Canada). NaB[3H]4 was from Amersham (Oakville, Ontario, Canada). Hydroxylapatite (Bio-Gel HTP) was obtained from Bio-Rad Laboratories (Richmond, Calif.). CytoScint ES was from ICN Biomedicals (Montreal, Canada). Commercial lanosterol was treated with borane-tetrahydrofuran followed by protonolysis with propionic acid to give 24,25-dihydrolanosterol. 4,4-Dimethyl-5a-cholesta-8,14-dien-3p-ol was prepared as previously described (31). Cell growth and protein purification. S. cerevisiae Y222 (Fleishman's yeast collection) was grown in 1- or 3-liter batches as previously described (54). Dexamethasone, dbcAMP, and phenobarbital were dissolved in 1 ml of sterile water and passed through a 45-p,m-pore-size filter prior to being added to culture flasks. Lanosterol, benzo[a]pyrene, 3-methylcholanthrene, and ,B-naphthoflavone were dissolved in 1 ml of sterile dimethyl sulfoxide and added in a similar manner. Late-log-phase cells (approximately 24 h of growth) were harvested by centrifugation at 3,000 x g for 10 min (all centrifugation steps were carried out at 4°C); washed with
Corresponding author. 1035
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distilled water; suspended in a solution containing 50 mM potassium phosphate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, and 500 mM sorbitol (pH 7.5); and disrupted in a Bead Beater (Biospec Products, Bartsville, Okla.) at 0°C for 2.5 min (10-s pulses with 20-s cooling intervals). Cell debris was removed by centrifugation at 3,000 x g for 10 min, and microsomes were collected by high-speed centrifugation at 110,000 x g for 90 min or by the addition of polyethylene glycol (molecular weight, 8,000) to 7.5% (wt/vol), which was followed by centrifugation at 30,000 x g for 25 min. Microsomes were suspended in a solution containing 10 mM potassium phosphate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, 1.0% (wt/vol) sodium cholate, and 20% (vol/vol) glycerol (pH 7.2), stirred for 1 h at 4°C, and then centrifuged at 100,000 x g for 60 min. The soluble enzymes were dialyzed overnight against a solution containing 10 mM potassium phosphate, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0). The dialysate was applied to a hydroxylapatite column (10 by 1.5 cm) equilibrated with a solution containing 10 mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0) and washed with 2 volumes of equilibration buffer. P-45014DM and NADPH-cytochrome P-450 reductase (P-450 reductase) were eluted simultaneously with a solution containing 200 mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0). The eluant was concentrated with a Minicon B15 concentrator unit (Amicon) to give a final P-45014DM concentration of 2 FLM (estimated by reduced-CO difference spectrum). Enzyme assays. (i) Synthesis of 3-oxo-24,25-dihydrolanosterol. Chromium trioxide (1.8 g, 18 mmol) was slowly added to 60 ml of ice-cold anhydrous pyridine over several minutes. A solution of 24,25-dihydrolanosterol (500 mg, 12 mmol) in pyridine (10 ml) was added dropwise to the stirring solution. The mixture was incubated under a nitrogen atmosphere at 0°C for 1 h followed by 8 h at 23°C with constant stirring. The reaction was terminated by the addition of 100 ml of ice water and extracted with ethyl acetate (three times, 100 ml each); the organic fractions were combined and dried over Na2SO4, and the volume was reduced to one-third the original. Silica gel (2 g, 70-230 mesh) was added, and the slurry was evaporated to dryness. The brown powder was applied to a plug of silica in a sintered glass funnel and washed with 250 ml of 2.5% ethyl acetate in hexane. The eluant was evaporated to give the desired ketone as a fine white powder (230 mg, 46%): mp, 106 to 108.5°C (literature values, 119.5 to 120.5°C [36]); Rf (20% ethyl acetate in hexane), 0.73; IR (NaCl), 3,393, 2,950, 2,339, 2,322, 1,708, 1,464, and 1,372 cm- '; 'H-nuclear magnetic resonance (CDCl3, 250 MHz), 8 2.85 to 0.6 ppm (methylene envelope); 13C-nuclear magnetic resonance (CDC13, 250 mHz), 8 216.7 (C-3) and 135.4 and 133.2 (C-8 and C-9, respectively). (ii) Synthesis of [3a_3H]24,25-dihydrolanosterol. 3-Oxo24,25-dihydrolanosterol (5 mg, 0.012 mmol) was dissolved with stirring in 1 ml of anhydrous CH2Cl2 and 0.5 ml of anhydrous methanol. NaB[3H]4 (1.9 mg, 0.05 mmol, 500 mCi/mmol) was added with 0.5 ml of methanol, and the mixture was stirred at 20°C for 60 min. The volume was reduced to 0.25 ml by passing dry N2 over the flask. The residue was applied to a silica gel thin-layer chromatography plate and developed with hexane-ethyl acetate (5:1). The
J. BACTERIOL.
appropriate band was recovered and eluted with CH2Cl2, and the solvent was evaporated. The crude sterol was further purified by reverse-phase fast protein liquid chromatography (FPLC) over PEP-RPC (5 by 50 mm; Pharmacia) by using 90% aqueous methanol as a mobile phase. The fraction corresponding to 24,25-dihydrolanosterol was collected and evaporated to give 3 mg of solid with a specific radioactivity of 16.2 mCi/mmol which behaved as authentic 24,25-dihydrolanosterol by thin-layer and gas chromatography (Pierce SE-54; 25-m capillary column at 250°C, He carrier gas). The sterol was suspended in benzene and kept frozen at -65°C.
(iii) P-45014DM assay. The P-45014DM assay procedure was similar to the method of Trzaskos et al. (41). A 0.5 mM solution of [3a-3H]24,25-dihydrolanosterol (diluted with cold sterol to a specific activity of 3,600 dpm/nmol) and 3.3 mM dilaurylphosphatidylcholine in benzene (50 IlI) was added to a test tube and evaporated under dry N2, and the residue was taken up in 100 RI of 100 mM potassium phosphate, pH 7.0, with sonication for 5 min. Partially purified enzyme solution (100 ,lI, approximately 0.2 nmol of P-450) was added and mixed thoroughly. To this mixture was added KCN (1 ,umol), AY-9944 (0.15 ,umol), glucose-6-phosphate (20 ,umol), glucose-6-phosphate dehydrogenase (1 U), and 100 mM potassium phosphate, pH 7.0, to give a final volume of 1.0 ml. The solution was preincubated for 2 min with shaking at 30°C, and the reaction was initiated with NADPH (2 ,umol). Assays were quenched after 60 min with 3 ml of 15% KOH in methanol (wt/vol), 50 RI of carrier sterols (24,25dihydrolanosterol and 4,4-dimethyl-5oa-cholesta-8,14-dien3,-ol; 0.5 mg/ml) was added, and the mixture was saponified for 30 min and extracted with petroleum ether-ether (9:1, 3 x 5 ml). The combined organic extracts were dried over Na2SO4 and evaporated under a stream of dry N2. The residue was taken up in 100 ,ul of ethanol, a 10-pul aliquot was removed to determine recovery, and 80 pA was analyzed by reverse-phase high-pressure liquid chromatography (HPLC, Waters ,uBondapak C18, 0.45 by 25 cm), with 90% aqueous methanol as the mobile phase, monitored at 248 nm. The 4,4-dimethyl-5a-cholesta-8,14-dien-3,-ol fraction was collected and counted in the presence of CytoScint ES scintillation cocktail. Recovery was typically greater than 80%. (iv) Other enzyme assays. P-450 and P-420 (denatured form of P-450) were determined from the reduced-CO difference spectrum, as previously described (11). P-450 levels reported are a sum of P-450 and P-420 levels. P-450 reductase activity was monitored by the NADPH-cytochrome c reductase assay as previously described (24); 1 U is defined as the amount of enzyme required to reduce 1 pumol of cytochrome c in 1 min. 7-Ethoxycoumarin and 7-ethoxyresorufin were prepared by previously described methods (33). All compounds were suspended in dilaurylphosphatidylcholine micelles and contained an NADPH regenerating system as described above, 2 mM NADPH, and partially purified enzyme (0.2 nmol of P-45014DM). Benzo[a]pyrene hydroxylase (52), 7-ethoxycoumarin deethylase (10), and 7-ethoxyresorufin deethylase (32) activities were monitored by established methods. Aminoantipyrene, p-nitroanisole, and N,Ndimethylaniline demethylase activities were monitored for formaldehyde production by using the Nash reagent (46). Assays with microsomal enzyme preparations (10 mg of protein per assay) used substrates delivered in dimethyl sulfoxide (50 RI per assay).
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P-45014DM INDUCTION AND SUBSTRATE SPECIFICITY
VOL. 173, 1991
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JOURNAL OF BACTERIOLOGY, Feb. 1991, p. 1035-1040 0021-9193/91/031035-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Induction and Substrate Specificity of the Lanosterol 14OxDemethylase from Saccharomyces cerevisiae Y222 GERARD D. WRIGHT AND JOHN F. HONEK*
Guelph-Waterloo Center for Graduate Work in Chemistry, Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3GJ Received
5
July 1990/Accepted 26 November 1990
The potential inducibility of the lanosterol 14at-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37°C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[alpyrene.
The lanosterol 14a-demethylase (P-45014DM) in yeasts and fungi is the target for the clinically and agriculturally important imidazole and triazole antifungal agents (5, 43, 55). Inhibition of this membrane-bound cytochrome P-450 monooxygenase (P-450) results in decreased biosynthesis of the major fungal sterol ergosterol and accumulation of C-14methylated sterol precursors. Ergosterol depletion results in altered membrane permeability and decreased activity of membrane enzymes such as chitin synthetase (42), which may contribute to inhibition of fungal growth. P-45014DM from the yeasts Saccharomyces cerevisiae and Candida albicans has been purified (15, 56) and cloned (18, 25). Many mammalian P-450s can be induced by a variety of compounds. In particular, these chemicals induce a specific enzyme, or group of enzymes, which catalyze distinct reactions (47, 53). There are several different classes of compounds which are known to induce mammalian P-450s; these include the phenobarbital class of compounds (12), polyaromatic hydrocarbons (e.g., 3-methylcholanthrene) (28), steroids (e.g., dexamethasone) (35), pituitary hormones (44), and ethanol (37), among others. P-450s in S. cerevisiae have been shown to catalyze a number of reactions, including lanosterol demethylation (2), A22-sterol desaturation (13), hydroxylation of benzo[a]pyrene (52), deethylation of 7-ethoxycoumarin (8), and activation of promutagens such as dimethylnitrosamine (6). Little is known about the induction of P-450 in yeasts. Ishidate and coworkers (17) and others (50) have shown that P-450 could be detected in S. cerevisiae cultures grown only at a low oxygen concentration (semianaerobic growth) and high glucose content (1 to 20%). Wiseman and Lim (49) indicated that S. cerevisiae P-450 was induced by phenobarbital, while in studies by Karenlampi et al. (20), P-450 in various yeast strains was not induced by a number of chemicals, including phenobarbital and 3-methylcholanthrene. King et al. (22) have shown that a benzo[a]pyrene hydroxylase can be induced in a particular strain of S. cerevisiae. Previous studies with this strain demonstrated a negative correlation between cellular cyclic AMP (cAMP) content and P-450 concentration (51), which is unlike the hormone-inducible mammalian P-450s involved in mammalian steroidogenesis in which cAMP is thought to be the second messenger (47). *
We have previously noted a relationship between iron and P-450 content in S. cerevisiae Y222 (54). While it seems probable that the various reactions catalyzed by P-450s are performed by distinct enzymes, only one P-450 locus has yet been identified in S. cerevisiae (18). Because of the small quantities of P-450 in yeast and fungi compared with those in mammalian sources, extensive studies on these enzymes are hampered by the availability of P-450. In this study we have investigated the induction of P-45014DM in S. cerevisiae Y222. It is shown that this enzyme is the only P-450 produced by this organism and that it does not metabolize a number of mammalian P-450 substrates.
MATERIALS AND METHODS Materials. trans-1,4-Bis(2-chlorobenzylaminomethylcyclohexane dihydrochloride) (AY-9944) was a gift from WyethAyerst Research (Princeton, N.J.). Benzo[a]pyrene, 3-methylcholanthrene, dilaurylphosphatidylcholine, Tergitol 15S-12, and lanosterol were from Sigma (St. Louis, Mo.). ,B-Naphthoflavone and N6,0-2'-dibutyryl (db)-cAMP were purchased from Aldrich (Milwaukee, Wis.). Dexamethasone was obtained from Fluka (Ronkonkoma, N.Y.). Phenobarbital was from Fisher (Toronto, Canada). NaB[3H]4 was from Amersham (Oakville, Ontario, Canada). Hydroxylapatite (Bio-Gel HTP) was obtained from Bio-Rad Laboratories (Richmond, Calif.). CytoScint ES was from ICN Biomedicals (Montreal, Canada). Commercial lanosterol was treated with borane-tetrahydrofuran followed by protonolysis with propionic acid to give 24,25-dihydrolanosterol. 4,4-Dimethyl-5a-cholesta-8,14-dien-3p-ol was prepared as previously described (31). Cell growth and protein purification. S. cerevisiae Y222 (Fleishman's yeast collection) was grown in 1- or 3-liter batches as previously described (54). Dexamethasone, dbcAMP, and phenobarbital were dissolved in 1 ml of sterile water and passed through a 45-p,m-pore-size filter prior to being added to culture flasks. Lanosterol, benzo[a]pyrene, 3-methylcholanthrene, and ,B-naphthoflavone were dissolved in 1 ml of sterile dimethyl sulfoxide and added in a similar manner. Late-log-phase cells (approximately 24 h of growth) were harvested by centrifugation at 3,000 x g for 10 min (all centrifugation steps were carried out at 4°C); washed with
Corresponding author. 1035
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WRIGHT AND HONEK
distilled water; suspended in a solution containing 50 mM potassium phosphate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, and 500 mM sorbitol (pH 7.5); and disrupted in a Bead Beater (Biospec Products, Bartsville, Okla.) at 0°C for 2.5 min (10-s pulses with 20-s cooling intervals). Cell debris was removed by centrifugation at 3,000 x g for 10 min, and microsomes were collected by high-speed centrifugation at 110,000 x g for 90 min or by the addition of polyethylene glycol (molecular weight, 8,000) to 7.5% (wt/vol), which was followed by centrifugation at 30,000 x g for 25 min. Microsomes were suspended in a solution containing 10 mM potassium phosphate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, 1.0% (wt/vol) sodium cholate, and 20% (vol/vol) glycerol (pH 7.2), stirred for 1 h at 4°C, and then centrifuged at 100,000 x g for 60 min. The soluble enzymes were dialyzed overnight against a solution containing 10 mM potassium phosphate, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavin adenine dinucleotide, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0). The dialysate was applied to a hydroxylapatite column (10 by 1.5 cm) equilibrated with a solution containing 10 mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0) and washed with 2 volumes of equilibration buffer. P-45014DM and NADPH-cytochrome P-450 reductase (P-450 reductase) were eluted simultaneously with a solution containing 200 mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol) Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0). The eluant was concentrated with a Minicon B15 concentrator unit (Amicon) to give a final P-45014DM concentration of 2 FLM (estimated by reduced-CO difference spectrum). Enzyme assays. (i) Synthesis of 3-oxo-24,25-dihydrolanosterol. Chromium trioxide (1.8 g, 18 mmol) was slowly added to 60 ml of ice-cold anhydrous pyridine over several minutes. A solution of 24,25-dihydrolanosterol (500 mg, 12 mmol) in pyridine (10 ml) was added dropwise to the stirring solution. The mixture was incubated under a nitrogen atmosphere at 0°C for 1 h followed by 8 h at 23°C with constant stirring. The reaction was terminated by the addition of 100 ml of ice water and extracted with ethyl acetate (three times, 100 ml each); the organic fractions were combined and dried over Na2SO4, and the volume was reduced to one-third the original. Silica gel (2 g, 70-230 mesh) was added, and the slurry was evaporated to dryness. The brown powder was applied to a plug of silica in a sintered glass funnel and washed with 250 ml of 2.5% ethyl acetate in hexane. The eluant was evaporated to give the desired ketone as a fine white powder (230 mg, 46%): mp, 106 to 108.5°C (literature values, 119.5 to 120.5°C [36]); Rf (20% ethyl acetate in hexane), 0.73; IR (NaCl), 3,393, 2,950, 2,339, 2,322, 1,708, 1,464, and 1,372 cm- '; 'H-nuclear magnetic resonance (CDCl3, 250 MHz), 8 2.85 to 0.6 ppm (methylene envelope); 13C-nuclear magnetic resonance (CDC13, 250 mHz), 8 216.7 (C-3) and 135.4 and 133.2 (C-8 and C-9, respectively). (ii) Synthesis of [3a_3H]24,25-dihydrolanosterol. 3-Oxo24,25-dihydrolanosterol (5 mg, 0.012 mmol) was dissolved with stirring in 1 ml of anhydrous CH2Cl2 and 0.5 ml of anhydrous methanol. NaB[3H]4 (1.9 mg, 0.05 mmol, 500 mCi/mmol) was added with 0.5 ml of methanol, and the mixture was stirred at 20°C for 60 min. The volume was reduced to 0.25 ml by passing dry N2 over the flask. The residue was applied to a silica gel thin-layer chromatography plate and developed with hexane-ethyl acetate (5:1). The
J. BACTERIOL.
appropriate band was recovered and eluted with CH2Cl2, and the solvent was evaporated. The crude sterol was further purified by reverse-phase fast protein liquid chromatography (FPLC) over PEP-RPC (5 by 50 mm; Pharmacia) by using 90% aqueous methanol as a mobile phase. The fraction corresponding to 24,25-dihydrolanosterol was collected and evaporated to give 3 mg of solid with a specific radioactivity of 16.2 mCi/mmol which behaved as authentic 24,25-dihydrolanosterol by thin-layer and gas chromatography (Pierce SE-54; 25-m capillary column at 250°C, He carrier gas). The sterol was suspended in benzene and kept frozen at -65°C.
(iii) P-45014DM assay. The P-45014DM assay procedure was similar to the method of Trzaskos et al. (41). A 0.5 mM solution of [3a-3H]24,25-dihydrolanosterol (diluted with cold sterol to a specific activity of 3,600 dpm/nmol) and 3.3 mM dilaurylphosphatidylcholine in benzene (50 IlI) was added to a test tube and evaporated under dry N2, and the residue was taken up in 100 RI of 100 mM potassium phosphate, pH 7.0, with sonication for 5 min. Partially purified enzyme solution (100 ,lI, approximately 0.2 nmol of P-450) was added and mixed thoroughly. To this mixture was added KCN (1 ,umol), AY-9944 (0.15 ,umol), glucose-6-phosphate (20 ,umol), glucose-6-phosphate dehydrogenase (1 U), and 100 mM potassium phosphate, pH 7.0, to give a final volume of 1.0 ml. The solution was preincubated for 2 min with shaking at 30°C, and the reaction was initiated with NADPH (2 ,umol). Assays were quenched after 60 min with 3 ml of 15% KOH in methanol (wt/vol), 50 RI of carrier sterols (24,25dihydrolanosterol and 4,4-dimethyl-5oa-cholesta-8,14-dien3,-ol; 0.5 mg/ml) was added, and the mixture was saponified for 30 min and extracted with petroleum ether-ether (9:1, 3 x 5 ml). The combined organic extracts were dried over Na2SO4 and evaporated under a stream of dry N2. The residue was taken up in 100 ,ul of ethanol, a 10-pul aliquot was removed to determine recovery, and 80 pA was analyzed by reverse-phase high-pressure liquid chromatography (HPLC, Waters ,uBondapak C18, 0.45 by 25 cm), with 90% aqueous methanol as the mobile phase, monitored at 248 nm. The 4,4-dimethyl-5a-cholesta-8,14-dien-3,-ol fraction was collected and counted in the presence of CytoScint ES scintillation cocktail. Recovery was typically greater than 80%. (iv) Other enzyme assays. P-450 and P-420 (denatured form of P-450) were determined from the reduced-CO difference spectrum, as previously described (11). P-450 levels reported are a sum of P-450 and P-420 levels. P-450 reductase activity was monitored by the NADPH-cytochrome c reductase assay as previously described (24); 1 U is defined as the amount of enzyme required to reduce 1 pumol of cytochrome c in 1 min. 7-Ethoxycoumarin and 7-ethoxyresorufin were prepared by previously described methods (33). All compounds were suspended in dilaurylphosphatidylcholine micelles and contained an NADPH regenerating system as described above, 2 mM NADPH, and partially purified enzyme (0.2 nmol of P-45014DM). Benzo[a]pyrene hydroxylase (52), 7-ethoxycoumarin deethylase (10), and 7-ethoxyresorufin deethylase (32) activities were monitored by established methods. Aminoantipyrene, p-nitroanisole, and N,Ndimethylaniline demethylase activities were monitored for formaldehyde production by using the Nash reagent (46). Assays with microsomal enzyme preparations (10 mg of protein per assay) used substrates delivered in dimethyl sulfoxide (50 RI per assay).
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P-45014DM INDUCTION AND SUBSTRATE SPECIFICITY
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