[Sll
SITE O F A C T I O N O F b s - R E Q U I R I N G S U B S T R A T E S
523
[51] Site of Action of Substrates Requiring Cytochrome b s for Oxidation by Cytochrome P450 B y LucY A.
WASKELL,J. L. VIGNE, and G. VERGERES
Introduction Cytochrome b5 (bs) is a membrane-bound protein of the endoplasmic reticulum which in vivo provides electrons for the biosynthesis of a number of lipids including cholesterol? In addition to furnishing electrons for lipid biosynthesis, b5 can donate the second but not the first electron to cytochrome P450. ~The role of b5 in cytochrome P450-catalyzed oxidations has been investigated in detail using purified proteins in a reconstituted system. Using purified cytochrome P450, cytochrome-P450 reductase, and bs, investigators have demonstrated that b5 may inhibit, stimulate, or have no effect on the metabolism of various substrates. 3-7 Studies by Brsteding et al. 4 have helped to clarify these apparently conflicting results. A 200% stimulation of benzphetamine (BP) N-demethylation by b5 was observed at a P450 reductase-P450 ratio similar to that in microsomes (1 : 10) but only a 20% stimulation at a ratio of 1 : 1. The second conclusion of Brsterling et al. 4 was that at lipid-protein ratios less than 50: 1, the addition of b5 caused inhibition of benzphetamine metabolism. This finding was explained in terms of a model in which inhibition of cytochrome P450mediated reactions by b 5 results from changes of phospholipid-protein interactions. It has also been noted that b 5 can increase the coupling of N A D P H oxidation to substrate hydroxylation. 5,8 In addition to a facilitatory role, b 5 has an obligatory, that is, markedly stimulating, role in the cytochrome P450-mediated metabolism of a number of exogenous (methoxyflurane, halothane, p-nitroanisole, n-methylcarbazole, chlorobenzene, p-nitrophenetole, 7-ethoxycoumarin, benzo[a]py-
i N. Oshino, in "Hepatic Cytochrome P-450 Mono-OxygenaseSystem" (J. B. Schenkman and D. Kupfer, eds.), p. 407. Pergamon, New York, 1982. 2 A. Hildebrandt and R. W. Estabrook, Arch. Biochem. Biophys. 143, 66 (1971). 3 D. Gorsky and M. J. Coon, Drug Metab. Dispos. 14, 89 (1986). 4 B. Brsterling, J. R. Trudell, A. J. Trevor, and M. Bendix, J. Biol. Chem. 257, 4375 (1982). 5 M. Ingeiman-Sundbergand I. Johansson, Biochem. Biophys. Res. Commun. 97,582 (1980). 6 E. T. Morgan and M. J. Coon, Drug Metab. Dispos. 12, 358 (1984). 7 E. Canova-Davis and L. Waskell, J. Biol. Chem. 259, 2541 (1984). s y. Imai and R. Sato, Biochem. Biophys. Res. Commun. 75, 420 (1977). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
524
ENZYME ASSAYS
[51]
rene, and endogenous substrates (lauric acid, prostaglandins). 7,9,1° The requirement for b~ in the reconstituted drug oxidation system is very specific and is dependent on both the substrate and isozyme ofcytochrome P450. Thus, the compounds listed will be dependent on b5 for metabolism by only one of the several forms of cytochrome P450 which catalyze oxidation of the indicated compound. The in vivo function of the requirement for b5 for the metabolism of natural substrates may be a mechanism by which the redox state of the cell (as reflected by the concentration of NADPH and NADH) regulates the biodegradation and/or biosynthesis of vital endogenous compounds, whereas the physiological consequences of a requirement for b5 for the metabolism of some exogenous substrates may be disruption of lipid biosynthesis which ultimately results in hepatotoxicity. This chapter describes experiments aimed at elucidating the detailed molecular explanation for the observation that a given cytochrome P450 (LM2) may require b5 for the metabolism of one substrate (methoxyflurane) but not another (benzphetamine) and emphasizes the rationale of our experiments while providing only a minimum of the experimental details, which, in any case, would probably not apply to other laboratories owing to the marked variability in optimal conditions in the reconstituted system with different substrates and cytochromes P450. The experiments are designed to answer the question, Is the requirement for b 5 for the metabolism of some substrates due to their ability to be allosteric effectors of the reconstituted system or due to an interaction of the substrate at the substrate-binding site of cytochrome P450? This question is addressed with two series of experiments, essentially as previously described.11 The first experiments investigate whether the presence of the bs-dependent substrate can induce a requirement for b5 for the metabolism of a substrate which typically does not utilize b5. The second series of experiments investigate whether the bs-dependent and bsindependent substrates are competitive inhibitors in the absence of b5 and competing alternate substrates in the presence of bs, thereby eliminating the possibility that the bs-dependent substrate is an aUosteric effector of the reconstituted system. These experiments are performed in a reconstituted system using three purified proteins: cytochrome P450, cytochrome-P450 reductase, and bs .~l Cytochrome P450 LM2 and cytochrome-P450 reductase are purified according to previously described procedures.12-14 Cyto9 K. P. Vatsis, A. D. Theoharides, D. Kupfer, and M. J. Coon, J. Biol. Chem. 257, 11221 (1982). J0 H. A. Sasame, S. S. Thorgeirsson, J. R. Mitchell, and J. Gillette, Life Sci. 14, 35 (1974). II j. j. Lipka and L. A. Waskell, Arch. Biochem. Biophys. 268, 152 (1989). 12 D. A. Haugen and M. J. Coon, J. Biol. Chem. 251, 7929 (1976). 13 j. French and M. Coon, Arch. Biochem. Biophys. 195, 565 (1979).
[51]
SITE OF ACTION OF bs-REQUIRING SUBSTRATES
525
chrome b5 is currently purified in the authors' laboratory as a by-product from the same rabbit liver microsomal preparation used to purify cytochrome P450 LM2 and P450 reductase using a modification of the procedure of Carlsen, z4awhich in our laboratory did not yield homogeneous bs. The procedure to be described yields b 5 at a specific content of 50-55 nmol/mg protein versus other schemes yielding ---30 nmol/mg. Partial purification prior to loading the TSK column was found to be necessary to obtain b5 with the specific content indicated. Methods
Assays The concentration of purified b5 is ascertained from the absorbance at 413 nm using an extinction coefficient of 117 mM -1 c m - l ) 5 In cruder preparations the b5 content is estimated from the reduced versus oxidized spectra.16 Protein concentration is determined by the method of Lowry et a l ) 7 Formaldehyde resulting from benzphetamine and methoxyflurane metabolism is measured by the method of Nash) 8
Cytochrome b5 Purification: Microsome Preparation and DEAE Column Rabbits are induced with phenobarbital (PB), and the liver microsomes are isolated as previously described. ~2All procedures are carried out at 4°. The microsomes are solubilized by adding dropwise a 10% (w/v) Tergitol solution to a final detergent-to-protein (w/w) ratio of 1. The solution is stirred for 2 hr and then centrifuged at 10,000 rpm (17,500 g) for 1 hr in a JA-10 rotor of a Beckman (FuUerton, CA) centrifuge (Model J-21B). The red-brown solution is applied to a DE-52 Whatman (Clifton, N J) cellulose column (5 liters, 10 × 65 cm; 0.8 mg protein/ml DE-52) previously equilibrated with buffer A [10 mM Tris-acetate, pH 7.4, at 25°, 1 mM EDTA, 20% glycerol (v/v), and 0.5% Tergitol (w/v)]. The flow-through which contains several cytochromes P450 is collected, and the column is subsequently washed with the same buffer until the optical density (OD) at 413 14 M. J. Coon, T. A. van der Hoeven, S. B. Dahl, and D. A. Haugen, this series, Vol. 52, p. 109. 14a j. Carlsen, K. Christiansen, and H. M. Jensen, Biochem. J. 256, 1051 (1988). 15 p. Strittmatter and S. F. Velick, J. Biol. Chem. 221~ 253 (1956). J6 R. W. Estabrook and J. Werringloer, this series, Vol. 52, p. 212. 17 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). is T. Nash, Biochem. J. 55, 416 (1953).
526
ENZYME ASSAYS
[51]
TABLE I PURIFICATION OF CYTOCHROME b 5
Protein Specific concentration Volume contentof b5 Yield Purification (mg/ml) (ml) (nmol/mgprotein) (%) (-fold)
Sample PB-induced microsomes Solubilized microsomes 0.15 M KCI DE-52 eluate TSK DEAE eluate G-75 eluate
nm decreases chromes P450 tions with an against buffer (see Table I).
30
800
0.8
100
4
5000
0.9
94
1.1
1.2
9000
1.52
86
1.9
1 1.5
550 64
67 27
29.4 66.0
23.5 53.0
1
to 0. I. Cytochrome b5 as well as some additional cytoare then eluted with 0.15 M KCI in the same buffer. FracOD at 413 nm of 0.1 or greater are pooled and dialyzed B (10 mM Tris-acetate, pH 8.1, at 22° and 1 mM EDTA)
TSK-Gel Toyopearl DEAE-650 (S) Dialyzed bs-containing fractions (----11 gm protein) are loaded onto a 500-ml, 2.5 × 100 cm, 20-50 tzm TSK column (Supelco, Inc., Bellefonte, PA) previously equilibrated with buffer B. A pump is required to perform all chromatography steps on the TSK column. After loading the column is washed extensively with buffer C [5 mM HEPES, pH 7.4, 20 mM NaF, 20% glycerol, 0.05% Lubrol (w/v)] until no further protein can be detected (OD at 280 nm
SITE O F A C T I O N O F b s - R E Q U I R I N G S U B S T R A T E S
523
[51] Site of Action of Substrates Requiring Cytochrome b s for Oxidation by Cytochrome P450 B y LucY A.
WASKELL,J. L. VIGNE, and G. VERGERES
Introduction Cytochrome b5 (bs) is a membrane-bound protein of the endoplasmic reticulum which in vivo provides electrons for the biosynthesis of a number of lipids including cholesterol? In addition to furnishing electrons for lipid biosynthesis, b5 can donate the second but not the first electron to cytochrome P450. ~The role of b5 in cytochrome P450-catalyzed oxidations has been investigated in detail using purified proteins in a reconstituted system. Using purified cytochrome P450, cytochrome-P450 reductase, and bs, investigators have demonstrated that b5 may inhibit, stimulate, or have no effect on the metabolism of various substrates. 3-7 Studies by Brsteding et al. 4 have helped to clarify these apparently conflicting results. A 200% stimulation of benzphetamine (BP) N-demethylation by b5 was observed at a P450 reductase-P450 ratio similar to that in microsomes (1 : 10) but only a 20% stimulation at a ratio of 1 : 1. The second conclusion of Brsterling et al. 4 was that at lipid-protein ratios less than 50: 1, the addition of b5 caused inhibition of benzphetamine metabolism. This finding was explained in terms of a model in which inhibition of cytochrome P450mediated reactions by b 5 results from changes of phospholipid-protein interactions. It has also been noted that b 5 can increase the coupling of N A D P H oxidation to substrate hydroxylation. 5,8 In addition to a facilitatory role, b 5 has an obligatory, that is, markedly stimulating, role in the cytochrome P450-mediated metabolism of a number of exogenous (methoxyflurane, halothane, p-nitroanisole, n-methylcarbazole, chlorobenzene, p-nitrophenetole, 7-ethoxycoumarin, benzo[a]py-
i N. Oshino, in "Hepatic Cytochrome P-450 Mono-OxygenaseSystem" (J. B. Schenkman and D. Kupfer, eds.), p. 407. Pergamon, New York, 1982. 2 A. Hildebrandt and R. W. Estabrook, Arch. Biochem. Biophys. 143, 66 (1971). 3 D. Gorsky and M. J. Coon, Drug Metab. Dispos. 14, 89 (1986). 4 B. Brsterling, J. R. Trudell, A. J. Trevor, and M. Bendix, J. Biol. Chem. 257, 4375 (1982). 5 M. Ingeiman-Sundbergand I. Johansson, Biochem. Biophys. Res. Commun. 97,582 (1980). 6 E. T. Morgan and M. J. Coon, Drug Metab. Dispos. 12, 358 (1984). 7 E. Canova-Davis and L. Waskell, J. Biol. Chem. 259, 2541 (1984). s y. Imai and R. Sato, Biochem. Biophys. Res. Commun. 75, 420 (1977). METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
524
ENZYME ASSAYS
[51]
rene, and endogenous substrates (lauric acid, prostaglandins). 7,9,1° The requirement for b~ in the reconstituted drug oxidation system is very specific and is dependent on both the substrate and isozyme ofcytochrome P450. Thus, the compounds listed will be dependent on b5 for metabolism by only one of the several forms of cytochrome P450 which catalyze oxidation of the indicated compound. The in vivo function of the requirement for b5 for the metabolism of natural substrates may be a mechanism by which the redox state of the cell (as reflected by the concentration of NADPH and NADH) regulates the biodegradation and/or biosynthesis of vital endogenous compounds, whereas the physiological consequences of a requirement for b5 for the metabolism of some exogenous substrates may be disruption of lipid biosynthesis which ultimately results in hepatotoxicity. This chapter describes experiments aimed at elucidating the detailed molecular explanation for the observation that a given cytochrome P450 (LM2) may require b5 for the metabolism of one substrate (methoxyflurane) but not another (benzphetamine) and emphasizes the rationale of our experiments while providing only a minimum of the experimental details, which, in any case, would probably not apply to other laboratories owing to the marked variability in optimal conditions in the reconstituted system with different substrates and cytochromes P450. The experiments are designed to answer the question, Is the requirement for b 5 for the metabolism of some substrates due to their ability to be allosteric effectors of the reconstituted system or due to an interaction of the substrate at the substrate-binding site of cytochrome P450? This question is addressed with two series of experiments, essentially as previously described.11 The first experiments investigate whether the presence of the bs-dependent substrate can induce a requirement for b5 for the metabolism of a substrate which typically does not utilize b5. The second series of experiments investigate whether the bs-dependent and bsindependent substrates are competitive inhibitors in the absence of b5 and competing alternate substrates in the presence of bs, thereby eliminating the possibility that the bs-dependent substrate is an aUosteric effector of the reconstituted system. These experiments are performed in a reconstituted system using three purified proteins: cytochrome P450, cytochrome-P450 reductase, and bs .~l Cytochrome P450 LM2 and cytochrome-P450 reductase are purified according to previously described procedures.12-14 Cyto9 K. P. Vatsis, A. D. Theoharides, D. Kupfer, and M. J. Coon, J. Biol. Chem. 257, 11221 (1982). J0 H. A. Sasame, S. S. Thorgeirsson, J. R. Mitchell, and J. Gillette, Life Sci. 14, 35 (1974). II j. j. Lipka and L. A. Waskell, Arch. Biochem. Biophys. 268, 152 (1989). 12 D. A. Haugen and M. J. Coon, J. Biol. Chem. 251, 7929 (1976). 13 j. French and M. Coon, Arch. Biochem. Biophys. 195, 565 (1979).
[51]
SITE OF ACTION OF bs-REQUIRING SUBSTRATES
525
chrome b5 is currently purified in the authors' laboratory as a by-product from the same rabbit liver microsomal preparation used to purify cytochrome P450 LM2 and P450 reductase using a modification of the procedure of Carlsen, z4awhich in our laboratory did not yield homogeneous bs. The procedure to be described yields b 5 at a specific content of 50-55 nmol/mg protein versus other schemes yielding ---30 nmol/mg. Partial purification prior to loading the TSK column was found to be necessary to obtain b5 with the specific content indicated. Methods
Assays The concentration of purified b5 is ascertained from the absorbance at 413 nm using an extinction coefficient of 117 mM -1 c m - l ) 5 In cruder preparations the b5 content is estimated from the reduced versus oxidized spectra.16 Protein concentration is determined by the method of Lowry et a l ) 7 Formaldehyde resulting from benzphetamine and methoxyflurane metabolism is measured by the method of Nash) 8
Cytochrome b5 Purification: Microsome Preparation and DEAE Column Rabbits are induced with phenobarbital (PB), and the liver microsomes are isolated as previously described. ~2All procedures are carried out at 4°. The microsomes are solubilized by adding dropwise a 10% (w/v) Tergitol solution to a final detergent-to-protein (w/w) ratio of 1. The solution is stirred for 2 hr and then centrifuged at 10,000 rpm (17,500 g) for 1 hr in a JA-10 rotor of a Beckman (FuUerton, CA) centrifuge (Model J-21B). The red-brown solution is applied to a DE-52 Whatman (Clifton, N J) cellulose column (5 liters, 10 × 65 cm; 0.8 mg protein/ml DE-52) previously equilibrated with buffer A [10 mM Tris-acetate, pH 7.4, at 25°, 1 mM EDTA, 20% glycerol (v/v), and 0.5% Tergitol (w/v)]. The flow-through which contains several cytochromes P450 is collected, and the column is subsequently washed with the same buffer until the optical density (OD) at 413 14 M. J. Coon, T. A. van der Hoeven, S. B. Dahl, and D. A. Haugen, this series, Vol. 52, p. 109. 14a j. Carlsen, K. Christiansen, and H. M. Jensen, Biochem. J. 256, 1051 (1988). 15 p. Strittmatter and S. F. Velick, J. Biol. Chem. 221~ 253 (1956). J6 R. W. Estabrook and J. Werringloer, this series, Vol. 52, p. 212. 17 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). is T. Nash, Biochem. J. 55, 416 (1953).
526
ENZYME ASSAYS
[51]
TABLE I PURIFICATION OF CYTOCHROME b 5
Protein Specific concentration Volume contentof b5 Yield Purification (mg/ml) (ml) (nmol/mgprotein) (%) (-fold)
Sample PB-induced microsomes Solubilized microsomes 0.15 M KCI DE-52 eluate TSK DEAE eluate G-75 eluate
nm decreases chromes P450 tions with an against buffer (see Table I).
30
800
0.8
100
4
5000
0.9
94
1.1
1.2
9000
1.52
86
1.9
1 1.5
550 64
67 27
29.4 66.0
23.5 53.0
1
to 0. I. Cytochrome b5 as well as some additional cytoare then eluted with 0.15 M KCI in the same buffer. FracOD at 413 nm of 0.1 or greater are pooled and dialyzed B (10 mM Tris-acetate, pH 8.1, at 22° and 1 mM EDTA)
TSK-Gel Toyopearl DEAE-650 (S) Dialyzed bs-containing fractions (----11 gm protein) are loaded onto a 500-ml, 2.5 × 100 cm, 20-50 tzm TSK column (Supelco, Inc., Bellefonte, PA) previously equilibrated with buffer B. A pump is required to perform all chromatography steps on the TSK column. After loading the column is washed extensively with buffer C [5 mM HEPES, pH 7.4, 20 mM NaF, 20% glycerol, 0.05% Lubrol (w/v)] until no further protein can be detected (OD at 280 nm
