Human Cytochrome P450 3A4: Enzymatic Properties of a Purified

Home , Erythromycin

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11748-11752, December 1993 Pharmacology Human cytochrome P450 3A4: Enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase (drug metabolism/erythromycin/steroid 63-hydroxylation) MANJUNATH S. SHET, CHARLES W. FISHER, PRISCILLA L. HOLMANS, AND RONALD W. ESTABROOK Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9038 Contributed by Ronald W. Estabrook, September 9, 1993

ABSTRACT Human cytochrome P450 3A4is recognized as Recombinant CYP3A4 has been expressed in Escherichia the catalyst for the oxygen-dependent metaboism of a diverse coli (12, 13), yeast (14), and various cell lines (15) and shown group of medicafly important chemicals, inWuding the immu- to function in the metabolism of aflatoxin B1, testosterone, nosuppressive agent cyclosporin; macrolide antibiotics, such as nifedipine, and lidocaine, to name but a few of the substrates erythromycin; drugs such as benzphetamine, nifedipine, and tested. P450 3A4 expressed in human lymphoblastoid cells cocaine; and steroids, such as cortisol and testosterone to name has been used to evaluate cytotoxic and mutagenic responses but a few. We have engineered the cDNA for human cytochrome to various chemicals (15). P450 3A4 by linkage to the cDNA for the rat or human We describe here results of experiments designed to eval- flavoprotein, NADPH-P450 reductase (NADPH:ferrihemopro- uate the enzymatic properties of a purified recombinant tein oxidoreductase, EC 1.6.2.4). An enzymaticafly active fusion fusion protein containing the heme domain of human protein (rF450[mHum3A4/mRatOR]Ll) has been expressed at CYP3A4 and the flavoprotein domain of rat NADPH-P450 high levels in Escherichia coli and purified to homogeneity. reductase (NADPH:ferrihemoprotein oxidoreductase, EC Enzymatic studies show a requirement for lipid, detergent, and 1.6.2.4). This fusion protein is active in the metabolism of cytochrome bs for the 63-hydroxylation of steroids and the many steroids and drugs in a reaction dependent on the N-oxidation of nifedipine. In contrast, these additions are not presence of lipid, detergent, and cytochrome b5. The N-de- required for the N-demethylation oferythromycin or benzphet- methylation of the antibiotic erythromycin, as well as drugs amine. A spectrophotometricafly detectable metabolite complex such as benzphetamine and imipramine, does not require of P450 3A4 is formed during the metabolism of triacetylolean- these additives. In vitro studies designed to measure inhibi- domycin, and this has a pronounced inhibitory effect on the tion by a number of compounds reported as substrates for These re- CYP3A4 were carried out to determine their influence on the metabolim of both testosterone and erthromycin. 63-hydroxylation of testosterone or the N-demethylation of suits relate to the interpretation of current methods used to erythromycin. These studies revealed a number of unex- assess the in vivo activity of P450 3A4. plained differences in this in vitro measure of potential drug-drug interactions. Formation of a metabolite-inhibitor One ofthe most versatile ofthe cytochrome P450s is the form complex during the metabolism of low levels of TAO is present in human liver called CYP3A4, which catalyzes the inhibitory to the metabolism of both testosterone and eryth- oxidative metabolism of a wide array of different chemicals romycin. These results demonstrate the usefulness of this with markedly different structural characteristics (1). Interest recombinant fusion enzyme in evaluating the profiles of drug has centered on this P450 since it is reported to be one of the and steroid metabolism by this versatile human cytochrome more abundant P450s in human liver (2); it is inducible by a P450. Furthermore, these studies relate to the interpretation variety of agents including glucocorticoids as well as pheno- of the erythromycin breath test as an indicator of the in vivo barbital (3); it appears to play a central role in the metabolism function of CYP3A4 in the metabolism of other drugs. of the immunosuppressive cyclic peptide cyclosporin A as well as macrolide antibiotics, such as erythromycin (4); it also catalyzes the 63-hydroxylation of a number of steroids in- MATERIALS AND METHODS cluding testosterone, progesterone, and cortisol (5). Clinical Construction of Plasmids. The plasmid pCWori+: :bovl7A- interest relates to the measurement of erythromycin metab- rORfus described for the expression of the fusion protein olism by a breath test (6) and the presence of6f-hydroxylated rF450[mBovl7A/mRatOR]L1 (r, recombinant; F, fused; steroids in urine (7) as indicators of CYP3A4 function for 450, P450; m, modified cDNA; Bov or Rat, species; 17A, evaluation of transplant recipients. family name of P450; OR, NADPH-P450 reductase; Li, P450s ofthe 3A family were first characterized by Guzelian linker type), containing the cDNA of P450 17A linked to the and colleagues (8) based on the ability ofthe catabolic steroid cDNA of rat NADPH-P450 reductase, was modified in a pregnenolone-16a-carbonitrile to induce a unique form of manner similar to that described for the construction of the P450 (which they called P-450p). They also recognized the plasmid used for expression of the fusion protein containing ability of the macrolide antibiotic triacetyloleandomycin a rat liver w-hydroxylase, rF450[mRat4Al/mRatOR]L1 (16). (TAO) to serve as a powerful inducer ofthis type of P450-a A Agtll clone (NF-25) containing the nucleotide sequence for result of interest because of the known ability of TAO and the open reading frame ofhuman P450 3A4 was obtained from other compounds to form stable, metabolite-inhibitor com- F. P. Guengerich (Vanderbilt University, Nashville, TN) and plexes with P450 (9). Reconstitution studies using purified used for PCR mutagenesis with the oligonucleotide GT- P450s of the 3A family have shown the need to include CATATGGCTCTGTTATTAGCAGTTTTTCTGGTGC- phospholipids, detergents, and cytochrome b5 when testing TCCTC as the 5' primer and CCTCTAGACTAGTCAGGC- enzymatic activities (10, 11). TCCACTTACGGTGCC as the 3' primer. A portion of the coding sequence for the N terminus of P450 3A4 was deleted The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: TAO, triacetyloleandomycin; CHAPS, 3-[(3- in accordance with 18 U.S.C. §1734 solely to indicate this fact. cholamidopropyl)dimethylammoniol-l-propanesulfonate. 11748 Downloaded by guest on September 30, 2021 Pharmacology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11749 and replaced with a 27-bp fragment of the bovine 17A RESULTS modified by Barnes et al. (17) as described by Fisher et al. (12). The C terminus of P450 3A4 was modified by incorpo- Effect of Lipid, Detergent, and Cytochrome b5 on the rating a Sal I site encoding Ser-Thr as a dipeptide linker Metabolism of Nifedipine and the 6(-Hydroxylation of Testos- replacing the TGA stop codon to allow fusion with the terone. The in vitro metabolism of many chemicals by P450s modified DNA sequence of NADPH-P450 reductase as de- ofthe 3A family require the addition of a number ofadditives scribed (16). The plasmid encoding rF450[mBovl7A/ mixed together in an appropriate sequence. Incubation ofthe mRatOR]Ll was digested with Nde I and Sal I to remove the diluted purified fusion protein, rF450[mHum3A4/mRa- P450 17A domain and this digested plasmid, containing only tOR]L1, in the same manner as used with other P450- the rat reductase domain, was ligated with the Nde I/Sal I containing fusion proteins (16), resulted in little or no me- PCR amplified human P450 3A4 domain. Restriction digests tabolism of testosterone or nifedipine (Fig. 1 A and B). As confirmed that the 3A4 segment was incorporated in the described by Imaoka et al. (10) and Eberhart and Parkinson correct orientation. This plasmid is called (11), preincubation of the undiluted purified enzyme with pCWori+::hum3A4-rORfus. Recombinant human and rat cy- purified cytochrome b5, detergent, and phospholipid (in this tochrome b5, containing a histidine domain, were purified sequence), followed by dilution in the buffer mixture, results from E. coli membranes in which the cDNAs were expressed in optimal enzymatic activities. A systematic study of the by using a T7 expression system (ref. 18; unpublished re- requirement for phospholipid indicates a greater effective- sults). ness with those phospholipids containing unsaturated fatty Cell Growth, Disruption, and Purification of the Enzyme. E. acids-e.g., dioleoylphosphatidylserine (11). Most effective coli DH5a was transformed, plated, selected, and grown at was a sonicated suspension of a lipid extract prepared from 26°C as described (16) (with the omission of the rare salts rat liver microsomes (14). We find that a small amount of mixture in the growth medium). Cells were generally har- detergent (0.05% CHAPS) also must be included in the vested after 72 hr of growth and expressed 150-200 nmol of reaction mixture. This requirement for phospholipid and spectrophotometrically detectable P450 per liter of growth detergent is in sharp contrast to our results obtained when medium. It was noted that the expression of this fusion studying the enzymatic properties of other P450-containing protein is very sensitive to variation in growth conditions. fusion proteins (e.g., rF450[mBovl7A/mRatOR]L1 or Cells were disrupted by sonic treatment and membranes rF450[mRat4A1/mRatOR]L1), which showed no effect or an containing the fusion protein were prepared by differential inhibition of their catalytic activities when either phospho- centrifugation (-0.25 nmol of P450 per mg of membrane lipids or detergent were added to the reaction mixture. protein). The fusion protein was solubilized by addition ofthe Fig. 1A demonstrates the >20-fold stimulation in the rate detergent Emulgen 911 and purified by chromatography with of 6,B3hydroxylation of testosterone obtained by addition of a 2',5'-ADP Sepharose affinity column as described (16). The the purified membrane-bound form of recombinant cy- purified fusion protein contained 6.5-7.0 nmol ofP450 per mg tochrome b5. Maximal stimulation was obtained when a 1:1 of protein. ratio of P450/cytochrome b5 was used. Little stimulation of Enzymatic Assays. Rates of steroid hydroxylation were metabolism was seen when using the low molecular weight determined by incubating the designated radioactive steroid (truncated proteolytic product) form of cytochrome b5. In with the purified fusion enzyme diluted in 50 mM Tris HCl, contrast to the recent report by Gillam et al. (13), we find little pH 7.5/10 mM MgCl2 buffer containing 8 mM sodium iso- or no metabolism of testosterone or nifedipine when 50 mM citrate and 0.1 unit of isocitrate dehydrogenase per ml as an potassium phosphate (pH 7.4) replaced Tris HCl, pH 7.5/ NADPH-regenerating system, unless otherwise stated. The MgCl2 as reaction buffer and we failed to see a stimulatory reactions were started by the addition of NADPH (final effect of glutathione on the overall reaction rate. The mech- concentration, 1 mM). Samples (0.5 ml) were removed at the anism(s) by which lipid, detergent, and cytochrome b5 stim- times designated, added to methylene chloride (5.0 ml), ulate cytochrome P450-catalyzed reactions and the vagaries extracted, and prepared for HPLC analysis as described (19). associated with the use of other buffers or additions such as When other chemicals were tested as competitive inhibitors glutathione remain unexplained. they were added together with the steroid substrate. For Effect of Lipid, Detergent, and Cytochrome bs on the experiments designed to test the effects of lipid and deter- Metabolism ofErythromycin. When similar experiments were gent, an aliquot of concentrated fusion protein (20 nmol/ml) carried out to measure the rate of N-demethylation of eryth- was mixed with an aliquot of concentrated cytochrome b5 (60 romycin, benzphetamine, or imipramine, we noted that the nmol/ml), detergent, and lipid; mixed; incubated for 10 min presence of lipid, detergent, or cytochrome b5 was not at 37°C; and then diluted with the Tris/MgCl2 buffer mixture. required. Indeed, addition of these agents inhibited the rate Unless stated otherwise the final concentrations of reagents of erythromycin N-demethylation (Fig. 1C). This result sug- in the diluted reaction mixture were 1 nmol of P450 per ml, gests a possible difference in the mechanistic pathways of 1 nmol of cytochrome b5 per ml, 500 ,ug of 3-[(3- metabolism of erythromycin or benzphetamine compared to cholamidopropyl)dimethylammonio]-1-propanesulfonate other substrates, such as testosterone and nifedipine. Studies (CHAPS) per nmol of P450, and 625 pg of lipid extract with the P450 inhibitors showed a >50%o inhibition by 1 uM prepared from rat liver microsomes per nmol of P450 using ketoconazole (25) of both the 6f-hydroxylation of testoster- the procedure described by Folch et al. (20). Measurements one and the formation of formaldehyde from erythromycin. ofnifedipine metabolism were carried out in a similar manner Inhibition by Other Substrates. If separate pathways of using reversed-phase HPLC, and product formation was metabolism were functioning for different substrates, one test monitored spectrophotometrically at 254 nm. would be the determination of substrate inhibition. A series Rates of erythromycin, benzphetamine and imipramine ofexperiments were carried out to determine the influence of metabolism were determined by measuring the rate of form- a variety of different compounds reported to be substrates of aldehyde formation using the Nash reagent (21). P450 3A in order to assess their inhibition of the 6f- The rate of cytochrome c reduction was determined spec- hydroxylation of testosterone or the N-demethylation of trophotometrically at 550 nm as described (22). The content erythromycin (Fig. 2). For these experiments, optimal con- of P450 was measured with an Aminco DW2 wavelength ditions for the metabolism oftestosterone (i.e., preincubation scanning differential spectrophotometer as described (23). with cytochrome b5, detergent, and lipid prior to dilution of Protein content was estimated by the Bradford method the fusion protein) or erythromycin (i.e., initial dilution ofthe using bovine serum albumin as the standard (24). fusion protein in the absence of added lipid extract, deter- Downloaded by guest on September 30, 2021 11750 Pharmacology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) A 200 8 A

LO) 150 I'l-

0 E C) 0it 100 C

50 (0 0 0 E E N4 0 E

0 0 10 20 30 B 200 50 C N E L O E2 6 B 1500 LO) 0~ 0 X 100 a)

0 0 r 2

0 0 10 20 30 C E 0

C N T L 0 E2 :- FIG. 2. Inhibition of the metabolism of (A) 0.2 mM testosterone C~ 0.2mM 0 (T) or (B) 0.2 mM erythromycin (E) by 0.2 mM nifedipine (N), C:a1) lidocaine (L), 0.2 mM quinidine (Q), and 0.2 mM estradiol (E2). C, control, no inhibitor. Substrates were added simultaneously to the 0 0 reaction mixture containing 1 nmol of P450 per ml. Assays were 0 carried out as described. I 0 gent, or cytochrome b5) were used. Equimolar concentrations (200 ,uM) of added inhibitory compounds to the substrate tested (testosterone or erythromycin) were used. As shown in Fig. 2, erythromycin is a poor inhibitor of testosterone metabolism and vice versa. In contrast nifedipine is a 0 10 20 30 potent inhibitor of both reactions. Lidocaine, quinidine, and Time, min estradiol are intermediary in their inhibitory properties. We FIG. 1. Effect of cytochrome b5 and other agents on the rate of interpret the degree of inhibition to be a reflection ofthe rate metabolism of testosterone (A), nifedipine (B), and erythromycin of metabolism of these compounds by this fusion enzyme. (C). Reaction mixtures contained 1 nmol of pure fusion protein Formation of the Metabolite-Inhibitor Complex Formed rF450[mHum3A4/mRatOR]L1 per ml as determined by the P450 During TAO Metabolism. It is known that P450s of the 3A content and 1 nmol of purified recombinant high molecular weight family readily form spectrophotometrically detectable me- form of rat cytochrome b5 per ml. Samples were preincubated for 10 min with the indicated additions before dilution with Tris.HCl/MgCl2 tabolite-inhibitor complexes during the metabolism of some buffer followed by addition of substrate and NADPH. Initial sub- substrates (26). The time-dependent formation of an absor- strate concentrations were 0.2 mM testosterone (T), 0.2 mM nifed- bance band located at -456 nm is a measure of the rate of ipine (NIF), and 0.5 mM erythromycin. o, Without addition of generation of the metabolite-inhibitor complex and results cytochrome b5, lipid, or detergent; o, with cytochrome b5 but without from the interaction of a reactive metabolite with the ferrous lipid or detergent; *, with cytochrome b5, lipid, and detergent; A, and form of the hemoprotein (27). Such complexes slowly disso- v, metabolites formed during metabolism of testosterone (2, and serve to inhibit the of the P450. 6fT) or nifedipine [designated MET. (8.5 min) and MET. (4.5 min), ciate and enzymatic activity signifying two metabolites with HPLC retention times of 4.5 and 8.5 Of interest is the ability of metabolites formed during me- min, respectively]. tabolism of many substrates by P450s of family 3A to form Downloaded by guest on September 30, 2021 Pharmacology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11751 these metabolite-inhibitor complexes, such as TAO and erythromycin (9, 28). Incubation of rF450[mHum3A4/mRatOR]L1 with micro- molar concentrations of TAO and NADPH showed the time-dependent formation of such a spectrophotometrically measurable complex (Fig. 3). The subsequent addition of the reducing agent sodium dithionite further increases the 0 amount ofmetabolite-inhibitor complex formed. As reported n.__ earlier (26), the P450 metabolite-inhibitor complex interferes with the binding of carbon monoxide to the reduced hemo- O protein. Parallel experiments in which various concentrations of TAO were first incubated with rF450[mHum3A4/ mRatOR]L1 and NADPH followed then by the addition of 200 ,uM testosterone showed pronounced inhibition of the 6f3-hydroxylation reaction (Fig. 4). A similar inhibition of testosterone 6f-hydroxylation was obtained when the P450 fusion enzyme was preincubated with low concentrations of 0 2 4 6 8 10 erythromycin in the same way (data not shown). Of interest TAO, ,uM is the observation (Fig. 4) that erythromycin N-demethylation is also strongly inhibited by the metabolite-inhibitor FIG. 4. Inhibition oftestosterone or erythromycin metabolism by TAO metabolism. the metabolite-inhibitor complex formed during metabolism ofTAO. complex formed during Purified rF450[mHum3A4/mRatOR]L1 was preincubated for 10 min at 37°C with cytochrome b5, detergent, and lipid extract as described 450 followed by dilution with Tris.HCl/MgCl2 buffer and addition of the concentrations of TAO indicated. NADPH (final concentration, 1 \ mM) was added and the mixture was incubated for a further 20 min. I Testosterone (final concentration, 200 iLM) was then added and samples were removed at the times indicated in Fig. 1A. The rate of I . 63-hydroxylation oftestosterone in the absence ofTAO was 9.0 nmol . . . per min per nmol of P450. Experiments to measure the effect on erythromycin metabolism were carried out in a similar manner but omitting the preincubation step and the addition of cytochrome bs, 'I detergent, and lipid. The rate of formaldehyde formed during N-de- C methylation oferythromycin in the absence ofTAO was 5.2 nmol per .456 min per nmol of P450. DISCUSSION The ability to engineer the cDNAs for human proteins and express these in bacteria to serve as a source of functional enzymes that participate in the metabolism of xenobiotics offers new opportunities to evaluate pathways of drug metabolism in humans. Extension of this approach to the generation of self-sufficient artificial fusion proteins opens avenues of chemistry of direct application to the pharmaceu- tical sciences and toxicology. One example is illustrated by the work presented here. The great variety of substrate types metabolized by human P450 3A4 illustrates the need to better A ABSORBANCE understand the biochemical events occurring in order to = 0.02 evaluate interpatient differences and the probability of drug- drug interactions during the in vivo function of this unique hemoprotein. In vitro measurement of inhibition is one approach for answering these questions. Considerable interest has focused on establishing a means of measuring the func-

I I i I i a I I I I I tioning of CYP3A4 in potential organ transplant patients I I I I I I 1 because of the role of this enzyme in the metabolism of 400 450 500 immunosuppressive agents such as cyclosporin. It is recognized that agents like cyclosporin are of great benefit for WAVELENGTH (nm) organ transplantation but "its optimal use is difficult because FIG. 3. Spectrophotometric measurement of fonnation of the of the highly variable pharmacokinetics that to date remains metabolite-inhibitor complex generated during metabolism of TAO unsatisfactorily explained" (29). To measure the rate of by the fusion protein. Purified fusion protein was preincubated with cyclosporin metabolism, it was necessary to identify cyclo- cytochrome b5, lipid extract, and CHAPS as described. The mixture sporin metabolites by collecting bile from patients receiving was diluted with Tris HCl/MgCl2 buffer to give 1 nmol of P450 per this drug (30) as well as monitoring metabolites in blood and ml for the fusion enzyme and 1 nmol/ml for cytochrome b5. Differ- urine. In fact, the immunosuppressive properties of cyclo- ence spectra were recorded (curves A) by repetitive scanning (1 scan sporin may be associated with the hydroxylated metabolites per min) after addition of TAO (final concentration, 5 '"M) to the Watkins and his have the contents of the sample cuvette and NADPH to the contents of both (31). colleagues (32) pioneered the sample and reference cuvettes. After 30 min, a few crystals of development of a breath test to measure 14CO2 formation sodium dithionite were added and the spectrum was recorded (curve after the intravenous administration of [N-methyl-14C]eryth- B) followed by gassing ofthe sample for 30 sec with carbon monoxide romycin to patients as a means of estimating the patients' (curve C). ability to metabolize cyclosporin. They provided evidence Downloaded by guest on September 30, 2021 11752 Pharmacology: Shet et al. Proc. Natl. Acad. Sci. USA 90 (1993) (32) correlating the content of CYP3A4, as measured immu- 11. Eberhart, D. C. & Parkinson, A. (1991) Arch. Biochem. Bio- nochemically in microsomes of liver samples obtained at phys. 291, 231-240. surgery, with results of the erythromycin breath test. 12. Fisher, C. W., Caudle, D. L., Martin-Wixtrom, C., Quattro- The present study was undertaken to examine the enzy- chi, L. C., Tukey, R. H., Waterman, M. R. & Estabrook, R. W. (1992) FASEB J. 6, 759-764. matic properties of a recombinant artificial fusion protein 13. Gillam, E. M. J., Baba, T., Kim, B.-R., Ohmori, S. & Guenger- containing the functional elements ofhuman P450 3A4 linked ich, F. P. (1993) Arch. Biochem. Biophys. 305, 123-131. to the required electron transport flavoprotein NADPH-P450 14. Brian, W. R., Sari, M. A., Iwasaki, M., Shimada, T., Kamin- reductase. We found differences in the conditions required sky, L. S. & Guengerich, F. P. (1990) Biochemistry 29, 11280- for the in vitro metabolism of different drugs and steroids, in 11292. particular when comparing the metabolism of erythromycin 15. Crespi, C. L., Penman, B. W., Steimel, D. T., Gelboin, H. V. with the conditions for 6f-hydroxylation oftestosterone. The & Gonzalez, F. J. (1991) Carcinogenesis 12, 355-359. 16. Fisher, C. W., Shet, M. S., Caudle, D. L., Martin-Wixtrom, latter is a second reaction, which has been proposed as a C. A. & Estabrook, R. W. (1992) Proc. Natl. Acad. Sci. USA measure ofthe in vivo function of CYP3A4 (7). The ability of 89, 10817-10821. macrolide antibiotics, such as erythromycin, to influence the 17. Barnes, H. J., Arlotto, M. P. & Waterman, M. R. (1991) Proc. pharmacokinetics ofother drugs is exemplified by the recent Natl. Acad. Sci. USA 88, 5597-5601. report (33) of the "undesirably severe and excessively long- 18. Tabor, S. & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. lasting hypnotic effects" seen when young adults were pre- USA 82, 1074-1078. treated with erythromycin to oral administration of the 19. Arlotto, M. P., Trant, J. M. & Estabrook, R. W. (1991) Meth- prior ods Enzymol. 206, 454-461. 1,4-benzodiazepine short-acting hypnotic agent midazolam. 20. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) J. Biol. Midazolam is metabolized by P450 3A4 (34). Formation of a Chem. 226, 497-509. metabolite-inhibitor complex CYP3A4 during pretreatment 21. Werringloer, J. (1978) Methods Enzymol. 52, 297-302. with erythromycin, in a manner similar to that illustrated here 22. Yasukochi, Y., Okita, R. T. & Masters, B. S. S. (1980) Arch. with TAO, would explain this result. The present study Biochem. Biophys. 202, 491-498. points to the value of recombinant enzymes for predicting 23. Estabrook, R. W., Peterson, J. A., Baron, J. & Hildebrandt, A. such negative interactions. (1971) in Methods in Pharmacology, ed. Chignell, C. F. (Ap- pleton-Century-Crofts, New York), Vol. 2, pp. 303-350. 24. Bradford, M. (1976) Anal. Biochem. 72, 248-254. This study was supported in part by Grant GM16488-24 from the 25. Sheets, J. J. & Mason, J. I. (1984) Drug Metab. Dispos. 12, National Institutes of Health and a Sponsored Research Agreement 603-606. from Dallas Biomedical, Inc. 26. Werringloer, J. & Estabrook, R. W. (1973) Life Sci. 13, 1319- 1330. 1. Guengerich, F. P. (1992) FASEB J. 6, 745-748. 27. Pershing, L. K. & Franklin, M. R. (1982) Xenobiotica 12, 2. Wrighton, S. A. & Stevens, J. C. (1992) Crit. Rev. Toxicol. 22, 687-699. 1-21. 28. Pessayre, D., Descatoire, V., Konstantinova-Mitcheva, M., 3. Hostetler, K. A., Wrighton, S. A., Kremers, P. & Guzelian, Wandscheer, J. C., Cobert, B., Level, R., Benhamou, P. J., P. S. (1987) Biochem. J. 245, 27-33. Jaouen, M. & Mansuy, D. (1981) Biochem. Pharmacol. 30, 4. Kronbach, T., Fischer, V. & Meyer, U. A. (1988) Clin. Phar- 553-558. macol. Ther. 43, 630-635. 29. Tan, K. K., Trull, A. K., Hue, K. L., Best, N. G., Wallwork, 5. Waxman, D. J., Attisano, C., Guengerich, F. P. & Lapenson, J. & Higenbottam, T. W. (1993) Clin. Pharmacol. Ther. 53, D. P. (1988) Arch. Biochem. Biophys. 263, 424-436. 544-554. 6. Watkins, P. B., Murray, S. A., Winkelman, L. G., Heuman, 30. Wang, C. P., Hartman, N. R., Venkataramanan, R., Jardine, D. M., Wrighton, S. A. & Guzelian, P. S. (1989) J. Clin. Invest. I., Lin, F. T., Knapp, J. E., Starzl, T. E. & Burckart, G. J. 83, 688-697. (1989) Drug Metab. Dispos. 17, 292-2%. 7. Watkins, P. B., Turgeon, D. K., Saenger, P., Lown, K. S., 31. Kunzendorf, U., Brockmoller, J., Jochimsen, F., Roots, I. & Kolars, J. C., Hamilton, T., Fishman, K., Guzelian, P. S. & Offermann, G. (1990) Transplant Proc. 22, 1697-1699. Voorhees, J. J. (1992) Pharmacol. Ther. 52, 265-273. 32. Lown, K., Kolars, J., Turgeon, K., Merion, R., Wrighton, 8. Elshourbagy, N. A. & Guzelian, P. S. (1980) J. Biol. Chem. S. A. & Watkins, P. B. (1992) Clin. Pharmacol. Ther. 51, 255, 1279-1285. 229-238. 9. Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., 33. Olkkola, K. T., Aranko, K., Luurila, H., Huller, A., Saar- Young, B. & Guzelian, P. S. (1985) Biochemistry 24, 2171- nivaara, L., Himberg, J. J. & Neuvonen, P. J. (1993) Clin. 2178. Pharmacol. Ther. 53, 298-305. 10. Imaoka, S., Imai, Y., Shimada, T. & Funae, Y. (1992) Bio- 34. Kronbach, T., Mathys, D., Umeno, M., Gonzalez, F. J. & chemistry 31, 6063-6069. Meyer, U. A. (1989) Mol. Pharmacol. 36, 89-96. Downloaded by guest on September 30, 2021