Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

ARCHIVES

Vol.

OF BIOCHEMISTRY

291, No. 1, November

AND

BIOPHYSICS

15, pp. 176-186,199l

Functional Expression of Mammalian P45OllB in the Yeast Saccharomyces Karen

M. Kedzie,*l’

Richard

M. Philpot,?

and James

Cytochromes cerevisjae’ R. Halpert*

*Department of Pharmacology and Toxicology, College of Phurmucy, University of Arizona, and TLaboratory of Pharmacology, Nationul Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Received

June 10,1991,

and in revised

form

July

Tucson, Arizona

85721;

29, 1991

Three mammalian cytochromes P450 from the IIB subfamily, P45OIIBll from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomycee cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gall0 promoter. Cytochromes P450IIB4 and P45OIIB5 are closely related proteins, with only 11 amino acid substitutions between them. P45OIIBll is a homologous protein, likely orthologous with IIB4 or IIBB, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the PISOIIBS protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P45OIIBll hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIBS was observed, with IIB4 producing almost exclusively the 16@-hydroxy metabolite and IIB5 producing the 16a-hydroxy and 15a-hydroxy products. This is the first time that 15a-hydroxylase activity has been associated with IIB4/IIBS. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes. 0 1991 Academic Press, Inc.

i This research was supported by Grants ROl ES04995 and F32 ES05502 from the National Institutes of Health. J.R.H. was the recipient of Research Career Development Award ES00151 (1985-1990). ’ To whom correspondence should be addressed.

The cytochrome P4503 superfamily contains a large number of highly related proteins that exhibit characteristic and often overlapping substrate specificities and are responsible for the metabolism of a wide variety of endogenous and exogenous compounds. The cytochromes P450 have been classified into 27 families based on amino acid sequenceidentity (1). Multiple P450 subfamilies may be present in a single species, and allelic variants may occur among individuals. The presence of multiple forms of cytochromes P450 in mammalian microsomes makes the evaluation of the activities of individual P45Osdifficult and may complicate protein purification. We are investigating the functional properties of cytochrome P45011B44 (commonly known as LM2 (2,4, 73 Abbreviations used: PB, phenobarbital; bp, base pair(s); P450, cytochrome P450; YPD, yeast extract-peptone-dextrose (rich growth medium); SD, synthetic medium with dextrose (yeast minimal growth medium); LB, Luria broth; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; HCl, hydrochloric acid; TI’BS, TweenTris-buffered saline; PAGE, polyacrylamide gel electrophoresis; OH, hydroxy; PVDF, polyvinylidene difluoride; NBT, nitro blue tetraxolium; BCIP, 5-bromo-4-chloro-3-indolyl phosphate, dNTP, deoxyribonucleotide triphosphate; IgG, immunoglobin G; androstenedione, androst-4ene-3,17dione; TLC, thin-layer chromatography; DLPC, dilauroyl-la-phosphatidylcholine; uv, ultraviolet. 4 Nomenclature: The P450IIB gene subfamily is composed of cytochromes P450 from different species which have been grouped together on the basis of amino acid sequence identity, in accordance with the recently suggested nomenclature of Nebert et al. (1). The rat, mouse, rabbit, and human express multiple cytochromes P450 within this subfamily, some of which are inducible with phenobarbital. Seven cDNAs have been isolated from PB-induced rabbits. They are all greater than 95% identical in deduced amino acid sequence and have been classified as belonging to either of two genetic loci, IIB4 or IIB5 (l-3). Some of these cDNAs may be allelic variants, since they exhibit only a few nucleotide differences. The cytochrome P450IIB4 that we are studying is the BO form (2). The deduced amino acid sequence of this cDNA is the same as the protein sequence of P450 LM2 (4). Reported variants of P450IIB4 are ~54, P4501, b15 and b46 (3), and Bl (2). The P450IIB5 that we are studying is the B2 form (2). Reported variants are b52 and HP1 (3). The IIB5 form that has recently been expressed using bacu-

176 All

Copyright 0 1991 rights of reproduction

0003-9861/91 $3.00 by Academic Press, Inc. in any form reserved.

CYTOCHROME

P450IIB

11)) and cytochrome P450IIB5 (2) from rabbit, and cytochrome P450IIBll (commonly known as PBD-2 (6,12, 13)) from canine. The rabbit P45Os are encoded by two separate genes, although the proteins differ by only 11 amino acid residues (2). The P450IIBll protein is homologous to the rabbit IIB enzymes and exhibits 79 and 78% amino acid sequence identity with IIB4 and IIB5, respectively (6). Studies with purified, reconstituted P450IIB4 (10, 11,14) and P450IIBll (12,13) have indicated differences in the regio- and stereoselectivity of androstenedione hydroxylation by these enzymes. However, the evaluation of the activity of IIB4 is complicated by conflicting reports as to the identity of the androstenedione metabolites produced (14). This may reflect differences in the LM2 preparations, as recent studies indicate microheterogeneity of some preparations of the purified protein (3). To define the steroid metabolite profiles of IIB4, IIB5, and IIBll, we decided to express the cDNAs in the yeast Saccharomyces cerevisiae and evaluate the activities of the expressed proteins. The P450IIB5 protein has not been purified to homogeneity, although it may be a contaminant of some IIB4 preparations. One form of IIB5 has been expressed in a heterologous system (5), although the deduced amino acid sequence differs at 17 positions from the IIB5 considered here. P450IIB4 and P450IIBll have not previously been expressed in a heterologous system. S. cerevisiae has been used successfully to express a wide variety of mammalian cytochromes P450 (15-28). The expressed proteins are targeted to the endoplasmic reticulum of the yeast and are recovered in the microsomal fraction. Yeast-expressed cytochromes P450 are active, both in the whole cells and in the microsomal extracts. Although other types of expression systems have been utilized for expression of active cytochromes P450 (5,2931), the yeast system yields relatively high levels of the expressed proteins, and yeast propagation requires no special equipment or containment facilities. In the present study, the cDNAs encoding the cytochromes P450IIB4, P4501IB5, and P450IIBll were inserted into the autonomously replicating yeast expression vector YEp51 (32), under control of the galactose-inducible gall0 promoter, and the resulting constructs used to transform yeast 334 (33). Transformed yeast were analyzed for their ability to produce these cytochromes P450, and the activities of the heterologously produced proteins were analyzed using androstenedione, progesterone, and 7-ethoxycoumarin as substrates.

lovirus is HP1 (5), which differs in sequence from the B2 form (2) by 17 ammo acid residues. Only one P45OIIB, P45OIIBl1, has been identified in canine (6). The sequences in the P45OIIB subfamily are similar enough within each species that it is impossible to identify orthologous forms across species lines.

EXPRESSION

MATERIALS

IN

177

YEAST

AND

METHODS

Materials. Restriction endonucleases, DNA-modifying enzymes, and the PhotoBlot Kit were obtained from Bethesda Research Laboratories (Bethesda, MD). The Gene Clean Kit was purchased from BiolOl (La Jolla, CA). Growth media were obtained from Difco Co. (Detroit, MI). Bluescript KS- was obtained from Stratagene (San Diego, CA). Reagents for gel electrophoresis were obtained from Bio-Rad (Richmond, CA). Glass beads, NBT, BCIP, goat anti-rabbit IgG conjugated to alkaline phosphatase, 16a-OH-androstenedione, DLPC, NADH, and fl-hydroxysteroid dehydrogenase (from Pseudomonas testosteroni) were purchased from Sigma Chemical Co. (St. Louis, MO). 6j3-OH Androstenedione was purchased from Steraloids (Wilton, NH). 15a-OH Androstenedione was obtained from D. N. Kirk (University of London, London, England). [I-“ClProgesterone (57.2 mCi/mmol) and [4-i4C]androst-4-ene-3,17dione were purchased from NEN Research Products (Boston, MA). TLC plates (Baker silica gel, 250 pm, Si 250 PA (19C)) were obtained from Baker Chemicals (Phillipsburg, NJ). All chemicals and reagents not specifically mentioned were obtained from standard commercial sources. Strains and media. Yeast S. cerevisioc strain 334 (Mata, pep4-3, p&I-1122, ur&52, .!Qu~-3,112, regl-501, gaU) was obtained from R. A. Sclafani (University of Colorado Health Sciences Center, Denver, CO) (33). Strain 334 was grown in either rich YPD or minimal SD medium supplemented with uracil and leucine (34). Transformed 334 strains were grown in SD supplemented with uracil. Induction of gene expression was obtained by the addition of galactose, to a final concentration of 2% (33). All yeast cultures were grown at 28’C. The Escherichia coli strain utilized for all plasmid isolations and genetic constructions was DHSa (3536). DH5a was cultivated in LB (37); for those strains containing a plasmid, LB was supplemented with 50 pg/ml ampicillin. All bacterial cultures were grown at 37°C. E. coli were transformed by the CaClz method (37). S. cerevisioc were transformed by the lithium acetate method (38). Plasmids and cDNAs. The E. coli plasmid used for all genetic constructions was Bluescript II KS. The yeast expression vector utilized was YEp51 (32), obtained from C. Dieckmann (University of Arizona, Tucson, AZ). The cDNAs encoding P450IIBll (6) and P450IIB4 and P450IIB5 (2) were isolated and characterized previously. All restriction fragments isolated from agarose gels were purified with the use of the Gene Clean Kit. All ligations were performed by standard procedures. Yeast microsome preparation. Yeast were grown overnight at 28’C in 5 ml SD supplemented with uracil. Two milliliters of the overnight culture was used to seed 1 liter of SD supplemented with uracil. Galactose was added immediately. The large culture was grown for 36 h at 28OC with shaking. After 18 h, the culture was supplemented with additional glucose (20 g), uracil (20 mg), and galactose (10 g). After 36 h, the yeast cells were harvested by centrifugation at 4500g. Yeast microsomes were prepared by modifications of a published method (39). The cells were washed once with water, collected, and resuspended in 25 ml Breaking Buffer (10 mM Tris, pH 7.5,0.65 M sorbitol, 0.1 mM EDTA). A total of 25 g glass beads (cl50 am) and DTT to 1 mM were added, and the cells were broken mechanically by homogenization on ice with an Omni-mixer at top speed (approximately 16,900 rpm). The yeast were broken in 30-s bursts, followed by 30 s rest. This cycle was repeated 10 times, for a total homogenization time of 5 min. The mixture was poured into a beaker and the yeast suspension separated from the glass beads. Differential centrifugation was used to isolate microsomes. Cell debris was removed with a low speed spin (10 min, 3OOOg). Nuclei and mitochondria were removed by centrifugation at 10,000g for 20 min. Microsomes were collected by ultracentrifugation at 150,OOOg for 90 min. The microsomal pellet was resuspended in 50 mM Tris, pH 7.4, 1 mM EDTA, and 25% glycerol and stored at -70°C. Preparation of total yea&proteins. Various yeast cultures were grown to saturation in 10 ml SD supplemented with uracil. When expression of the plasmid-encoded cytochrome P450 was desired, 2% galactose was added.

178

KEDZIE,

PHILPOT,

Yeast proteins were prepared by modification of a published method (17). Yeast cells were harvested by centrifugation and washed with water. The pellet was resuspended in 300 ~1 lysis buffer (0.62 M Tris, pH 8.0, 1% SDS). Glass beads (0.3 g) were added and the yeast cells broken by vortexing three times for 30 s. Between breakings, the mixture was cooled on ice. The samples were boiled for 5 min, and then microfuged for 5 min. The supernatant was recovered and the proteins were precipitated by the addition of 10% TCA. The mixture was microfuged and the resultant pellet resuspended in 10 pl water. SDS gel electrophoresis buffer containing tracking dye was added and the mixture was boiled for 10 min prior to loading the entire volume onto a 7.5% SDS-PAGE gel. Steroid hydroxykwe assays. Microsomal protein was incubated with 1 mM NADPH and either 25 pM [4-“C]androst-4-ene-3,17-dione or 25 pM [4-“C]progesterone in a final volume of 0.1 ml of 0.05 M Hepes, pH 7.6, 1.5 mM MgC&, and 0.1 mM EDTA. Incubations were carried out at 37°C for 30 min (yeast microsomes) or 5 min (mammalian microsomes). When exogenous flavoprotein was utilized, yeast microsomes were incubated with 25 pmol rat NADPH-cytochrome P450 reductase (40) at room temperature for 10 min prior to addition to the reaction mixtures. The reaction was stopped by the addition of 0.05 ml tetrahydrofuran. Aliquots (50 ~1) of the final 0.15-ml reaction were spotted onto the preadsorbent loading zone of a TLC plate. The plate was developed twice in chloroform/ethyl acetate (l/2, v/v) for androstenedione or three times in benzene/ethyl acetate/acetone (10/1/l, v/v/v) for progesterone. When further resolution of androstenedione metabolite bands was necessary, the solvent system utilized was dichloromethane/acetone (4/l, v/v) (41). Metabolites were localized by autoradiography and identified by comparison to unlabeled standards (42, 43). The radioactive areas from the plate were scraped into scintillation vials and the metabolites quantitated by liquid scintillation counting. p-Hydroxysteroid dehydrogenase assay. Specific androstenedione metabolite bands were recovered from the TLC plate by elution with ethyl acetate. The metabolites were dried under nitrogen and reduced to the corresponding testosterone metabolites with B-hydroxysteroid dehydrogenase (44). The loo-p1 reaction, containing up to 10 nmol unlabeled steroid, was carried out at room temperature in 30 mM potassium phosphate, pH 6.5, in the presence of 0.2 mM NADH and 0.04 U /3hydroxysteroid dehydrogenase. Aliquots (50 ~1) were taken after reaction times of 0 and 30 min. The entire 50-~1 aliquot was loaded onto the preadsorbent loading zone of a TLC plate. The plate was developed once in dichloromethane/acetone (4/l). Radiolabeled metabolites were localized by autoradiography and unlabeled standards under uv light. Determination of 7-ethoxycoumarin deethylase activity. Yeast microsomal proteins were incubated with 0.3 mM 7-ethoxycoumarin and 1 mM NADPH in a final volume of 1 ml 50 mM Hepes, pH 7.6,1.5 mM and 0.1 mM EDTA. Incubations were carried out for 30 min. M&la The yeast-derived microsomes were incubated with exogenous rat NADPH-cytochrome P450 reductase for 10 min at room temperature prior to addition to the reaction mixture. The reaction was stopped by the addition of 100 pl 2 N HCl. Metabolites were extracted with chloroform, back-extracted with 30 mM sodium borate, and detected fluorometrically using an excitation wavelength of 366 nm and an emission wavelength of 454 nm (45,46). SDS-polyacrylamide gel electroZmmunochemical determinations. phoresis (7.5%) was conducted as described (47). Proteins were electropboretically transferred to nitrocellulose in 25 mM Tris, pH 8.2,192 mM glycine, and 20% methanol. Minigels were transferred in 2 h at 250 mA, large gels were transferred overnight at 100 mA. After transfer, membranes were blocked for 30 min using 3% nonfat dry milk in TTBS (20 mM Tris base, 0.5 M NaCl, 0.15% Tween 20). All washes were with ‘ITBS. After blocking, the membranes were incubated with primary antibody (rabbit anti-rat P450IIBl IgG, 10 pg/ml) (12) in 3% nonfat dry milk in TTBS for 1 h. After washing three times for 5 min, the membranes were incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase as the secondary antibody. Protein bands

AND

HALPERT

were visualized with NBT and BCIP as substrates. The membrane was rinsed with water to stop the color reaction and air dried. Other methods. Proteins concentrations were estimated by the method of Lowry (48). Liver microsomes were prepared as previously described (12,49). Purified P450IIBl1, P45OIIB4 (lung), and goat antirabbit P450IIB4 IgG were prepared previously. (12, 50). RESULTS

Plusmid

Constructions

The ability of a certain plasmid construct to direct the production of a foreign protein in yeast depends upon the correct placement of the cDNA in relation to transcriptional and translational signals, such as the promoter, transcriptional initiation point, ribosome binding site, and polyadenylation signal. To analyze the effects of some of these factors, the expression of cytochrome P450IIBll from various plasmid constructs was investigated. Construction

of YEpDl

and YEpD2

The effect of distance from the promoter to the start codon of P450IIBll was evaluated by positioning the cDNA either 38 bp (YEpDl) or 290 bp (YEpD2) downstream from the transcriptional start point. The cDNA encoding P450IIBll is contained on a 2600-bp EcoRI fragment which includes approximately 1100 bp of 3’ noncoding cDNA. Plasmid pPBD2 (D39) containing the full-length cDNA encoding P450IIBll was digested with Sal1 and BamHI, and the fragment containing the full length cDNA was recovered. Ligation of this fragment into SalI-BamHI-digested YEp51 yielded YEpDl (Fig. 1). Plasmid pPBD2 (B41), containing the full-length cDNA encoding P450IIBll in the opposite orientation from pPBD2 (D39), was digested with BamHI and HindIII, and the fragment containing the full-length cDNA was recovered. Ligation of this fragment into BamHI-HindIII-digested YEp51 yielded YEpD2 (Fig. 1). Construction

of Truncated

cDNAs

Truncated cDNAs, in which approximately 1050 bp of 3’ noncoding cDNA was deleted, including the native polyadenylation signals, were constructed in Bluescript. Plasmid pPBD2 (D39) was digested with SalI and PvuII, and the 1550-bp fragment was recovered. Ligation of this fragment into SalI-Sk&-digested Bluescript yielded pPBDBt1, containing the truncated cytochrome P45011Bll cDNA (Fig. 2). Plasmid pPBD2 (B41) was digested with BamHI and PuuII, and the 1550-bp fragment was recovered. Ligation of this fragment into BamHI-EcoRVdigested Bluescript yielded pPBD2t2, containing the truncated cytochrome P450IIBll cDNA (Fig. 2). Construction

of YEpD21

and YEpD22

The effect of distance from the promoter to the start codon of P450IIBll and the effect of removal of native

CYTOCHROME BarnHI

Sal1 11-

ECCRI

ECORI pPBD2

P450IIB Hlndll

BunHI CORI

(D29)

EcoRl

P-f)2

v-

(841) YBps1

YEPSl

EXPRESSION

IN

179

YEAST

to create strains 33451, 334:Dl, 334:D2, 334:D21, 334: D22, 334:BO, and 334:B2, respectively (Table I). Intact yeast cells were transformed using the lithium acetate procedure, and selection was based on the ability of transformants containing the plasmid to complement the chromosomal Zeu2-3,112 mutation. Colonies were visible 3-5 days after plating. Negative controls, which underwent the entire transformation procedure in the absence of plasmid DNA, yielded no colonies on the selective medium.

HIndIll

---.

EcoRl YBpD1

FIG. 1. Construction of YEpDl and YEpD2. The full-length cDNA encoding cytochrome P450IIBll from canine was originally subcloned into the EcoRI site of Bluescript in both orientations, resulting in pPBD2 (D39) and pPBD2 (B41). The full-length P450IIBll cDNA was removed from pPBD2 (D39) by digestion with San and BarnHI, and ligated into YEp51 digested with San and BamHI to form YEpDl. The full-length P450IIBll cDNA was removed from pPBD2 (B41) by digestion with BamHI and HindIII, and ligated into YEp51 digested with BamHI and HindI to form YEpD2. In YEpDl, the start codon for the cDNA is 38 bp downstream of the transcriptional start for the mRNA, in YEpD2 the distance is 290 bp. Due to the mode of subcloning, the regions of YEpDl between SoLI and EcoRI and between EcoRI and BamHI are derived from Bluescript. At the 5’ end, this includes restriction sites C&I, HindIII, and EcoRV, at the 3’ end this includes restriction sites PstI and SmuI. In YEpD2, the regions between BamHI and EcoRI and between EcoRI and HkdIII are derived from Bluescript. At the 5’ end, this includes restriction sites SnaI and P&I; at the 3’ end this includes restriction site EcoRV.

polyadenylation signals were evaluated by positioning the truncated P450IIBll cDNA either 38 bp (YEpD21) or 290 bp (YEpD22) downstream from the transcriptional start point. Plasmid pPBD2tl was digested with S&I and BamHI, and the 1550-bp fragment was recovered. Ligation of this fragment into SalI-BamHI-digested YEp51 yielded YEpD21 (Fig. 2). Plasmid pPBD2t2 was digested with BamHI and HindIII, and the 1550-bp fragment was recovered. Ligation of this fragment into BamHI-HindUIdigested YEp51 yielded YEpD22 (Fig. 2). Construction

Expression

Optimization

YEpD2

of YEpB2 and YEpBO

Plasmid 2-211 contains the cDNA encoding P450IIB5 in PBS. A 1750-bp fragment was removed from 2-211 by digestion with NcoI and PuuII, and was inserted into NcoI-SmaI-digested YEpDl. The resultant plasmid is named YEpB2 (Fig. 3). Plasmid pB0 contains the P450IIB4 cDNA in pGEM2. Plasmid pB0 was digested with EcoRI, end filled with the Klenow fragment and dNTP’s, and then digested with NcoI. The 2000-bp fragment was recovered and inserted into NcoI-SmuI-digested YEpDl, to yield YEpBO (Fig. 3). Yeast Transformation Yeast 334 was transformed with plasmids YEp51, YEpDl, YEpD2, YEpD21, YEpD22, YEpBO, and YEpB2

Yeast strains 334:51,334:D1,334:D2,334:D21, and 334: D22 were grown in 10 ml SD supplemented with uracil and galactose. Additionally, yeast 334:Dl was grown in the same medium lacking galactose and 334 was grown in SD supplemented with uracil, leucine, and galactose.

,

I

EC&

ECORI ppsw

yEpo21

(841)

Epop

FIG. 2. Construction of YEpD21 and YEpD22. Truncated cDNAs were generated with the use of the PuuII site present in the cDNA, approximately 50 bp downstream of the stop codon. pPBD2 (D39) was truncated by digestion with SalI and PudI, followed by ligation of the fragment into SalI-SmoI-digested Bluescript. The resulting plasmid, pPBD2t1, contains the truncated P450IIBll cDNA in the same orientation as the parental D39 construct. pPBD2 (B41) was truncated by digestion with BamHI and PuuII, followed by ligation of the fragment into BamHI-EcoRV-digested Bluescript. The resulting plasmid, pPBD2t2, contains the truncated P450IIBll cDNA in the same orientation as the parental B41 construct. pPBD2tl was digested with San and BamHI, and the recovered fragment ligated into similarly digested YEP51, to form YEpD21. pPBD2t2 was digested with BamHI and HindIII, and the fragment ligated into similarly digested YEP51, to form YEpD22. In YEpDPl, the start codon is 38 bp downstream from the transcriptional initiation site of the mRNA, in YEpD22, this distance is 290 bp. In YEpD21, the regions between San and EcoRI and between the PuuII/SmaI region and the BamHI site are derived from Bluescript due to the subcloning process. In YEpD22, the regions between BamHI and EcoRI and between the PvuII/ EcoRV region and HindI are derived from Bluescript.

180

KEDZIE. EcoRl

ECORI

NWI

2.211

Pvull

EIXRI , ) Ncol

PHILPOT.

HALPERT

EWRI

TABLE

I

Ncol EcoRl/flll

,

EcoRl/llll/Smal

Pvull/Smal SamHI

I

Yeast Strains Utilized for Expression of Mammalian Cytochrome P450IIB Proteins

Pso

NC01 Pvull

YEp82

AND

SamHI YEW0

FIG. 3. Construction of YEpB2 and YEpBO. 2-211 contains the cDNA encoding cytochrome P450IIB5 in the EcoRI site of PBS. The cDNA is 2500 bp in length and contains approximately 500 bp of 5’ and 500 bp of 3’ noncoding sequences. The P450IIB5 cDNA was removed from 2211 by digestion with NcoI and PuuII and subcloned into the yeast expression vector by using NcoI-SmaI-digested YEpDl, to form YEpB2. The NcoI site is at the start codon of the IIB5 cDNA, while the PuuII site is approximately 250 bp downstream of the stop codon. YEpDl was utilized due to the presence of extra restriction sites obtained from Bluescript during subcloning, which are not present in the parental YEp51 vector. The region between EcoRI and NcoI is derived from the canine cDNA, the region between Sal1 and EcoRI and the region from PuuII/SmaI to BamHI are derived from Bluescript. pB0 contains the cDNA encoding cytochrome P450IIB4 in the EcoRI site of pGEM2. The cDNA is 2000 bp in length and contains approximately 500 bp of the 3’ noncoding region. No convenient restriction site for truncation exists in the 3’ noncoding region of the cDNA. The P450IIB4 cDNA was removed from pB0 by digestion with EcoRI and end filling with Klenow and dNTP’s, followed by digestion with NcoI. The NcoI site is at the start codon of the IIB4 cDNA. This fragment was subcloned into the yeast expression vector by using NcoI-SmaI-digested YEpDl, to form YEpBO. YEpDl was utilized due to the presence of extra restriction sites obtained from Bluescript during subcloning which are not present in the parental YEp51 vector. The region between EcoRI and NcoI is derived from the canine cDNA; the region between Sal1 and EcoRI and the region from PuuII/SmoI to BamHI are derived from Bluescript.

The yeast were lysed, and their proteins were TCA precipitated and resolved on an SDS-polyacrylamide gel. After electrotransfer to a membrane, the P450IIBll proteins were visualized using specific antibodies and a chemiluminescent detection method (Fig. 4). 334 and 334~51 (Lanes 4 and 5) do not contain the cDNA encoding the mammalian cytochrome P450IIBll protein and do not produce a cross-reacting protein, even in the presence of galactose. 334:Dl produces P450IIBll (Lane 7) when induced with galactose; in the absence of galactose (Lane 6), the protein is not made. The only other yeast strain to produce the P450IIBll protein is 334:D21 (Lane 9); 334:D2 and 334:D22 do not (Lanes 8 and 10). Expression of P450IIBll in this system is thus dependent on the presence of an inducer (galactose) and the proximity of the start codon of the cDNA to the promoter.

Yeast

P450

strain

Plasmid

334” 33451 334:Dl 334:D2 334:D21 334:D22 334:BO 334:B2

YEp51 YEpDl YEpD2 YEpD21 YEpD22 YEpBO YEpB2

encoded

P450 produced? No No Yes No Yes No Yes Yes

P450IIBll P450IIBll P450IIBll P450IIBll P450IIB4 P450IIB5

a The parental strain in all cases is 334. The number follow indicate the plasmid construct carried.

and letters

which

Large-Scale Yeast Expression Yeasts 334:51, 334:Dl, 334:BO, and 334:B2 (Table I) were grown in 1 liter of SD supplemented with uracil and galactose, and microsomes were prepared. The microsomal proteins were analyzed for the presence of the various cytochrome P450 proteins expressed from the plasmid. Microsomal proteins were resolved on SDS-polyacrylamide gels and electrotransferred to a membrane. The proteins were detected with the use of specific antiP450IIBl antibodies and a color reaction catalyzed by alkaline phosphatase (Fig. 5). Microsomes from 334:Dl (Lane 3) produce a protein that apparently comigrates with the protein detected in control and PB-induced canine liver microsomes. Microsomes from 334:BO (Lane 5) and 334:B2 (Lane 6) produce single bands which exhibit greater mobility than the protein(s) detected in the PB-

12345678910

FIG. 4. Chemiluminescent detection of heterologously expressed P450IIB proteins in yeast. TCA-precipitated yeast proteins were resolved on an SDS-polyacrylamide gel. After electrotransfer to a PVDF membrane, proteins were detected with the use of specific antibodies and a chemiluminescent detection method. Incubations were carried out according to the directions with the PhotoBlot Kit. The primary antibody was rabbit anti-rat P450IIBl IgG (10 pg/ml) and the second antibody was biotinylated goat anti-rabbit IgG. Protein bands were detected by incubation with a streptavidin-alkaline phosphatase complex followed by exposure to Lumi-Phos 530. A standard X-ray film was exposed for 45 min to obtain the optimal signal. Lane 1,0.4 pmol P450IIBll; Lane 2,0.1 pmol P450IIBll; Lane 3,0.05 pmol P45OIIBll; Lane 4,334; Lane 5,334:51; Lane 6,334:Dl; Lane 7,334:Dl; Lane 8,334:D2; Lane 9,334: D21; Lane 10, 334:D22: All cultures were grown in SD supplemented with uracil and galactose, with the exception of 334 (Lane 4), which was also supplemented with leucine, and 334:Dl (Lane 6), in which galactose was omitted.

CYTOCHROME

1

23

45

67

P450IIB

EXPRESSION

IN

181

YEAST

0

detection of 16B-OH androstenedione. This assay utilized 10 times the amounts of protein used in the conventional assay. After stopping the reaction, the metabolites were extracted, concentrated, and resolved on a TLC plate. This readily permitted the visualization of 16&OH androstenedione (Fig. 6B, Lane 2). FIG. 5. Immunological detection of heterologously produced P45OIIB 334:B2 microsomes, containing cytochrome P45OIIB5, proteins in yeast microsomes. Yeast microsomel proteins were separated produce 16a-OH and 15~OH, but no 16/3-OH androon an SDS-polyacrylamide gel followed by electrotransfer to a nitrostenedione (Fig. 6A, Lane 6). Androstenedione 15a-hycellulose membrane. Proteins were detected with anti-rat P450IIBl IgC and an enzymatic color reaction, as described under Materials and droxylase activity has not been previously associated with Methods. Lane 1,l fig control canine liver microsomes; Lane 2, 0.3 pg IIB5 or IIB4 (14). To identify this metabolite, two differPB-induced canine liver microsomes; Lane 3,40 pg 334:Dl microsomes; ent TLC solvent systems were used, chloroform/ethyl acLane 4, 40 pg 334~51 microsomes; Lane 5, 100 pg 334:BO microsomes; etate (l/2) and dichloromethane/acetone (4/l). These Lane 6, 100 pg 334:B2 microsomes; Lane 7, 0.3 pg PB-induced rabbit liver microsomes; Lane 8, 1 pg control rabbit liver microsomes. make it possible to distinguish 15~OH androstenedione from 12 of 13 other hydroxylated metabolites, the exception being 6a-OH androstenedione (41). Since 15~OH induced rabbit liver microsomes. The reason for the dif- testosterone can be clearly resolved from 6a-OH testosterone by TLC in dichloromethane/acetone (4/l) (41), pference in mobility is unknown. hydroxysteroid dehydrogenase was utilized to reduce the By comparison with appropriate standards, the cytochrome P450 content of the yeast microsomes was esti- putative 15~OH androstenedione to the corresponding testosterone metabolite for identification. mated by immunoblotting to be approximately 50 pmol/ The 15~OH androstenedione band was recovered and mg for 334:Dl microsomes and approximately 10 pmol/ mg for 334:BO and 334:B2 microsomes (data not shown). reduced by @-hydroxysteroid dehydrogenase at pH 6.5 in Despite the presence of the same 5’ noncoding sequence the presence of unlabeled 6a-OH androstenedione and in all three constructs, differential expression occurs. This 15~OH androstenedione. The reduced radioactive proddifference in expression levels (Fig. 5) appears greater uct corn&rated with the 15~OH testosterone (Rf = 0.08) than it really is, due to the greater cross-reactivity of the and was clearly separated from the 6~OH testosterone anti-rat IIBl IgG with IIBll than with IIB4 (data not (Rf = 0.26). shown). Monooxygenase Activities of Expressed Cytochromes P&O Androstenedione Steroid hydroxylase assays were carried out with microsomes from 334:51, 334:Dl, 334:BO, and 334:B2. Androstenedione hydroxylase activity is observed with microsomes from 334:Dl, 334:BO, and 334:B2 (Fig. 6A), although the specific products formed and their ratios vary. All three proteins exhibit catalytic activity in yeast microsomes, indicating a productive interaction with the endogenous yeast cytochrome P450 reductase. 334:Dl microsomes, containing cytochrome P45OIIBl1, hydroxylate androstenedione at the 16a!and 16p positions in approximately a 1:l ratio (Lane 3). The same metabolites and ratio of metabolites are seen with PB-induced canine liver microsomes (Lane 2) and have also been observed with the purified, reconstituted cytochrome P450IIBll protein (12) (Table II). 334:BO microsomes, containing cytochrome P450IIB4, hydroxylate androstenedione at the 16/3 position (Lane 5). The purified, reconstituted P450IIB4 protein also exhibits 16fl hydroxylation of androstenedione (Table II). However, the levels of androstenedione hydroxylation by 334:BO microsomes were low, and additional verification of activity was required. An androstenedione hydroxylase assay was carried out to maximize the production and

A

1234

567

8

B

’

2

16a

168

FIG. 6. (A) Autoradiogram of androstenedione hydroxylation products. Microsomes were analyzed for their ability to hydroxylate androstenedione, as described under Materials and Methods. One third of each incubation mixture was applied to the TLC plate. Lane 1, 5 fig control canine liver microsomes; Lane 2,5 pg PB-induced canine liver microsomes; Lane 3,200 pg 334:Dl microsomes; Lane 4,400 I.cg 334:51 microsomes; Lane 5,400 pg 334:BO microsomes; Lane 6,200 pg 334:B2 microsomes; Lane 7,5 gg PB-induced rabbit liver microsomes; Lane 8, 5 pg control rabbit liver microsomes. (B) Autoradiogram of androstenedione hydroxylation products. Microsomes from 334~51 and 334:BO were incubated with 25 nmol of radiolabeled substrate and the appropriate buffers in a volume of 1 ml. After the reaction was stopped, the metabolites were extracted twice with 2 vol of chloroform and concentrated by evaporation. The metabolites were resuspended in 75 ~1 of methanol, and 50 ~1 was applied to a TLC plate. Lane 1,4.3 mg 33451 microsomes; Lane 2,4.4 mg 334:BO microsomes. The additional metabolites observed are present in both the sample and the control lanes. These may be metabolites produced by yeast enzymes when the protein is present in large amounts or may be contaminants in the substrate.

182

KEDZIE,

PHILPOT,

Progesterone Progesterone hydroxylase activity is observed with yeast microsomes from strains 334:Dl and 334:B2 (Fig. 7). The three major metabolites induced by PB in canine liver microsomes (Lane 2) are 16a-OH progesterone, 21OH progesterone, and an unidentified metabolite (Ul) of progesterone. These metabolites are also produced by the purified, reconstituted P450IIBll protein (13, data not shown). 334:Dl microsomes also exhibit these same activities, although at much lower levels. Microsomes from 334:B2 produce 16a-OH progesterone and two unidentified metabolites (Ul and U2). 334:BO microsomes appear unable to metabolize progesterone. Buffer Effects In the course of investigating the monooxygenase activities of the yeast microsomes, it was noted that the observed activity of an individual preparation varied widely with the assay conditions utilized. A variety of assay buffers was analyzed (Table III) for the buffers’ effects on monooxygenase activity. Depending on the buffer utilized, the activity of the microsomal P45Oscould differ by as much as lo-fold. Of the buffers tested, 50 mM Hepes, pH 7.6, 1.5 mM MgClz, and 0.1 mM EDTA appeared optimal. This buffer was utilized for all enzyme assays and activity determinations.

AND

HALPERT

123

45

676

FIG. 7. Autoradiogram of progesterone hydroxylation products. Microsomes were incubated with the radiolabeled substrate and the products resolved on a TLC plats as described under Materials and Methods. One third of each incubation mixture was applied to the TLC plate. Lane 1,5 pg control canine liver microsomes; Lane 2,5 fig PB-induced canine liver microsomes; Lane 3, 300 pg 334:Dl microsomes; Lane 4, 300 pg 334:51 microsomes; Lane 5,300 pg 334:BO microsomes; Lane 6, 300 pg 334:B2; Lane 7,5 pg PB-induced rabbit liver microsomes; Lane 8, 5 pg control rabbit liver microsomes. Bands detected in the 334:Dl or 334:B2 microsomes but not in the 334 control are indicated.

marized in Table II, along with the activities of reconstituted P450IIB4 and P450IIBll. Yeast microsomes utilized for these activity measurements were preincubated with Summary of Catalytic Activities exogenous rat NADPH-cytochrome P450 reductase prior The activities of 334:Dl, 334:BO and 334:B2 are sum- to the start of the reaction, and all reactions were carried out in 50 mM Hepes, pH 7.6, 1.5 mM MgC&, and 0.1 mM TABLE II EDTA. The addition of exogenous P450 reductase approximately doubles the monooxygenase activity of these Activities of Mammalian Cytochrome P450IIB Proteins in microsomes. When normalized for P450 content deterYeast Microsomesand in a Reconstituted System’ mined by immunoblotting, the activities of the heterologously expressed P45OIIB proteins are approximately Androstenedione hydroxylation 7-Ethoxycoumarin & to i those of the reconstituted proteins. In the 334:Dl 16n 15a deethylation W microsomes, for which reliable CO-difference spectra were obtained, at least half of the cytochrome P450 was present pmol/30 min/mg pmol/30 min/mg as the holoenzyme. Thus, the presence of apocytochrome P450 can only partially account for the lower turnover b 334:Dl 294 283 293 numbers of the yeast microsomal samples relative to the c 334:B2 115 56 9 c reconstituted system. 334:BO ’ 7 58 7-Ethoxycoumarin deethylase activity was also analyzed (Table II). This assay was mainly utilized to verify nmol/min/nmol nmol/min/nmol the activity of the 334:BO microsomes, since they are not active in the hydroxylation of progesterone and exhibit b IIBlld 1.86 2.42 5.4 only low levels of androstenedione hydroxylation. YeastIIB4 0.03 0.44
out as described under Materials and Methods. Purified P45OIIB4 pmol) was reconstituted with a 1.5 M excess of NADPH-cytochrome P450 rsductase and DLPC, as described previously for P450IIBll * Not determined. ’ Not detectable. dFrom Ref. (12).

(25 (12).

Investigation of 15a-Androstenedine Hydroxyluse Activity of Rabbit Liver Microsomes The association of androstenedione 15cY-hydroxylase activity with 334:B2 microsomes, but not with the sample

CYTOCHROME

P450IIB

EXPRESSION

of PB-induced rabbit liver microsomes initially analyzed, led to the examination of liver microsomes from other rabbits. Liver microsomes were prepared from four PBtreated rabbits, and the androstenedione hydroxylation products were analyzed (Fig. 8). Three of these rabbits had been phenotyped by Northern blot analysis for the presence of BO, Bl, and B2 mRNAs (Table IV) using specific oligonucleotide probes (51); the fourth rabbit had not been phenotyped. Liver microsomes from three rabbits demonstrate readily detectable androstenedione 16cuand 15cu-hydroxylase activity (Lanes l-6). In the fourth individual, the 16cu-hydroxylase activity is much lower and the 15a-hydroxylase activity is virtually absent (Lanes 7 and 8). The identity of the 15c&H androstenedione metabolite generated by rabbit liver microsomes was verified by the same procedure used for the 15a-OH androstenedione generated by transformed yeast. Antibody inhibition experiments were carried out to determine whether the observed androstenedione 15ahydroxylase activity was due to a rabbit P450IIB protein (Table IV). Rabbit microsomes were incubated with a single concentration of either control IgG or goat antirabbit IIB4 IgG prior to evaluation of androstenedione hydroxylase activities. The goal of the experiment was to generate a profile of anti-IIB4 inhibitable activities, rather

TABLE

III

Buffer Effects on Androstenedione Hydroxylase Activity of Mammalian Cytochromes P450 in Yeast Microsomes” Androstenedione hydroxylase (pmol/30 min/mg) 334:Dl Buffer 50 mM Hepes, pH 7.6, 15 mM MgClz 0.1 mM EDTA 50 mM Hepes, pH 7.6, 1.5 mM MgCl, 0.1 mM EDTA 50 mM Hepes, pH 7.6, 0.1 mM EDTA 50 mM Tris, pH 7.4, 0.1 mM EDTA 100 mM potassium phosphate, pH 7.4,0.1 mM EDTA

activity

334:BZ

1601

163

16a!

15a

42

39

17

14

176

174

67

43

144

121

b

b

65

43

b

b

14

22

b

IN

183

YEAST

12345676

FIG. 8. Autoradiogram of androstenedione hydroxylation products from rabbit liver microsomes. Microsomes from livers of four individual PB-induced rabbits were incubated with radiolabeled substrate, and f of the reaction mixture was resolved on a TLC plate as described under Materials and Methods. Lanes 1 and 2, PB3 microsomes (BO/Bl/BZ); Lanes 3 and 4, PB4 microsomes (BO); Lanes 5 and 6, PB6 microsomes (Bl/B2); Lanes 7 and 8, SWG microsomes.

than to completely titrate out each individual hydroxylase. Nonetheless, in those three sampleswith appreciable 15ohydroxylase activity, 70-90s inhibition was observed. With regard to the profile of inhibitable hydroxylase activities, a number of interesting observations were made. First, the inhibitable 16j%hydroxylase activity is remarkably constant among the sampies, regardless of phenotype. Second, the inhibitable 15cu-hydroxylase activity is highest in those two samples (PB3 and PB6) phenotyped as expressing the B2 form of IIB5. Third, the sample (PB4) phenotyped to contain only the BO form of IIB4 exhibits significant inhibitable 16o- and 15a-hydroxylase activity. These activities were not expected, since they are essentially absent in the purified and expressed P450IIB4 (Table II). The appearance of these additional inhibitable hydroxylase activities in the phenotyped BO microsomes strongly suggeststhe presence of an additional, highly related P450IIB4 or P450IIB5 protein.5 Fourth, the nonphenotyped sample (SWG) exhibits a predominantly 16P-hydroxylase activity, consistent with purified IIB4 and suggestive of a BO homozygote. Overall, these data confirm at the level of PB-induced liver microsomes, the functional heterogeneity of rabbit P450IIB4 and IIB5 inferred from the yeast expression studies. DISCUSSION

o Results represent the mean of assays performed twice in duplicate, using the same yeast microsome preparation for all assays. Incubations were carried out as described under Materials and Methods. * Not determined.

b

We have utilized S. cereuisiw to express mammalian cytochromes P450IIB4, IIB5, and IIBll. In the course of this investigation, some of the parameters necessary for ’ One additional form of P450IIB from sequenced (R. M. Philpot et al., manuscript

rabbits has been cloned in preparation).

and

184

KEDZIE,

PHILPOT, TABLE

Antibody

Inhibition

of Androstenedione

I& PB3’ PB4d PB6’ SWG

16~~ anti-IIB4 kG 653 292 407 145

1287 881 2126 165

HALPERT

IV

Hydroxylation

Androstenedione Control

AND

in PB-Induced

Rabbit Liver

hydroxylase (pmol/min/mg)

16P A* 652 589 1719 20

Control kG

anti-IIB4 W

508 430 545 502

226 211 277 256

’ Microsome were incubated with control or goat anti-rabbit P450IIB4 androstenedione hydroxylase activity, as described under Materials and b Difference in activity between control IgG-treated and anti-IIB4-treated activity of the microsomes.

IgG

A

to contain to contain

mRNA mRNA

encoding encoding

monooxygenase activity in yeast microsomes were defined. Successful functional expression of these three cytochromes P450IIB allowed us to confirm some of the catalytic properties observed with purified P450IIBll and P450IIB4 and to discover novel catalytic functions associated with P450IIB5. While the distance from the transcriptional start point to the start codon of the cDNA has been shown to be an important factor in the production of heterologous proteins in yeast, other, less well-defined components also influence expression. The expression levels of P450IIB4, P450IIB5, and P450IIBll differ, despite the use of the same 5’ noncoding region in all three constructs. It has been reported that differences in the coding sequence of the first 43 amino acids of cytochrome P450 proteins influence the amount of protein produced in yeast expression systems (18). The mechanism for this phenomenon has not been elucidated, although it may be a contributing factor to the differential expression observed by us. If the sequence at the amino terminus of the protein influences translation efficiency, it may be possible to increase the production of certain cytochromes P450 in yeast by the alteration of the amino terminus of the protein through cDNA splicing. However, this may also alter the enzymatic activities of the engineered protein. If such cDNA constructs are utilized for the purpose of increased protein production in the yeast system, experiments to verify the retention of the expected activities will be needed. The buffer effects on steroid hydroxylase activity in yeast microsomes are intriguing. Initially, all assays were carried out in 50 mM Hepes, pH 7.6, 15 mM MgClz, and 0.1 mM EDTA. This buffer has been optimized in our

15a anit-IIB4 W

357 144 525 30

35 40 51 26

(10 pg/pg

microsomal

protein)

for 20 min prior

A 322 104 476 4 to the assay of

Methods. Results represent the mean of duplicate assays. microsomes.

the BO form of P450IIB4 (2,51). Bl and B2 forms of P450IIB4 and IIB5

the successful production of these proteins were identified, such as proximity of the start codon to the translational initiation site and the use of an inducible promoter. In addition, buffer conditions required for optimal assay of

Control I&

282 219 268 246

Control

IgG did not afkt

’ Phenotyped by Northern blotting to contain mRNA encoding BO, Bl, and B2 forms of P450IIB4 d Phenotyped ’ Phenotyped

Microsomes”

and P450IIB5

the androstenedione

hydroxylase

(2,51).

(2, 51).

laboratory for use with mammalian microsomes and reconstituted systems. However, these conditions are apparently not appropriate for use with the 334-yeast-microsome system. After analyzing a variety of buffers, it was decided that all yeast microsomal enzyme assays would be carried out in 50 mM Hepes, pH 7.6, 1.5 mM MgC&, and 0.1 mM EDTA. It is interesting to note that buffers successfully utilized in other laboratories for the assay of yeast-expressed cytochromes P450 yielded lower activities than our optimal buffer in our system. In fact, some observed activities were lo-fold lower with these buffers. Use of these conditions would have led to very different conclusions as to the activities present in our yeast microsomes. Our results are consistent with other work indicating the inhibition of P450-related activities with increasing ionic strength (52), although this may not be the only buffer factor influencing the yeast microsomal system. It is possible that cytochromes P450 exhibit maximal activities under conditions that differ with the family of the enzyme, the expression system utilized, and the mode of microsome preparation. The expressed P450IIB4 and IIBll metabolize the substrates androstenedione, progesterone, and 7-ethoxycoumarin in a manner that reflects the regio- and stereoselectivity of the parental enzymes. However, the progesterone hydroxylase activity in 334:Dl microsomes is much lower than expected, based on androstenedione hydroxylase activity.6 This reduction in progesterone hydroxylase activity of the 334:Dl microsomes could reflect a deficiency in the yeast expression system, a need for ’ The rate of progesterone hydroxylation a reconstituted system is approximately stenedione hydroxylation. With 334:Dl gesterone hydroxylation is approximately stenedione hydroxylation.

catalyzed by P450IIBll in f that of the rate of andromicrosomes, the rate of pro3% that of the rate of andro-

CYTOCHROME

P45OIIB

altered conditions in the assay, or the presence of a second cytochrome P450IIB in canines. This second protein, if it does exist, would need to exhibit high levels of sequence identity with the P450IIBll protein, as it would have been copurified with the P450IIBll protein. It is premature at this point to assume that a second protein does exist, although this is a possibility since there is evidence for two genes in the canine P450IIB subfamily (6). In contrast to P450IIB4 and IIBll, a protein corresponding to cytochrome P450IIB5 has not been isolated. Thus, the yeast expression system allowed us to characterize the enzymatic properties of P450IIB5 for the first time. Perhaps most interesting was the association of an androstenedione 15cu-hydroxylase activity with P450IIB5 but not P450IIB4. The observation of this activity in the expression system led to a reinvestigation of androstenedione hydroxylation in rabbit liver microsomes. As a consequence, 15cu-hydroxylase activity that is largely inhibited by anCP450IIB IgG was subsequently detected, although not in all the individual rabbits tested. In contrast, the inhibitable 16/3-hydroxylase activity is remarkably constant among the individuals. It is interesting to speculate that the particular rabbit used for subsequent protein purification could influence the P450IIB protein isolated. Thus, the protein identified as P450IIB4 from one rabbit could exhibit different substrate specificities than the protein identified as P450IIB4 from another. This may explain the conflicting reports in the literature on the activities of P450 LM2, especially with regard to steroid metabolism (10, 11, 14, 53). These results also suggest that androstenedione 15a-hydroxylase activity could serve as a marker for IIB5 contamination of IIB4 preparations. In conclusion, we report the first successful expression of P450IIB4, P450IIB5, and P450IIBll in the yeast S. cerevisiae. Striking functional differences have been observed among these expressed cytochromes P450, especially between the highly related IIB4 and IIB5. By use of hybrid proteins and site-directed mutants, it should be possible to determine the specific regions and residues responsible for the observed metabolic differences. ACKNOWLEDGMENTS We thank Gina Escobar for help in subcloning and preparing plasmids and Celia Balfour for assistance with enzyme assays. We also thank Scott Grimm and Dr. Jeffrey Stevens for help with enzyme assays, valuable discussions, and technical advice. We thank Dr. Andrew Parkinson for his kind gift of 6o-OH testosterone and purified P450IIA1, a rat liver steroid Go-hydroxylase. We also thank Dr. David Waxman for generously supplying Go-OH androstenedione and a copy of Ref. (41) prior to publication.

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