Vol. 173, No. 3

Induction and Substrate Specificity of the Lanosterol 14Ox-Demethylase 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 cerevisiaeY222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffectedby 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°Cresulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a numberof P-450 substrates including benzo[alpyrene.

The lanosterol 14a-demethylase (P-45014DM) in yeasts andfungi is the target for the clinically and agriculturally impor-tant imidazole and triazole antifungal agents (5, 43, 55).Inhibition of this membrane-bound cytochrome P-450 mono-oxygenase (P-450) results in decreased biosynthesis of themajor fungal sterol ergosterol and accumulation of C-14-methylated sterol precursors. Ergosterol depletion results inaltered membrane permeability and decreased activity ofmembrane enzymes such as chitin synthetase (42), whichmay contribute to inhibition of fungal growth. P-45014DMfrom the yeasts Saccharomyces cerevisiae and Candidaalbicans 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 specificenzyme, or group of enzymes, which catalyze distinct reac-

tions (47, 53). There are several different classes of com-

pounds which are known to induce mammalian P-450s; theseinclude the phenobarbital class of compounds (12), polyaro-matic hydrocarbons (e.g., 3-methylcholanthrene) (28), ste-roids (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]py-rene (52), deethylation of 7-ethoxycoumarin (8), and activa-tion of promutagens such as dimethylnitrosamine (6). Littleis known about the induction of P-450 in yeasts. Ishidate andcoworkers (17) and others (50) have shown that P-450 couldbe detected in S. cerevisiae cultures grown only at a lowoxygen concentration (semianaerobic growth) and high glu-cose content (1 to 20%). Wiseman and Lim (49) indicatedthat S. cerevisiae P-450 was induced by phenobarbital, whilein studies by Karenlampi et al. (20), P-450 in various yeaststrains was not induced by a number of chemicals, includingphenobarbital and 3-methylcholanthrene. King et al. (22)have shown that a benzo[a]pyrene hydroxylase can beinduced in a particular strain of S. cerevisiae. Previousstudies with this strain demonstrated a negative correlationbetween cellular cyclic AMP (cAMP) content and P-450concentration (51), which is unlike the hormone-induciblemammalian P-450s involved in mammalian steroidogenesisin which cAMP is thought to be the second messenger (47).

* Corresponding author.

We have previously noted a relationship between iron andP-450 content in S. cerevisiae Y222 (54).While it seems probable that the various reactions cata-

lyzed by P-450s are performed by distinct enzymes, only oneP-450 locus has yet been identified in S. cerevisiae (18).Because of the small quantities of P-450 in yeast and fungicompared with those in mammalian sources, extensive stud-ies on these enzymes are hampered by the availability ofP-450. In this study we have investigated the induction ofP-45014DM in S. cerevisiae Y222. It is shown that thisenzyme is the only P-450 produced by this organism and thatit does not metabolize a number of mammalian P-450 sub-strates.

MATERIALS AND METHODS

Materials. trans-1,4-Bis(2-chlorobenzylaminomethylcyc-lohexane dihydrochloride) (AY-9944) was a gift from Wyeth-Ayerst Research (Princeton, N.J.). Benzo[a]pyrene, 3-me-thylcholanthrene, dilaurylphosphatidylcholine, Tergitol 15-S-12, and lanosterol were from Sigma (St. Louis, Mo.).,B-Naphthoflavone and N6,0-2'-dibutyryl (db)-cAMP werepurchased from Aldrich (Milwaukee, Wis.). Dexamethasonewas obtained from Fluka (Ronkonkoma, N.Y.). Phenobar-bital was from Fisher (Toronto, Canada). NaB[3H]4 was

from Amersham (Oakville, Ontario, Canada). Hydroxylapa-tite (Bio-Gel HTP) was obtained from Bio-Rad Laboratories(Richmond, Calif.). CytoScint ES was from ICN Biomedi-cals (Montreal, Canada). Commercial lanosterol was treatedwith borane-tetrahydrofuran followed by protonolysis withpropionic acid to give 24,25-dihydrolanosterol. 4,4-Dimeth-yl-5a-cholesta-8,14-dien-3p-ol was prepared as previouslydescribed (31).

Cell growth and protein purification. S. cerevisiae Y222(Fleishman's yeast collection) was grown in 1- or 3-literbatches as previously described (54). Dexamethasone, db-cAMP, and phenobarbital were dissolved in 1 ml of sterilewater and passed through a 45-p,m-pore-size filter prior tobeing added to culture flasks. Lanosterol, benzo[a]pyrene,3-methylcholanthrene, and ,B-naphthoflavone were dissolvedin 1 ml of sterile dimethyl sulfoxide and added in a similarmanner. Late-log-phase cells (approximately 24 h of growth)were harvested by centrifugation at 3,000 x g for 10 min (allcentrifugation steps were carried out at 4°C); washed with

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1036 WRIGHT AND HONEK

distilled water; suspended in a solution containing 50 mMpotassium phosphate, 1 mM EDTA, 1 mM phenylmethylsul-fonyl fluoride, 2 ,uM flavin mononucleotide, 2 ,uM flavinadenine dinucleotide, and 500 mM sorbitol (pH 7.5); anddisrupted in a Bead Beater (Biospec Products, Bartsville,Okla.) at 0°C for 2.5 min (10-s pulses with 20-s coolingintervals). Cell debris was removed by centrifugation at3,000 x g for 10 min, and microsomes were collected byhigh-speed centrifugation at 110,000 x g for 90 min or by theaddition of polyethylene glycol (molecular weight, 8,000) to7.5% (wt/vol), which was followed by centrifugation at30,000 x g for 25 min. Microsomes were suspended in a

solution containing 10 mM potassium phosphate, 1 mMEDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ,uM flavinmononucleotide, 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 for60 min. The soluble enzymes were dialyzed overnightagainst a solution containing 10 mM potassium phosphate, 1mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 2 ,uMflavin 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 hydroxylapatitecolumn (10 by 1.5 cm) equilibrated with a solution containing10 mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol)Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0) andwashed with 2 volumes of equilibration buffer. P-45014DMand NADPH-cytochrome P-450 reductase (P-450 reductase)were eluted simultaneously with a solution containing 200mM potassium phosphate, 1 mM EDTA, 0.2% (vol/vol)Tergitol 15-S-12, and 20% (vol/vol) glycerol (pH 7.0). Theeluant was concentrated with a Minicon B15 concentratorunit (Amicon) to give a final P-45014DM concentration of 2FLM (estimated by reduced-CO difference spectrum).Enzyme assays. (i) Synthesis of 3-oxo-24,25-dihydrolano-

sterol. Chromium trioxide (1.8 g, 18 mmol) was slowly addedto 60 ml of ice-cold anhydrous pyridine over several min-utes. A solution of 24,25-dihydrolanosterol (500 mg, 12mmol) in pyridine (10 ml) was added dropwise to the stirringsolution. The mixture was incubated under a nitrogen atmo-sphere at 0°C for 1 h followed by 8 h at 23°C with constantstirring. The reaction was terminated by the addition of 100ml of ice water and extracted with ethyl acetate (three times,100 ml each); the organic fractions were combined and driedover Na2SO4, and the volume was reduced to one-third theoriginal. Silica gel (2 g, 70-230 mesh) was added, and theslurry was evaporated to dryness. The brown powder was

applied to a plug of silica in a sintered glass funnel andwashed with 250 ml of 2.5% ethyl acetate in hexane. Theeluant was evaporated to give the desired ketone as a finewhite powder (230 mg, 46%): mp, 106 to 108.5°C (literaturevalues, 119.5 to 120.5°C [36]); Rf (20% ethyl acetate inhexane), 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-Oxo-24,25-dihydrolanosterol (5 mg, 0.012 mmol) was dissolvedwith stirring in 1 ml of anhydrous CH2Cl2 and 0.5 ml ofanhydrous methanol. NaB[3H]4 (1.9 mg, 0.05 mmol, 500mCi/mmol) was added with 0.5 ml of methanol, and themixture was stirred at 20°C for 60 min. The volume was

reduced to 0.25 ml by passing dry N2 over the flask. Theresidue was applied to a silica gel thin-layer chromatographyplate and developed with hexane-ethyl acetate (5:1). The

appropriate band was recovered and eluted with CH2Cl2,and the solvent was evaporated. The crude sterol wasfurther purified by reverse-phase fast protein liquid chroma-tography (FPLC) over PEP-RPC (5 by 50 mm; Pharmacia)by using 90% aqueous methanol as a mobile phase. Thefraction corresponding to 24,25-dihydrolanosterol was col-lected and evaporated to give 3 mg of solid with a specificradioactivity of 16.2 mCi/mmol which behaved as authentic24,25-dihydrolanosterol by thin-layer and gas chromatogra-phy (Pierce SE-54; 25-m capillary column at 250°C, Hecarrier gas). The sterol was suspended in benzene and keptfrozen at -65°C.

(iii) P-45014DM assay. The P-45014DM assay procedure wassimilar to the method of Trzaskos et al. (41). A 0.5 mMsolution of [3a-3H]24,25-dihydrolanosterol (diluted with coldsterol to a specific activity of 3,600 dpm/nmol) and 3.3 mMdilaurylphosphatidylcholine in benzene (50 IlI) was added toa test tube and evaporated under dry N2, and the residue wastaken 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 andmixed 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 100mM potassium phosphate, pH 7.0, to give a final volume of1.0 ml. The solution was preincubated for 2 min with shakingat 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 (24,25-dihydrolanosterol and 4,4-dimethyl-5oa-cholesta-8,14-dien-3,-ol; 0.5 mg/ml) was added, and the mixture was saponifiedfor 30 min and extracted with petroleum ether-ether (9:1, 3 x5 ml). The combined organic extracts were dried overNa2SO4 and evaporated under a stream of dry N2. Theresidue was taken up in 100 ,ul of ethanol, a 10-pul aliquot wasremoved to determine recovery, and 80 pA was analyzed byreverse-phase high-pressure liquid chromatography (HPLC,Waters ,uBondapak C18, 0.45 by 25 cm), with 90% aqueousmethanol as the mobile phase, monitored at 248 nm. The4,4-dimethyl-5a-cholesta-8,14-dien-3,-ol fraction was col-lected and counted in the presence of CytoScint ES scintil-lation cocktail. Recovery was typically greater than 80%.

(iv) Other enzyme assays. P-450 and P-420 (denatured formof P-450) were determined from the reduced-CO differencespectrum, as previously described (11). P-450 levels reportedare a sum of P-450 and P-420 levels. P-450 reductase activitywas monitored by the NADPH-cytochrome c reductaseassay as previously described (24); 1 U is defined as theamount of enzyme required to reduce 1 pumol of cytochromec in 1 min. 7-Ethoxycoumarin and 7-ethoxyresorufin wereprepared by previously described methods (33). All com-pounds were suspended in dilaurylphosphatidylcholine mi-celles and contained an NADPH regenerating system asdescribed above, 2 mM NADPH, and partially purifiedenzyme (0.2 nmol of P-45014DM). Benzo[a]pyrene hydroxy-lase (52), 7-ethoxycoumarin deethylase (10), and 7-ethoxyre-sorufin deethylase (32) activities were monitored by estab-lished methods. Aminoantipyrene, p-nitroanisole, and N,N-dimethylaniline demethylase activities were monitored forformaldehyde production by using the Nash reagent (46).Assays with microsomal enzyme preparations (10 mg ofprotein per assay) used substrates delivered in dimethylsulfoxide (50 RI per assay).

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P-45014DM INDUCTION AND SUBSTRATE SPECIFICITY

0.8

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Volume (ml)FIG. 1. Chromatography of P-45014DM and P-450 reductase over hydroxylapatite. Arrows indicate elution with 200 (A) and 500 (B) mM

phosphate (see Materials and Methods for more details). P450 reductase units (0), A280 (El), and A410 (*) were monitored.

RESULTS

Protein purification and P-45014DM assay. We have par-

tially purified P-45014DM from S. cerevisiae Y222 grown inthe presence of a number of compounds. Passage of crude,solubilized membrane proteins through a hydroxylapatitecolumn allows the enrichment of P-45014DM and P-450 re-

ductase in the same fraction (Fig. 1). All of the P-450 wasobtained in the 200 mM phosphate elution step. We typicallyobtained P-45014DM in 46% yield with 1.6-fold purificationand P-450 reductase in 51% yield with 1.4-fold purification.Assays were conducted in the presence of the nonionicdetergent Tergitol 15-S-12 (0.02%), which did not adverselyaffect the demethylase reaction. Attempts to remove thedetergent with Biobeads SM-2 resulted in a decrease inactivity which we have attributed to the instability of thereductase in the absence of detergent (data not shown).Incubations were conducted in the presence of the A14-sterolreductase inhibitor AY-9944 (1) and KCN, which inhibitssterol C-4 demethylation (30). The addition of these com-

pounds was precautionary only since we have not found adistribution of label into other fractions in the absence ofthese compounds by using partially purified enzyme (deter-mined by HPLC; data not shown).

Induction studies. Table 1 summarizes the P-450 and P-450reductase content of cells grown in the presence of phe-nobarbital, 3-methylcholanthrene, and benzo[a]pyrene at a

concentration (50 ,uM) reported to induce yeast benzo[a]py-rene hydroxylase (22). No significant change in either P-450or P-450 reductase content was found. Furthermore, dexa-methasone, lanosterol, and P-naphthoflavone also do notinduce the yeast enzymes at the concentration tested.Lanosterol was included in this study even though yeastsreadily incorporate exogenous sterols only under anaerobicconditions (39). Nonetheless, lanosterol is a lipophilic com-

pound as well as the P-45014DM substrate and could poten-tially affect the levels of cellular P-450. A modest decrease inboth P-45014DM and P-450 reductase was found in cells

grown at 37°C, while db-cAMP (1 mM) had no effect. Theseresults indicate that P-450 and P-450 reductase levels in S.cerevisiae Y222 are not affected by xenobiotics.

Induction of the S. cerevisiae benzo[a]pyrene hydroxylasehas been demonstrated by a net decrease in the Km forbenzo[a]pyrene rather than an increase in overall P-450levels (22). Therefore, this activity along with the P-45014DMactivity of partially purified enzyme obtained from cellsgrown in the presence of potential inducers was also moni-tored (Table 2). Different batches of control growths (noadded compound) demonstrated considerable variation inP-45014DM activity. The addition of xenobiotics to thegrowth media did not result in significant change in thisactivity (within experimental error). Partially purified en-zyme preparations from each growth as well as the microso-mal fraction from cells grown in the presence of benzo[a]py-rene were monitored for benzo[a]pyrene hydroxylase

TABLE 1. P-450 and P-450 reductase content of S. cerevisiaeY222 grown in the presence of various compounds

P450 specific P-450 reductase spCompounda content act (U/mg of

(pmol/mg of protein) protein)

Control300C 27.4 ± 12.3 0.032 ± 0.016370C 9.0 ± 0.32 0.017 + 0.008

db-cAMP 17.9 ± 5.5 0.024 ± 0.001Dexamethasone 20.9 ± 11.7 0.035 ± 0.007Phenobarbital 18.7 ± 9.3 0.062 ± 0.033Dimethyl sulfoxide 19.7 ± 4.6 0.030 ± 0.008,-Naphthoflavone 11.1 ± 5.5 0.033 ± 0.0073-Methylcholanthrene 21.1 ± 14.3 0.058 ± 0.021Benzo[a]pyrene 23.9 ± 11.9 0.032 + 0.003Lanosterol 22.5 ± 8.7 0.017 ± 0.008

a All compounds were at 50 ,uM, except db-cAMP, which was at 1 mM; n

= 2 for all compounds.

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1038 WRIGHT AND HONEK

TABLE 2. P-45014DM activity of partially purified protein from S.cerevisiae Y222 grown in the presence of various compounds

P-45014DM activityCompounda (pmol/min/mg

of protein)

ControlExpt 1....................................... 1.58 ± 0.05Expt2....................................... 3.15 0.29Expt 3....................................... 6.56 ± 0.3

Dexamethasone ...................................... 7.55 ± 0.20Phenobarbital ...................................... 1.90 ± 0.01Dimethyl sulfoxide ....................................... 4.21 ± 0.743-Naphthoflavone ....................................... 5.05 ± 0.77

3-Methylcholanthrene ...................................... 7.56 ± 0.56Benzo[a]pyrene ...................................... 7.08 ± 2.35Lanosterol ...................................... 1.31 ± 0.40

a For all compounds, n = 2.

activity, with negative results in all cases (data not shown).The detection limit for the benzo[a]pyrene hydroxylaseactivity has been estimated to be 1 pmol/ml (27).

Substrate specificity. The ability of partially purifiedP-45014DM to catalyze typical mammalian P-450 reactionswas investigated. The partially purified enzyme obtainedfrom growths in the presence and absence of benzo[a]pyreneshowed no activity towards a number of potential substratesincluding 7-ethoxyresorufin, 7-ethoxycoumarin (detectionlimit, >10 pmollml [10, 32]), aminoantipyrene, N,N-dimeth-ylaniline, p-nitroanisole (detection limit, 0.5 ,umol/ml) andbenzo[a]pyrene. Identical results were obtained with mi-crosomal preparations.

DISCUSSION

P-45014DM is an important yeast enzyme and a target forantifungal agents. Extensive biochemical studies on thisenzyme are hampered by the low concentrations ofP-45014DM found in most yeasts (21). The presence ofalkane-inducible P-450s in some yeasts involved in alkanehydroxylation (38) and the knowledge that P-450s are ofteninduced to high levels in mammals by a number of com-

pounds (47) led us to examine the potential inducibility of theP-45014DM from S. cerevisiae. These studies required a

sensitive assay for P-45014DM* Yeast P-45014DM activity hasbeen previously monitored by gas chromatography (2) and,more recently, by 14C-labeled lanosterol obtained by incu-bation of a cell-free system with [2-14C]mevalonic acid (14).Confronted with difficulties in establishing a sensitive gaschromatography assay, we have developed a procedurebased on the determination of 3H-labeled 4,4-dimethyl-5c-cholesta-8,14-dien-3p-ol, the product of the demethylationreaction, by HPLC. A similar approach has been used tostudy the P-45014DM from rat liver by using 24,25-dihydro-lanosterol tritiated by catalytic reduction of the 24-25 doublebond in the presence of 3H2 (41). In our procedure, thelabeled substrate was easily prepared by chemical reductionfrom the known 3-oxo-24,25-dihydrolanosterol showing highspecific activity.Lindenmayer and Smith first reported that S. cerevisiae

produced P-450-like pigments when grown under anaerobicconditions (26). This phenomenon was later confirmed byIshidate et al., who found P-450 in yeast grown underanaerobic and semianaerobic conditions (17). Further stud-ies by Wiseman et al. (50) and Wiseman and Lim (49)demonstrated that P-450 was found only in S. cerevisiae

grown under high glucose concentrations (1 to 20%) or in thepresence of phenobarbital. Karenlampi et al. could notdetect the induction of P-450 by phenobarbital with the sameyeast strain grown under similar conditions (20). This samestudy examined the effects of a number of chemicals onP-450 production in S. cerevisiae and other yeasts; noevidence of induction was noted with most chemicals, in-cluding 3-methylcholanthrene, a potent inducer of mamma-lian P-450. Nevertheless, a modest increase in P-450 levelswas obtained with two compounds, lindane (hexachlorohex-ane) and Clophen A60 (a commercial mixture of polychlori-nated biphenyls), which increased P-450 levels 50 and 32%,respectively.

P-450s in S. cerevisiae have been shown to catalyze anumber of reactions, including lanosterol demethylation (2)and hydroxylation of benzo[a]pyrene (52). King et al. foundthat S. cerevisiae 240 grown in the presence of benzo[a]py-rene, dimethylnitrosamine, phenobarbital, or 3-methylchol-anthrene induced production of a P-450 with benzo[a]pyrenehydroxylase activity (22). In these studies the total cellularP-450 level did not increase over control values; rather, theinduction of the hydroxylase was indicated by a decrease inthe Km for benzo[a]pyrene. In our study, S. cerevisiae Y222grown in the presence of benzo[a]pyrene, phenobarbital, or3-methylcholanthrene, at levels which induce benzo[a]py-rene in S. cerevisiae 240, did not produce a benzo[a]pyrenehydroxylase, nor were the levels of P-450, P-450 reductase,or P-45014DM affected. Identical results were obtained withcells grown in the presence of dexamethasone and P-naph-thoflavone, which induce different mammalian P-450 en-zymes, and lanosterol. These results demonstrate thatP-45014DM and P-450 reductase are not induced by thesexenobiotics.

Previous studies had attributed activation of promutagens(6), 7-ethoxycoumarin deethylase (8), biphenyl hydroxylase(48), and benzo[a]pyrene hydroxylase (52) activities to S.cerevisiae P-450. Our results show that at least two of thesereactions are not catalyzed by the biosynthetic enzymeP-45014DM and imply that there exist several distinct en-zymes in some yeasts or isozymes of P-45014DM capable ofcatalyzing these reactions. The molecular weight and aminoacid composition for the benzo[a]pyrene hydroxylase havebeen determined (23) and are clearly distinct from themolecular weight of P-45014DM (56; our unpublished results)and the amino acid composition determined from the cDNAsequence for P-45014DM (19). This supports the presence ofmultiple P-450s in yeasts; however, it is not known if thebenzo[a]pyrene hydroxylase will metabolize lanosterol.Negative results were obtained with purified benzo[a]pyrenehydroxylase (23). However, lanosterol demethylation wasmonitored by the Nash reagent, which detects formaldehydebut not the demethylation product formic acid.

Previous work has indicated that yeast P-45014DM willmetabolize only a limited number of lanosterol analogs (3, 4).Our work extends these results to incorporate benzo[a]py-rene, 7-ethoxycoumarin, and 7-ethoxyresorufin among thecompounds which are not recognized by P-45014DM. This isin agreement with recent studies on the P-45014DM from C.albicans (15). Furthermore, aminoantipyrene, which is me-tabolized by purified rat liver P-45014DM (40), is not asubstrate for the yeast enzyme, nor is p-nitroanisole orN,N-dimethylaniline.

Levels of cAMP decrease in yeasts grown at high glucoseconcentrations (34). Wiseman and Woods (51) showed thatthe presence of cAMP repressed the de novo production ofP-450 in protoplasts of S. cerevisiae 240 (later shown to have

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P-45014DM INDUCTION AND SUBSTRATE SPECIFICITY 1039

benzo[a]pyrene hydroxylase activity [22]). In contrast,cAMP is the second messenger of mammalian P-450 en-zymes which are induced by pituitary hormones, and addi-tion of cAMP or cAMP analogs to cells often stimulatesP-450 production (57). In our study, S. cerevisiae Y222grown in the presence of high levels (1 mM) of db-cAMP, alipid-soluble cAMP analog which is known to penetrateyeast cells (29), showed no change in P-450 or P-450 reduc-tase, indicating that these enzymes may not be sensitive toregulation by cAMP.Temperature fluctuations affect a number of cellular fac-

tors, including transport, protein synthesis, and membranefluidity (45). Many organisms maintain membrane fluidity atvarious temperatures by altering the composition of cellmembranes, a process known as homeoviscous adaptation(7). The mesophilic yeast, S. cerevisiae, responds to lowtemperatures by increasing the content of unsaturated fattyacids (16), while at high temperatures these cells can incor-porate exogenous ergosterol to maintain fluidity (9). Weinvestigated the possibility that a potential homeoviscousresponse in yeasts would be an increase in ergosterol syn-thesis and the concomitant induction of sterol biosyntheticenzymes. An increase in the growth temperature from 30 to37°C results in a slight decrease in levels of both P-45014DMand P-450 reductase. This demonstrates that these enzymesmay be affected by temperature; it remains to be seen if thisresponse represents a decrease in transcription or translationor is due to degradation of the proteins.

This study clearly indicates that P-45014DM and P-450reductase are not inducible by xenobiotics in S. cerevisiaeY222. Furthermore, P-45014DM from this organism will notmetabolize a number of substrates previously shown to beacted on by P-450 in yeast. This provides further evidencethat there are at least two distinct S. cerevisiae P-450s.

ACKNOWLEDGMENTS

We thank Wyeth-Ayerst Research for the generous supply ofAY-9944.

This work was supported by a grant from the Natural Sciencesand Engineering Research Council of Canada (NSERC).

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3. Aoyama, Y., Y. Yoshida, Y. Sonoda, and Y. Sato. 1989. Role ofthe 8-double bond of lanosterol in the enzyme-substrate inter-action of cytochrome P-45014DM (lanosterol 14a-demethylase).Biochim. Biophys. Acta 1001:196-200.

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