- Email: [email protected]
[20] Reconstitution of the cytochrome P-450-containing mixed-function oxidase system of liver microsomes
200
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[20]
naphthalene 1,2-oxide, phenanthrene 9,10-oxide, benz[a]anthracene 5,6oxide, 3-methylcholanthrene ll,12-oxide, dibenz[a,h]anthracene 5,6oxide, and benzo[a]pyrene 4,5-, 7,8-, 9,10-, and ll,12-oxides. The assay of these substrates is facilitated by the development of a sensitive and rapid thin-layer chromatographic assay based on the separation of radioactive substrates and their dihydrodiol products on silica gel. 11 Thus, like the liver microsomal cyrochrome P-450-dependent hydroxylation system, epoxide hydrase also has a very broad substrate specificity. Activators and Inhibitors. Metal-chelating agents (EDTA and a,ot'dipyridyl) have no effect on purified epoxide hydrase activity while sulfhydryl-binding agents (iodoacetamide and p-hydroxymercuribenzoate) at 0.1 mM only slightly inhibit the reaction. Metyrapone and 1-(2isopropylphenyl)imidazole enhance the rate of the reaction; maximal activation (220%) is reached at 1.5 mM using styrene oxide as substrate. Ethanol at a final concentration of 5% and 10% inhibits the reaction 33% and 86%, respectively. High concentrations of Emulgen 911 also inhibit epoxide hydrase activity. For example, at a final concentration of 0.05% and 0.1%, Emulgen 911 inhibits the rate of reaction 25% and 53%, respectively. n D. M. Jerina, P. M. Dansette, A. Y. H. Lu, and W. Levin, Mol. Pharmacol. 13, 342 (1977).
[20] R e c o n s t i t u t i o n o f t h e C y t o c h r o m e P - 4 5 0 - C o n t a i n i n g Mixed-Function Oxidase System of Liver Microsomes B y M I N O R J . COON
Cytochrome P-450 is the predominant enzyme in the membranes of liver cells, for it occurs at levels of approximately 10% of the microsomal protein, and the endoplasmic reticulum from which the microsomes are derived represents as much as 90% of the total membranes. This pigment is an unusually versatile catalyst, as shown by its role in hydroxylation and other chemical reactions involving a variety of substrates: drugs, anesthetics, petroleum products, insecticides, carcinogens, and numerous other foreign compounds, as well as naturally occurring substances such as fatty acids and steroids.1 Liver microsomal cytochrome P-450 (P-450LM) has been characterized spectrally as a I j. R. Gillette, Adv. Pharmacol. 4, 219 (1966).
[20]
R E C O N S T I T U T I OOF N THE CYTOCHROME P-450
201
cytochrome of the b type, 2 but early attempts at solubilization and isolation yielded an altered heme protein, cytochrome P-420, without biological activity? During the course of studies on fatty acid to-hydroxylation by liver microsomes, the mixed-function oxidase system was solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable factor. 4"5 A reconstituted system prepared from these components and supplemented with NADPH was shown to be active under aerobic conditions in the hydroxylation of fatty acids, 4-6 d r u g s , 7 and a variety of other compounds. 8 Resolution of the Microsomal E n z y m e System Livers from male rabbits which have been fasted 24 hr are minced with scissors and homogenized with 4 volumes of 0.25 M sucrose in a Waring Blendor for 2 min at 4 °. The following operations are carried out at the same temperature. The homogenate is centrifuged for 25 min at 10,000 g, and the supernatant fluid is filtered through two layers of cheesecloth. The centrifugation at low speed is repeated, and the supernatant layer is then centrifuged at 105,000 g for 2 hr. The supernatant fluid is discarded, and the lipid adhering to the sides of the tubes is removed. The translucent red microsomal pellet is suspended in 0.25 M sucrose in a Potter-Elvehjem homogenizer and stored in the frozen state. In some experiments, particularly when it is desired to obtain a P-450LM preparation with minimal contamination by hemoglobin, the liver is homogenized in 1.15% KC1 and the microsomal pellet is resuspended in isotonic KCI and again precipitated by centrifugation. The resulting microsomes are then suspended in 0.25 M sucrose at a final protein concentration of about 30 mg per milliliter. A microsomal suspension containing 1.95 g of protein in 65 ml of sucrose solution is stirred at 4 ° for 20 min with a solution made by mixing 44.5 ml of glycerol, 14.8 ml of 1.0 M potassium citrate buffer (pH z T. Omura and R. Sato, J. Biol. Chem. 239, 2370 (1964). T. Omura and R. Sato, J. Biol. Chem. 237, PC1375 (1962). 4A. Y. H. Lu and M. J. Coon, J. Biol. Chem. 243, 1331 (1968). 5 M. J. Coon and A. Y. H. Lu, in "Microsomes and Drug Oxidations" (J. R. Gillette et al., eds.), p. 151. Academic Press, New York, 1969. 6A. Y. H. Lu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 244, 3714 (1%9). r A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Biochem. Biophys. Res. Commun. 36, 545 (1969). A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Mol. Pharmacol. 6, 213 (1970).
202
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[20]
7.6), 14.8 ml of 1.0 M KCI, 1.5 ml of 0.10 M dithiothreitol, and 7.6 ml of 10% sodium deoxycholate, and the precipitate formed upon centrifugation for 2 hr at 105,000 g is discarded. In typical experiments the supernatant layer contains at least 90% of the protein and 50% of the fatty acid to-hydroxylation activity originally present in the microsomes. Deoxycholate, glycerol, and citrate appear to be necessary for optimal solubilization of the enzyme system, whereas dithiothreitol is added as a precautionary measure to avoid the oxidation of enzyme sulfhydryl groups; KCI is added to raise the ionic strength, thereby enhancing the activity of the reductase. Other buffers, such as pyrophosphate, glycine, Tris, and phosphate are much less effective than citrate. The microsomal extract is filtered through glass wool to give a clear, faintly reddish solution having a protein concentration of 12 mg/ml, diluted with 3 volumes of deionized water, and put onto a DEAE-cellulose column (5 × 45 cm) previously equilibrated with 0.1 M Tris buffer, pH 7.7, containing 0.1 mM dithiothreitol and 0.05% deoxycholate. The column is washed with 880 ml of the same buffer mixture containing 0.10 M KCI, thereby removing hemoglobin. A reddish yellow fraction containing P450LM is then eluted with 1 liter of buffer mixture containing 0.20 M KCI, while NADPH-cytochrome P-450 reductase is eluted with 1 liter of a similar solution containing 0.30 M KCI. Finally, a fraction containing the heat-stable factor is eluted from the column with 1.5 liters of a similar solution containing 0.50M KCI. This stepwise elution gives better results than the salt gradient used previously. 4 This general procedure for solubilizing and separating cytochrome P450, the reductase, and the heat-stable component (subsequently shown to be phosphatidylcholine 9) has been used successfully for the resolution of the mixed function oxidase system of rat, s mouse, 1° and human, 1~ as well as rabbit liver microsomes. In addition, it has been used to resolve a similar cytochrome P-450-containing enzyme system in a yeast, Candida tropicalis. ~2
Purification and Characterization of Components of the Mixed Function Oxidase System Studies on the reconstitution of the mixed-function oxidase system have been aided greatly by the availability of purified, well-characterized 9 H. W. Strobel, A. Y. H. Lu, J. Heidema, and M. J. Coon, J. Biol. Chem. 245, 4851 (1970). lo D. W. Nebert, J. K. Heidema, H. W. Strobel, and M. J. Coon, J. Biol. Chem. 248, 7631 (1973). 11R. M. Kaschnitz and M. J. Coon, Biochem. Pharmacol. 24, 295 (1975). 12 W. Duppel, J. M. Lebeauit, and M. J. Coon, Fur. J. Biochem. 36, 583 (1973).
[20]
R E C O N S T I T U T I OOF N THE CYTOCHROME P-450
203
components. The chief benefit is that purified reductase (from rat liver) and synthetically prepared phospholipid may be made and stored for subsequent reconstitution experiments, either with various forms of P450LM or with cytochrome P-450 from other organelles, tissues, or species. As a result, optimal conditions need only be devised for the solubilization and stabilization of the cytochrome P-450 fraction from a particular source, rather than for the reductase and phospholipid fractions as well. These comments apply, of course, only to systems which, like that in liver microsomes, do not require a nonheme iron protein. C y t o c h r o m e P-450. Recently, evidence has been obtained for the occurrence of multiple forms of P-450LM. la Two forms of the cytochrome, phenobarbital-inducible P-450LM2 and 5,6-benzoflavone-inducible P-450LM4, have been purified from rabbit liver microsomes in the presence of Renex 690, a nonionic detergent. 14"1s These two cytochromes are homogeneous by the criteria of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, quantitative determination of the C-terminal amino acid residues, and Ouchterlony double-diffusion analysis carried out with the corresponding antisera. T M Highly purified P-450LM has also been isolated from 3-methylcholanthrene-treated rats and rabbits and from phenobarbital-treated rats by Ryan et al. 17 and Kawalek et al. ~8 and from phenobarbital- and 3-methylcholanthrenetreated rabbits by Imai and Sato TM and Hashimoto and Imai. 2° All these methods employ detergent solubilization of the cytochromes, but the purification precedures are different. In our experience, cytochrome P450 is stable at all stages of purification in the presence of 20% glycerol. Excess detergent (Renex 690) is removed from highly purified P450LMz and LM4 by treatment with Amberlite XAD-2 and calcium phosphate gel. TM The resulting preparations are soluble in aqueous buffers, though P-450LM4 requires a higher ionic strength (for example, phosphate buffer at least 0.15 M) than does P-450LM2. N A D P H - - C y t o c h r o m e P-450 R e d u c t a s e . The detergent-solubilized liver microsomal NADPH--cytochrome c reductase retains the ability to reduce cytochrome P-450, a its natural substrate, and therefore to funcla D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, J. Biol. Chem. 250, 3567 (1975). ~4 M. J. Coon, T. A. van der Hoeven, S. B. Dahl, and D. A. Haugen, this volume [10]. 1~ D. A. Haugen and M. J. Coon, J. Biol. Chem. 251, 7929 (1976). 16 W. L. Dean, Doctoral thesis, University of Michigan, Ann Arbor, 1976. lr D. Ryan, A. Y. H. Lu, J. Kawalek, S. B. West, and W. Levin, Biochem. Biophys. Res. Commun. 64, 1134 (1975). 18 j. C. Kawalek, W. Levin, D. Ryan, P. E. Thomas, and A. Y. H. Lu, Mol. Pharmacol. 11,874 (1975). 19 y . Imai and R. Sato, Biochem. Biophys. Res. Commun. 60, 8 (1974). z0 C. Hashimoto and Y. Imai, Biochem. Biophys. Res. Commun. 68, 821 (1976).
204
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[20]
tion in the reconstituted hydroxylation system. 6 In contrast, N A D P H cytochrome c reductase preparations purified after solubilization with a protease such as bromelain 21 or with a lipase ~2 believed to contain a protease 2a are not active toward cytochrome P-450. z4 We have obtained this flavoprotein in a highly purified state from rat liver microsomes after solubilization with detergent, 25 as have Dignam and Strobe126"2r and Yasukochi and Masters. zs Such preparations may be used to reconstitute an active hydroxylating system with cytochrome P-450 from a variety of sources, including rabbit, mouse, and human, as well as rat liver microsomes, and also yeast, thereby demonstrating the broad specificity of this flavoprotein. 7.10-~2
Phospholipid. After the heat-stable component had been identified as phosphatidylcholine, a variety of synthetically prepared compounds were tested for their ability to function in the reconstituted system, a Dilauroylglyceryl-3-phosphorylcholine proved to be one of the most effective and is presently used routinely because it does not readily undergo peroxidation, unlike phospholipids containing polyunsaturated fatty acids. The dilauroyl compound has been used to reconstitute the cytochrome P-450 containing enzyme systems of a variety of species. r'8"1°-~2 The phospholipid may be partially replaced for this purpose by synthetic detergents, and for optimal results we use both, as indicated in the procedure given below. Procedure for Reconstitution and Assay of Hydroxylation Activity
Principle. The assay is based on spectrophotometric determination of the rate of NADPH oxidation in the reconstituted system in the presence of a compound such as cyclohexane, which is one of the most active substrates for P - 4 5 0 L M 2. Saturating concentrations of phosphatidylcholine and purified NADPH-cytochrome P-450 reductase are added so that the cytochrome is the rate-limiting component. (As an alternative assay 21 S. D. Aust, D. L. Rorrig, and T. C. Pederson, Biochem. Biophys. Res. Commun. 47, 1133 (1972). 22 B. S. S. Masters, C. H. Williams, Jr., and H. Kamin, this series, Vol. 10, p. 565. 23 j. A. Buege and S. D. Aust, Biochim. Biophys. Acta 286, 433 (1972). 24 M. J. Coon, H. W. Strobel, and R. F. Boyer, Drug Metab. Disp. 1, 92 (1973). z5 j. L. Vermilion and M. J. Coon, Biochem. Biophys. Res. Commun. 60, 1315 (1974). 26 j. D. Dignam and H. W. Strobel, Biochem. Biophys. Res. Commun. 63, 845 (1975). 27 j. D. Dignam and H. W. Strobel, Biochemistry 16, 1116 (1977). 28 y . Yasukochi and B. S. S. Masters, J. Biol. Chem., 251, 5337 (1976).
[20]
RECONSTITUTION OF THE CYTOCHROME P - 4 5 0
205
procedure, the formaldehyde liberated by the demethylation of benzphetamine may be determined by a colorimetric method.2~'3°)
Reagents Sodium HEPES buffer, 0.5 M, pH 7.4 MgClz, 0.1 M Sodium deoxycholate, 0.5% Cyclohexane, 1.0 M in methanol NADPH, 10 mM P-450LM2 (from rabbit liver), 5 /zM solution (as determined by spectral assay of the reduced CO complex TM) NADPH-cytochrome P-450 reductase (from rat or rabbit liver), 20 units/ml Dilauroylglyceryl-3-phosphorylcholine, 0.1% aqueous suspension (sonicated)
Procedure. The assay components are added to 1-ml cuvettes with a 1-cm light path. The sample of P-450LM2 (0.02 ml), NADPH-cytochrome P-450 reductase (0.03 ml), and the phosphatidylcholine (0.03 ml) are first added, mixed, and allowed to stand for 1 min at room temperature. 31 The remaining components are then added in the order given: 0.10 ml of HEPES buffer, 0.15 ml of MgC12, 0.62 ml of water, 0.02 ml of deoxycholate, and 0.01 ml of the cyclohexane solution (replaced by 0.01 ml of methanol in the blank cell). The mixture is incubated for 3 min at 30 °, and the reaction is then initiated by the addition of 0.02 ml of NADPH to each cell. The rate of disappearance of NADPH is determined at 30 ° in a Gilford spectrophotometer equipped with a multiple sample absorbance recorder. The rate of the reaction remains constant for at least 5 min. Hydroxylation Activity. The activities are expressed as turnover numbers, i.e., moles of substrate hydroxylated per mole of cytochrome P-450 per minute. Such values are readily determined in the reconstituted system, in which cytochrome P-450 is easily made the rate-limiting component. In contrast, the apparent turnover numbers reported for various substrates in liver microsomal suspensions are usually lower, which suggests that cytochrome P-450 may not be the rate-limiting ~9 T. N a s h , Biochem. J. 55, 416 (1953). 3o j. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther. 125, 105 (1959). :~1 If the order of addition is c h a n g e d so the P-450LM, N A D P H - c y t o c h r o m e P-450 reductase, or phosphatidylcholine is added after the reaction mixture has been diluted with water and the other reagents, the activity m a y be reduced to lfr% or less.
206
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[21]
component in the membrane. A typical turnover number for cyclohexane with P-450LM2 in the reconstituted system under the conditions described is 59. The activity of several purified forms of rabbit liver microsomal cytochrome P-450 toward a variety of substrates, including drugs, aniline, p-nitroanisole, biphenyl, testosterone, and benzo[a]pyrene, indicates that these cytochromes apparently hydroxylate all such compounds, but that the relative rates and positional specificities are somewhat different. 13"32 32 F. J. Wiebel, J. K. Selkirk, H. V. Geiboin, D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 72, 3917 (1975).
[21] I n c o r p o r a t i o n o f M i c r o s o m a l E l e c t r o n - T r a n s f e r C o m p o n e n t s into L i p o s o m e s : C o n s i d e r a t i o n s for Diffusion-Limited Reactions B y PHILIPP STRITTMATTER, HARRY
G. E N O C H , and PATRICK F L E M I N G
The isolation of the three enzyme components of the stearyl-CoA desaturase system, i.e., cytochrome b5 reductase, 1 cytochrome b5 (see this volume [8]) and the desaturase (see this volume [18]), provided the protein components for studies on the binding of each to microsomes or vesicles of defined phospholipid composition, and the reconstitution of a partial or complete electron transport system from NADH to the terminal desaturase. In all cases, the rates of binding of the proteins to vesicles appears to be limited by the fact that these relatively nonpolar enzymes form stable aggregates in solution, thereby producing an extremely low concentration of monomeric species, the forms that appear to insert into the bilayer structure. The procedures described below have therefore employed either relatively lengthy incubations of the proteins with preformed phospholipid vesicles, incubation of vesicles with combinations of two proteins to produce less-stable mixed-protein aggregates, or binding in the presence of detergent followed by removal of the detergent by gel filtration. With such vesicle preparations, both partial and complete electrontransport sequences have been prepared to examine protein-protein, protein-lipid, and diffusion-dependent interactions. 1 L. Spatz and P. Strittmatter, J. Biol. Chem. 248, 793 (1973).
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[20]
naphthalene 1,2-oxide, phenanthrene 9,10-oxide, benz[a]anthracene 5,6oxide, 3-methylcholanthrene ll,12-oxide, dibenz[a,h]anthracene 5,6oxide, and benzo[a]pyrene 4,5-, 7,8-, 9,10-, and ll,12-oxides. The assay of these substrates is facilitated by the development of a sensitive and rapid thin-layer chromatographic assay based on the separation of radioactive substrates and their dihydrodiol products on silica gel. 11 Thus, like the liver microsomal cyrochrome P-450-dependent hydroxylation system, epoxide hydrase also has a very broad substrate specificity. Activators and Inhibitors. Metal-chelating agents (EDTA and a,ot'dipyridyl) have no effect on purified epoxide hydrase activity while sulfhydryl-binding agents (iodoacetamide and p-hydroxymercuribenzoate) at 0.1 mM only slightly inhibit the reaction. Metyrapone and 1-(2isopropylphenyl)imidazole enhance the rate of the reaction; maximal activation (220%) is reached at 1.5 mM using styrene oxide as substrate. Ethanol at a final concentration of 5% and 10% inhibits the reaction 33% and 86%, respectively. High concentrations of Emulgen 911 also inhibit epoxide hydrase activity. For example, at a final concentration of 0.05% and 0.1%, Emulgen 911 inhibits the rate of reaction 25% and 53%, respectively. n D. M. Jerina, P. M. Dansette, A. Y. H. Lu, and W. Levin, Mol. Pharmacol. 13, 342 (1977).
[20] R e c o n s t i t u t i o n o f t h e C y t o c h r o m e P - 4 5 0 - C o n t a i n i n g Mixed-Function Oxidase System of Liver Microsomes B y M I N O R J . COON
Cytochrome P-450 is the predominant enzyme in the membranes of liver cells, for it occurs at levels of approximately 10% of the microsomal protein, and the endoplasmic reticulum from which the microsomes are derived represents as much as 90% of the total membranes. This pigment is an unusually versatile catalyst, as shown by its role in hydroxylation and other chemical reactions involving a variety of substrates: drugs, anesthetics, petroleum products, insecticides, carcinogens, and numerous other foreign compounds, as well as naturally occurring substances such as fatty acids and steroids.1 Liver microsomal cytochrome P-450 (P-450LM) has been characterized spectrally as a I j. R. Gillette, Adv. Pharmacol. 4, 219 (1966).
[20]
R E C O N S T I T U T I OOF N THE CYTOCHROME P-450
201
cytochrome of the b type, 2 but early attempts at solubilization and isolation yielded an altered heme protein, cytochrome P-420, without biological activity? During the course of studies on fatty acid to-hydroxylation by liver microsomes, the mixed-function oxidase system was solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable factor. 4"5 A reconstituted system prepared from these components and supplemented with NADPH was shown to be active under aerobic conditions in the hydroxylation of fatty acids, 4-6 d r u g s , 7 and a variety of other compounds. 8 Resolution of the Microsomal E n z y m e System Livers from male rabbits which have been fasted 24 hr are minced with scissors and homogenized with 4 volumes of 0.25 M sucrose in a Waring Blendor for 2 min at 4 °. The following operations are carried out at the same temperature. The homogenate is centrifuged for 25 min at 10,000 g, and the supernatant fluid is filtered through two layers of cheesecloth. The centrifugation at low speed is repeated, and the supernatant layer is then centrifuged at 105,000 g for 2 hr. The supernatant fluid is discarded, and the lipid adhering to the sides of the tubes is removed. The translucent red microsomal pellet is suspended in 0.25 M sucrose in a Potter-Elvehjem homogenizer and stored in the frozen state. In some experiments, particularly when it is desired to obtain a P-450LM preparation with minimal contamination by hemoglobin, the liver is homogenized in 1.15% KC1 and the microsomal pellet is resuspended in isotonic KCI and again precipitated by centrifugation. The resulting microsomes are then suspended in 0.25 M sucrose at a final protein concentration of about 30 mg per milliliter. A microsomal suspension containing 1.95 g of protein in 65 ml of sucrose solution is stirred at 4 ° for 20 min with a solution made by mixing 44.5 ml of glycerol, 14.8 ml of 1.0 M potassium citrate buffer (pH z T. Omura and R. Sato, J. Biol. Chem. 239, 2370 (1964). T. Omura and R. Sato, J. Biol. Chem. 237, PC1375 (1962). 4A. Y. H. Lu and M. J. Coon, J. Biol. Chem. 243, 1331 (1968). 5 M. J. Coon and A. Y. H. Lu, in "Microsomes and Drug Oxidations" (J. R. Gillette et al., eds.), p. 151. Academic Press, New York, 1969. 6A. Y. H. Lu, K. W. Junk, and M. J. Coon, J. Biol. Chem. 244, 3714 (1%9). r A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Biochem. Biophys. Res. Commun. 36, 545 (1969). A. Y. H. Lu, H. W. Strobel, and M. J. Coon, Mol. Pharmacol. 6, 213 (1970).
202
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[20]
7.6), 14.8 ml of 1.0 M KCI, 1.5 ml of 0.10 M dithiothreitol, and 7.6 ml of 10% sodium deoxycholate, and the precipitate formed upon centrifugation for 2 hr at 105,000 g is discarded. In typical experiments the supernatant layer contains at least 90% of the protein and 50% of the fatty acid to-hydroxylation activity originally present in the microsomes. Deoxycholate, glycerol, and citrate appear to be necessary for optimal solubilization of the enzyme system, whereas dithiothreitol is added as a precautionary measure to avoid the oxidation of enzyme sulfhydryl groups; KCI is added to raise the ionic strength, thereby enhancing the activity of the reductase. Other buffers, such as pyrophosphate, glycine, Tris, and phosphate are much less effective than citrate. The microsomal extract is filtered through glass wool to give a clear, faintly reddish solution having a protein concentration of 12 mg/ml, diluted with 3 volumes of deionized water, and put onto a DEAE-cellulose column (5 × 45 cm) previously equilibrated with 0.1 M Tris buffer, pH 7.7, containing 0.1 mM dithiothreitol and 0.05% deoxycholate. The column is washed with 880 ml of the same buffer mixture containing 0.10 M KCI, thereby removing hemoglobin. A reddish yellow fraction containing P450LM is then eluted with 1 liter of buffer mixture containing 0.20 M KCI, while NADPH-cytochrome P-450 reductase is eluted with 1 liter of a similar solution containing 0.30 M KCI. Finally, a fraction containing the heat-stable factor is eluted from the column with 1.5 liters of a similar solution containing 0.50M KCI. This stepwise elution gives better results than the salt gradient used previously. 4 This general procedure for solubilizing and separating cytochrome P450, the reductase, and the heat-stable component (subsequently shown to be phosphatidylcholine 9) has been used successfully for the resolution of the mixed function oxidase system of rat, s mouse, 1° and human, 1~ as well as rabbit liver microsomes. In addition, it has been used to resolve a similar cytochrome P-450-containing enzyme system in a yeast, Candida tropicalis. ~2
Purification and Characterization of Components of the Mixed Function Oxidase System Studies on the reconstitution of the mixed-function oxidase system have been aided greatly by the availability of purified, well-characterized 9 H. W. Strobel, A. Y. H. Lu, J. Heidema, and M. J. Coon, J. Biol. Chem. 245, 4851 (1970). lo D. W. Nebert, J. K. Heidema, H. W. Strobel, and M. J. Coon, J. Biol. Chem. 248, 7631 (1973). 11R. M. Kaschnitz and M. J. Coon, Biochem. Pharmacol. 24, 295 (1975). 12 W. Duppel, J. M. Lebeauit, and M. J. Coon, Fur. J. Biochem. 36, 583 (1973).
[20]
R E C O N S T I T U T I OOF N THE CYTOCHROME P-450
203
components. The chief benefit is that purified reductase (from rat liver) and synthetically prepared phospholipid may be made and stored for subsequent reconstitution experiments, either with various forms of P450LM or with cytochrome P-450 from other organelles, tissues, or species. As a result, optimal conditions need only be devised for the solubilization and stabilization of the cytochrome P-450 fraction from a particular source, rather than for the reductase and phospholipid fractions as well. These comments apply, of course, only to systems which, like that in liver microsomes, do not require a nonheme iron protein. C y t o c h r o m e P-450. Recently, evidence has been obtained for the occurrence of multiple forms of P-450LM. la Two forms of the cytochrome, phenobarbital-inducible P-450LM2 and 5,6-benzoflavone-inducible P-450LM4, have been purified from rabbit liver microsomes in the presence of Renex 690, a nonionic detergent. 14"1s These two cytochromes are homogeneous by the criteria of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, quantitative determination of the C-terminal amino acid residues, and Ouchterlony double-diffusion analysis carried out with the corresponding antisera. T M Highly purified P-450LM has also been isolated from 3-methylcholanthrene-treated rats and rabbits and from phenobarbital-treated rats by Ryan et al. 17 and Kawalek et al. ~8 and from phenobarbital- and 3-methylcholanthrenetreated rabbits by Imai and Sato TM and Hashimoto and Imai. 2° All these methods employ detergent solubilization of the cytochromes, but the purification precedures are different. In our experience, cytochrome P450 is stable at all stages of purification in the presence of 20% glycerol. Excess detergent (Renex 690) is removed from highly purified P450LMz and LM4 by treatment with Amberlite XAD-2 and calcium phosphate gel. TM The resulting preparations are soluble in aqueous buffers, though P-450LM4 requires a higher ionic strength (for example, phosphate buffer at least 0.15 M) than does P-450LM2. N A D P H - - C y t o c h r o m e P-450 R e d u c t a s e . The detergent-solubilized liver microsomal NADPH--cytochrome c reductase retains the ability to reduce cytochrome P-450, a its natural substrate, and therefore to funcla D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, J. Biol. Chem. 250, 3567 (1975). ~4 M. J. Coon, T. A. van der Hoeven, S. B. Dahl, and D. A. Haugen, this volume [10]. 1~ D. A. Haugen and M. J. Coon, J. Biol. Chem. 251, 7929 (1976). 16 W. L. Dean, Doctoral thesis, University of Michigan, Ann Arbor, 1976. lr D. Ryan, A. Y. H. Lu, J. Kawalek, S. B. West, and W. Levin, Biochem. Biophys. Res. Commun. 64, 1134 (1975). 18 j. C. Kawalek, W. Levin, D. Ryan, P. E. Thomas, and A. Y. H. Lu, Mol. Pharmacol. 11,874 (1975). 19 y . Imai and R. Sato, Biochem. Biophys. Res. Commun. 60, 8 (1974). z0 C. Hashimoto and Y. Imai, Biochem. Biophys. Res. Commun. 68, 821 (1976).
204
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[20]
tion in the reconstituted hydroxylation system. 6 In contrast, N A D P H cytochrome c reductase preparations purified after solubilization with a protease such as bromelain 21 or with a lipase ~2 believed to contain a protease 2a are not active toward cytochrome P-450. z4 We have obtained this flavoprotein in a highly purified state from rat liver microsomes after solubilization with detergent, 25 as have Dignam and Strobe126"2r and Yasukochi and Masters. zs Such preparations may be used to reconstitute an active hydroxylating system with cytochrome P-450 from a variety of sources, including rabbit, mouse, and human, as well as rat liver microsomes, and also yeast, thereby demonstrating the broad specificity of this flavoprotein. 7.10-~2
Phospholipid. After the heat-stable component had been identified as phosphatidylcholine, a variety of synthetically prepared compounds were tested for their ability to function in the reconstituted system, a Dilauroylglyceryl-3-phosphorylcholine proved to be one of the most effective and is presently used routinely because it does not readily undergo peroxidation, unlike phospholipids containing polyunsaturated fatty acids. The dilauroyl compound has been used to reconstitute the cytochrome P-450 containing enzyme systems of a variety of species. r'8"1°-~2 The phospholipid may be partially replaced for this purpose by synthetic detergents, and for optimal results we use both, as indicated in the procedure given below. Procedure for Reconstitution and Assay of Hydroxylation Activity
Principle. The assay is based on spectrophotometric determination of the rate of NADPH oxidation in the reconstituted system in the presence of a compound such as cyclohexane, which is one of the most active substrates for P - 4 5 0 L M 2. Saturating concentrations of phosphatidylcholine and purified NADPH-cytochrome P-450 reductase are added so that the cytochrome is the rate-limiting component. (As an alternative assay 21 S. D. Aust, D. L. Rorrig, and T. C. Pederson, Biochem. Biophys. Res. Commun. 47, 1133 (1972). 22 B. S. S. Masters, C. H. Williams, Jr., and H. Kamin, this series, Vol. 10, p. 565. 23 j. A. Buege and S. D. Aust, Biochim. Biophys. Acta 286, 433 (1972). 24 M. J. Coon, H. W. Strobel, and R. F. Boyer, Drug Metab. Disp. 1, 92 (1973). z5 j. L. Vermilion and M. J. Coon, Biochem. Biophys. Res. Commun. 60, 1315 (1974). 26 j. D. Dignam and H. W. Strobel, Biochem. Biophys. Res. Commun. 63, 845 (1975). 27 j. D. Dignam and H. W. Strobel, Biochemistry 16, 1116 (1977). 28 y . Yasukochi and B. S. S. Masters, J. Biol. Chem., 251, 5337 (1976).
[20]
RECONSTITUTION OF THE CYTOCHROME P - 4 5 0
205
procedure, the formaldehyde liberated by the demethylation of benzphetamine may be determined by a colorimetric method.2~'3°)
Reagents Sodium HEPES buffer, 0.5 M, pH 7.4 MgClz, 0.1 M Sodium deoxycholate, 0.5% Cyclohexane, 1.0 M in methanol NADPH, 10 mM P-450LM2 (from rabbit liver), 5 /zM solution (as determined by spectral assay of the reduced CO complex TM) NADPH-cytochrome P-450 reductase (from rat or rabbit liver), 20 units/ml Dilauroylglyceryl-3-phosphorylcholine, 0.1% aqueous suspension (sonicated)
Procedure. The assay components are added to 1-ml cuvettes with a 1-cm light path. The sample of P-450LM2 (0.02 ml), NADPH-cytochrome P-450 reductase (0.03 ml), and the phosphatidylcholine (0.03 ml) are first added, mixed, and allowed to stand for 1 min at room temperature. 31 The remaining components are then added in the order given: 0.10 ml of HEPES buffer, 0.15 ml of MgC12, 0.62 ml of water, 0.02 ml of deoxycholate, and 0.01 ml of the cyclohexane solution (replaced by 0.01 ml of methanol in the blank cell). The mixture is incubated for 3 min at 30 °, and the reaction is then initiated by the addition of 0.02 ml of NADPH to each cell. The rate of disappearance of NADPH is determined at 30 ° in a Gilford spectrophotometer equipped with a multiple sample absorbance recorder. The rate of the reaction remains constant for at least 5 min. Hydroxylation Activity. The activities are expressed as turnover numbers, i.e., moles of substrate hydroxylated per mole of cytochrome P-450 per minute. Such values are readily determined in the reconstituted system, in which cytochrome P-450 is easily made the rate-limiting component. In contrast, the apparent turnover numbers reported for various substrates in liver microsomal suspensions are usually lower, which suggests that cytochrome P-450 may not be the rate-limiting ~9 T. N a s h , Biochem. J. 55, 416 (1953). 3o j. Cochin and J. Axelrod, J. Pharmacol. Exp. Ther. 125, 105 (1959). :~1 If the order of addition is c h a n g e d so the P-450LM, N A D P H - c y t o c h r o m e P-450 reductase, or phosphatidylcholine is added after the reaction mixture has been diluted with water and the other reagents, the activity m a y be reduced to lfr% or less.
206
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[21]
component in the membrane. A typical turnover number for cyclohexane with P-450LM2 in the reconstituted system under the conditions described is 59. The activity of several purified forms of rabbit liver microsomal cytochrome P-450 toward a variety of substrates, including drugs, aniline, p-nitroanisole, biphenyl, testosterone, and benzo[a]pyrene, indicates that these cytochromes apparently hydroxylate all such compounds, but that the relative rates and positional specificities are somewhat different. 13"32 32 F. J. Wiebel, J. K. Selkirk, H. V. Geiboin, D. A. Haugen, T. A. van der Hoeven, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 72, 3917 (1975).
[21] I n c o r p o r a t i o n o f M i c r o s o m a l E l e c t r o n - T r a n s f e r C o m p o n e n t s into L i p o s o m e s : C o n s i d e r a t i o n s for Diffusion-Limited Reactions B y PHILIPP STRITTMATTER, HARRY
G. E N O C H , and PATRICK F L E M I N G
The isolation of the three enzyme components of the stearyl-CoA desaturase system, i.e., cytochrome b5 reductase, 1 cytochrome b5 (see this volume [8]) and the desaturase (see this volume [18]), provided the protein components for studies on the binding of each to microsomes or vesicles of defined phospholipid composition, and the reconstitution of a partial or complete electron transport system from NADH to the terminal desaturase. In all cases, the rates of binding of the proteins to vesicles appears to be limited by the fact that these relatively nonpolar enzymes form stable aggregates in solution, thereby producing an extremely low concentration of monomeric species, the forms that appear to insert into the bilayer structure. The procedures described below have therefore employed either relatively lengthy incubations of the proteins with preformed phospholipid vesicles, incubation of vesicles with combinations of two proteins to produce less-stable mixed-protein aggregates, or binding in the presence of detergent followed by removal of the detergent by gel filtration. With such vesicle preparations, both partial and complete electrontransport sequences have been prepared to examine protein-protein, protein-lipid, and diffusion-dependent interactions. 1 L. Spatz and P. Strittmatter, J. Biol. Chem. 248, 793 (1973).