THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 9, Issue of May 10, pp. 5257-5262, 1982 Prcnted in U.S.A.

Multiple Forms of Cytochrome P-450 in Liver Microsomes from P-Naphthoflavone-pretreated Rats SEPARATION, PURIFICATION, AND CHARACTERIZATION OF FIVE FORMS*

(Received for publication, July 24, 1981)

Paul P. Lau and Henry W. Strobe14 From the Department of Biochemistry and Molecular Biology, The University of Texas Medical School a t Houston, Houston, Texas 77025

Five forms of cytochrome P-450 have been purified from liver microsomes of fi-naphthoflavone-pretreated rats by chromatography on DEAE-Sephadex, DEAE- cellulose, and hydroxylapatite or CM-Sepharose col- umns. Over 50% of the starting cytochrome P-450 con- tent can be accounted for in these five forms after resolution on the DEAE-cellulose column, and after further purification, the combined total recovery is 308. The five forms have the following M,: 47,000, 50,500, 51,500, 53,500, and 56,500. The absorption max- ima in reduced carbon monoxide difference spectra are 452.5, 449, 449, 447.5, and 447.5 nm, respectively. Anti- body has been prepared in rabbits to each of the five forms; each antibody reacts with the antigen for which it was prepared, but not with the other four heterolo- gous antigens. In addition, each form gives a unique peptide map pattern when partially digested with Staphylococcus aureus V-8 protease and electropho- resed in sodium dodecyl sulfate gels. Each form also shows an individual pattern of catalytic activities when tested with benzphetamine, ethylmorphine, p-nitroan- isole, benzo[a]pyrene, and 7-ethoxycoumarin as sub- strates. By all criteria examined, these five forms ap- pear to be distinct forms of cytochrome P-450.

Cytochrome P-450 is the terminal oxidase of the hepatic microsomal mixed function oxidase system which metabolizes endogenous and exogenous chemicals including steroids, fatty acids, drugs, pesticides, carcinogens, and precarcinogens (1,2).

The existence of the multiple forms of cytochrome P-450 has been amply demonstrated (1-6). Some of the forms have been purified to homogeneity from rats, rabbits, humans, and other organisms, including bacteria (1, 6-10). However, the exact number of forms of cytochrome P-450 in a tissue or an organism remains uncertain.

Purification and separation procedures for cytochrome P- 450 often take advantage of the fact that different prominent forms are induced by particular drugs. Therefore, certain forms can be more readily purified when a specific inducer is used. Purification of one to three forms has been accomplished

* This research was supported by Grant CA19621 from the Na- tional Cancer Institute, United States Public Health Service, Depart- ment of Health, Education and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

#Recipient of Research Career Development Award 1-K04- CAW311 from the Nation Cancer Institute, United States Public Health Service, Department of Health, Education and Welfare. To whom reprint requests should be addressed.

in rat liver microsomes treated with phenobarbital, 3-meth- ylcholanthrene, Aroclor 1254, P-naphthoflavone, polychlori- nated biphenyl, pregnenolone-l6-~arbonitrile, isosafrole, and cholestyramine (11-21). Although there are some outstanding exceptions (3, 15, 38, 39), most resolution procedures yield a single form in relatively low yield. We therefore undertook to resolve the total cytochrome P-450 content of liver micro- somes from P-naphthoflavone-induced rats into constituent forms in order to be able to account for as much as possible of the starting cytochrome P-450 content as discrete forms. Recently we reported the purification from P-naphthoflavone- treated rats of a form of liver cytochrome P-450 with a high turnover number for benzo[a]pyrene (17).

In this report we describe, for the first time, a procedure that can separate and purify from liver microsomes of /3- naphthoflavone-treated rats a total of five forms of cyto- chrome P-450 which appear to be electrophoretically homo- geneous, as well as immunochemically unique. Each form also appears distinct as demonstrated by molecular weight deter- mination, reactivity with antibodies, peptide mapping, and drug metabolism with the reconstituted system.

MATERIALS AND METHODS

Preparation of Microsomes-Sprague-Dawley male rats weighing less than 100 g were used in all experiments. The age of the rats is a very important factor for the purification procedure described below. Injections of P-naphthoflavone (80 mg/kg in corn oil) were given intraperitoneally for 3 days. The rats were fasted overnight on the 4th day before killing by decapitation. The microsomes were prepared by differential centrifugation using detailed procedures described earlier (17, 18).

Solubilization andPurification-The microsomes were solubilized with 1.5% (w/v) Renex 690 as described (17, 21, 22), except that the solution contained 0.1 M Tris-HC1, pH 7.7, instead of 7 mM Tris-HC1 buffer. A DEAE-Sephadex A-25 column was used to remove NADPH- cytochrome c reductase and most of the cytochrome b5 from the cytochrome P-450 pool fraction. The fraction containing 90% of the cytochrome P-450 was eluted with the equilibration buffer (0.1 M Tris-HCI, pH 7.7,30% (v/v) glycerol, 0.1 mM dithiothreitol, 0.15% (w/ v) Renex 690). This fraction was concentrated by treatment with 16% (w/v) polyethylene glycol (Mr = 6,OOO-7,500) and centrifuged for 1 h at 100,OOO X g. The pellet was resuspended in a minimal volume of DE53 buffer (10 m~ Tris-HCI, pH 7.7, 30% glycerol, 0.2% (w/v) Renex, 0.1 mM dithiothreitol, 1 nm EDTA, 0.5% (w/v) sodium cho- late). For older rats, some insoluble material was pelleted at 9,OOO x g. The total cytochrome P-450 content in the supernatant fraction remained relatively unchanged. Only a clear and fully solubilized solution was applied to the DE53 column as described below.

Separation of the Multiple Forms of Cytochrome P-450-DE53, a high ion capacity (2 meq/dry g) anion exchanger, was used to resolve the cytochrome P-450 containing fractions into its constituents. The resin must be treated as described by the manufacturer, i.e. it must be fully equilibrated with an excessive amount of Tris-HC1 buffer before it is packed, and then equilibrated with the starting buffer (DE53 buffer). Chromatography from this level onward was per-

5257

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5258 Resolution of Cytochrome P-450 Multiple Forms

formed at room temperature. The procedure at this stage is thus similar to that of Ryan et al. (15) and Warner et al. (26). Tris buffer, instead of phosphate buffer, was used to lower the initial ionic strength in the column. The pH was increased from 7.4 to 7.7 to facilitate stronger binding to this anion exchanger. The flow rate was adjusted to 0.5-1.0 ml/min. 1000 nmol of cytochrome P-450 were loaded to a fully packed column (2.4 X 60 cm). Two peaks containing hemoproteins were eluted with starting buffer, while other reddish bands on the column remained in the middle of the column. 100 ml of 50 mM buffer were then applied to the column, followed by an 800-ml linear gradient of 0-0.3 NaCl in 50 m~ Tris buffer. Four more fractions of hemoproteins were then eluted with the gradient. Some reddish material still remained on the top of the column after the gradient. A total of 6 fraction peaks was eluted, all of which contained some cytochrome P-450. The specific contents of 5 of these fractions ranged between 6 and 9 nmol/mg. Further purification of these fractions was achieved by chromatography on CM-Sepharose or hydroxylapatite columns.

Further Purification-Each of fractions 1 through 3 was applied to a CM-Sepharose column (3 X 7 cm) which was equilibrated with 10 mM potassium phosphate buffer, pH 6.5, 20% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol, and 0.2% Renex 690. Before loading, the sample was dialyzed into this buffer for 2 h a t room temperature. A 300-ml gradient (0-0.2 M NaC1) was applied. Only one major fraction was eluted from each column. The pooled fractions were stored at -70 "C. Fractions 4 and 5 were purified on a hydroxylapatite column as previously described (17).

Other Methods-Protein fractions were concentrated by reverse osmosis (Amicon). Removal of detergent was achieved by treatment with Bio-Beads SH-2 (17) or by hydroxylapatite column as described previously (17). SDSI-gel electrophoresis was performed as described by Laemmli (27), using a slab gel (10 X 15 cm) at room temperature.

Peptide mapping of the purSed forms was accomplished using a procedure similar to that described by Cleveland et al. (28). 3.3 nmol/ ml of cytochrome P-450 in buffer (0.5% (w/v) SDS, 0.125 M Tris-HC1, pH 6.8, 20% glycerol) were heated in a boiling water bath. Protease was added at 6 pg/nmol of cytochrome P-450. The reaction was stopped by addition of 2% SDS and 10% dithiothreitol and heating in a boiling water bath for 2 min. The sample was run in a 15% SDS gel (as above).

NADPH-cytochrome P-450 reductase was purified and assayed according to the procedure described by Dignam and Strobel (24,25).

Immunological analysis was performed by radial immunodiffusion (29). Ouchterlony diffusion plates contained 0.1% agarose, 0.2% Renex, 0.15 M NaC1,0.015 M sodium azide, and 1 M glycine, pH 7.3. Antibodies to the purified cytochromes P-450 were raised in rabbits by injecting 0.1 mg of protein in 1.0 ml of Freund's adjuvant once a week for 3 weeks before collecting serum. The antisera raised were used in the double diffusion experiments which were carried out at 4 "C for 3 days. Nonspecific precipitated material was washed away with phos- phate-buffered saline for 3 days before staining with 0.1% Amido black in 5% acetic acid.

Spectrophotometry was performed with a Cary 210 and a Beckman ACTA M-VI. Protein concentrations were determined by the proce- dure of Lowry et al. (30).

Drug Metabolism by the Reconstituted System-Metabolism was assayed in a reconstituted system (22, 23) which contained purified cytochrome P-450, purified NADPH-cytochrome P-450 reductase, and dilauroylphosphatidylcholine as previously described (17,22,23). For benzphetamine metabolism, formaldehyde formation was meas- ured by the method of Nash (31) as modified by Cochin and Axelrod (32). The 0-dealkylation ofp-nitroanisole was measured according to Netter and Seidel (33). Benzo[a]pyrene hydroxylation was measured according to the method of Nebert and Gelboin (34) . Ethoxycoumarin de-ethylation was measured according to Greenlee and Poland (35), as modified by Guengerich (3).

RESULTS AND DISCUSSION

Purification and Separation of the Multiple Forms of Cytochrome P-450

The procedure for the purification and separation contained several modifications of that described earlier (17, 24). Table I summarizes the purification scheme. DEAE-Sephadex A-25 was used to remove NADPH-cytochrome c reductase and

The abbreviation used is: SDS, sodium dodecyl sulfate.

TABLE I Purification of cytochromes P-4.50

The values reported here are averages of several preparations.

Step P-450 Protein Yield

nmol/mg mg/ml nmol/mg I

P-Naphthoflavone mi- 83.5 41.3 2.02 100 448

Solubilization 14.28 6.15 2.32 97 448 DEAE-Sephadex A-25 11.0 3.3 3.33 95 447.5 Polyethylene glycol 16% 34.06 7.45 4.57 92 447.5 DEAE-cellulose

crosomes

F- 1 2.53 0.477 5.3 9.2 450 F-2 2.75 0.43 6.45 10.9 447 F-3 0.99 0.145 6.8 4.1 449 F-4 0.77 0.144 5.34 4.6 451 F-5 3.07 0.38 8.05 21.6 446.5

50.4 F-1 CM-Sepharose" 6.04 0.27 22.3 3.9 449 F-2 CM-Sepharoseb 2.7 0.4 6.6 7.9 447.5 F-3 CM-Sepharose' 1.5 0.11 13 4.0 449 F-4 Hydroxylapatited 1.37 0.11 12.4 2.3 452.5 F-5 Hydroxylapatite' 1.23 0.07 17.5 10.0 447.5

28.1

Assigned as form 2 on the basis of M, = 50,500. Assigned as form 4 on the basis of M, = 53,500.

e Assigned as form 3 on the basis of M, = 51,500. Assigned as form 1 on the basis of M, = 47,000.

e Assigned as form 5 on the basis of M, = 56,500.

some of the cytochrome b6. The recovery of cytochrome P- 450 was increased from 55 to 90% due to the change in the buffer of the sample being loaded onto the column, as com- pared to the former procedure (17). When the solubilized sample contained only 5 m~ Tris buffer and was loaded onto the Sephadex column which was equilibrated with 0.1 M Tris- HC1 buffer at 4 "C, two peaks were obtained from the elution with the 0.1 M Tris-HC1 buffer used to wash the column. These two peaks, however, contained cross-contaminating cytochromes P-450. This procedure was not helpful for further separation and purification. Therefore, the solubilized sample was made 0.1 M in Tris-HC1, pH 7.7, and loaded onto the Sephadex column equilibrated in the same buffer. Only one fraction containing cytochrome P-450 was eluted with the 0.1 M Tris-HC1 buffer. This fraction contained no NADPH-cyto- chrome c reductase as determined both by SDS-gel electro- phoresis and by assay for activity according to Dignam and Strobe1 (24).

Sephadex ion exchangers swell and shrink with salt concen- tration and cannot be used when a steep salt gradient is applied. Thus, separation requires cellulose or Sepharose ion exchangers. Whatman DE53, which is a higher ion capacity anion exchanger than DE52, was used for resolution of the multiple forms of cytochrome P-450; under identical condi- tions, it was far superior to the DE52. Different forms of cytochrome P-450 have different PI values (4 ) and are stable in the range of pH 6.5 to pH 7.7 (36). For DE53, pH 7.7 was used and for CM-Sepharose, pH 6.5 was used. The nonionic detergent Renex 690 (0.2% w/v) and the ionic detergent so- dium cholate (0.5% w / ~ ) were used to make mixed micelles to minimize hydrophobic interactions among the cytochromes P-450 so that cross-contamination in the peaks resolved from the ion exchanger could be reduced or eliminated. The use of sodium cholate and nonionic detergent on an ion exchange column at room temperature to separate and purify cyto- chrome P-450 was f i s t described by Warner et al. (26) and used by Ryan et al. (15). The separation on DE53 of the multiple forms of cytochrome P-450 prepared from liver mi- crosomes of P-naphthoflavone-treated rats is shown in Fig. l.

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Resolution of Cytochrome P-450 Multiple Forms 5259

0.7 t

0.3 [ f i i5\ 0.1

10 20 30 40 50 60 70 80 90 Fraction Number

FIG. 1. Resolution of the multiple forms of cytochrome P- 450 by column chromatography on Whatman DE53 column. Fractions 1-3 were eluted with the starting buffer and a shallow step gradient of 30 and 50 m~ Tris buffer; fractions 4 and 5 were eluted by a linear, 1 liter, salt gradient of 0 to 0.3 M NaCI. Before the salt gradient, a step gradient of Tris-HC1 buffer was applied, 100 rnl of 35 m~ followed by 100 ml of 40 mM Tris-HC1, pH 7.7.

Each peak was partially pure at this step as judged by SDS- electrophoresis (data not shown). Further purification of each fraction resolved from DE53 was achieved by CM-Sepharose or hydroxylapatite column chromatography. Sodium cholate was omitted from the buffer solution used in the CM-Sepha- rose column chromatography step because it was found that fractions 1-3 from the DE53 column were unstable in the presence of sodium cholate at the acidic pH. Repeating the CM-Sepharose or hydroxylapatite chromatography steps did not improve the specific content of any of the five fractions.

Criteria for Purity and Distinctness of Each Form of Cytochrome P-450

1. Subunit Molecular Weight Determination-The purity of each form of cytochrome P-450 was examined by SDS-gel electrophoresis (Fig. 2). Each form appears as a single band with the exception of form 3, which shows the presence of trace contaminants. The molecular weight of each form was determined in calibrated SDS gels, and the results are shown in Fig. 3. Each form corresponds to a peptide present in the solubilized microsomes and has a molecular weight in the region of 47,000 to 56,000 as shown in the gel (Fig. 2). The different forms having different molecular weights appear not to arise from proteolysis during purification.

The IUPAC-IUB Commission on Biochemical Nomencla- ture recommends that the multiple forms of an enzyme should be differentiated on the basis of electrophoretic mobility, with the number 1 being assigned to that form having the highest mobility toward the anode. Therefore, form 1 corresponds to the peptide having the subunit M , = 46,000; form 2, 50,500, form 3, 51,500; form 4, 53,500, and form 5, 56,500. The promi- nent band immediately under form 1 in the solubilized micro- somes, as shown on the gel, may not be P-450. However, we cannot yet rule it out. The molecular weights of some of these 5 forms of cytochrome P-450 are similar to the molecular weights reported for some of the forms purified in other laboratories using other inducers (6). However, comparison by molecular weights between laboratories is difficult. The electrophoretic mobility of cytochrome P-450 can be affected by the gel system employed, the standards being used, the presence of salt, heating procedure, SDS concentration, and the volume of the sample loaded on the gel. Different subunit molecular weights of the same cytochrome P-450 have been reported by different laboratories and by the same laboratory.

Form 5 was reported earlier as having a M, = 53,500 (17); in this report, the subunit M, = 56,500. However, on the same gel, the molecular weights measured by the same operator are highly reproducible.

2. Spectral Properties-Differences in the absorption max- ima in reduced CO difference spectra of these five forms are shown in Fig. 4. The absorption maxima vary from 447 to 452.5 nm and are valid within a systematic error range of f0.5 nm. Nonionic detergent Renex 690 did not interfere with the spectra.

There have been reports which have shown two different cytochromes P-450 having the same CO-reduced spectrum maximum as measured by different laboratories (6). Since the

A B C @ 7

".-. - -

FIG. 2. SDS-gel electrophoresis of purified cytochrome P- 450. Lane A contained the fraction after Sephadex A-25 column chromatography (21 pg); B, form 5 (3.7 pg); C, form 1 (2.7 pg); D, form 3 (3.4 pg); E, form 4 (3.4 pg); and F, form 2 (3.4 pg). The gel used was a 7.5% Laemmli gel system.

10 - 1 1 I

9 -

Cytochrome P-450 Reductase

Bovine Serum Albumin

0 a - I" 4 - Ovalbumin

3 - 014 015 016 017 0:s

RI FIG. 3. Subunit molecular weight determination by SDS-gel

electrophoresis with standards. P-450 form 1 corresponds to frac- tion 4 from DE53 column; form 2, fraction 1; form 3, fraction 3; form 4, fraction 2; and form 5, fraction 5. Standards used are cytochrome P-450 reductase (76,500), bovine serum albumin (68,000). catalase (57,000), and ovalbumin (43,000).

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5260 Resolution of Cytochrome P-450 Multiple Forms

1 I I 400 450 500

Wavelength (nm) FIG. 4. Carbon monoxide-reduced difference spectra of pu-

rified forms (1-5) of cytochrome P-450. Each sample contained approximately 0.3 nmol/ml of cytochrome P-450 in 0.1 M potassium phosphate buffer, pH 7.4, 20% glycerol, and 0.2% Renex 690. Sodium dithionite was used as reducing agent.

differences in the CO maxima of the multiple forms of cyto- chrome P-450 varied moderately within instrumental errors, and are dependent on the purity and detergent conditions (6), the differentiation of each form by its CO maximum is not used here. For instance, the measured CO difference spectrum maximum of form 5 ranged from 446 to 447.5 nm a t various stages of its purification by the procedure reported here. However, the differences in CO maxima of the various forms, even though shown to be small (452 to 447.5), suggest that the forms are, in fact, distinct.

3. Immunological Analysis-Rabbit antibodies prepared to each purified form of rat liver cytochrome P-450 were found to be biospecific against their respective purified cytochrome P-450 antigens. Each cytochrome P-450 reacted specifically with its antibody raised in rabbits (Fig. 5). Form 2 reacted identically with a form purified from phenobarbital-pretreated rat microsomes (M, = 50,500), while form 5 reacted identically with the cytochrome P-448 (M, = 56,500) which we purified from 3-methylcholanthrene-pretreated rat microsomes.2 The purification procedures for phenobarbital-cytochrome and 3- methylcholanthrene-cytochrome P-448 were similar to that described in this paper. The presence of the nonionic detergent was found essential for eliminating artifacts due to nonspecific interaction.

Antibodies to each of the purified forms of cytochrome P- 450 from P-naphthoflavone-pretreated rat microsomes do not appear to cross-react with the four other heterologous forms of cytochrome purified from P-naphthoflavone-pretreated rat liver microsomes. Again, these data indicate the distinct na- ture of each of the five forms.

4. Peptide Mapping-Limited proteolytic digests of the purified forms of cytochrome P-450 showed marked differ- ences in the primary structure of the cytochromes P-450 (Fig. 6). Each form is distinct as shown on the peptide map. The distinct patterns of the cytochromes P-450, forms 1 to 5 on the peptide map, suggest that it is unlikely that the purified forms arose from a common and larger peptide degraded during purification, and that the differences in molecular weights, shown earlier, were simply due to proteolysis during solubilization and purification of the microsomes.

' P. P. Lau, C. E. Pickett, A. Y. H. Lu, and H. W. Strobel, manuscript submitted.

5. Catalytic Actiuity-Each cytochrome P-450, after it was reconstituted with the purified NADPH-cytochrome P-450 reductase and phospholipid, showed catalytic activity with various substrates as shown in Table 11.

Hydroxylation of benzo[a]pyrene, as determined fluoromet- rically, revealed that substrate specificities of the purified forms of cytochrome P-450 were different. Form 5 was highly active with benzo[a]pyrene, while the other forms showed low activities. The rate of hydroxylation of benzo[a]pyrene was linear only up to 4 min of incubation. The concentration of cytochrome P-450 for maximum hydroxylation activities was 2 pmol/ml. The hydroxylation activity also varied with lipid concentration. The activity increased more than 2-fold when 5 pg/ml of phosphatidylcholine were added and increased about %fold when 10 pg/ml of the phospholipid were added to the reconstitution system containing form 5 and reductase.

N-demethylation of benzphetamine, as determined colori- metrically (31), was not as sensitive as when it was determined fluorometrically (37). As shown in Table 11, all the forms did not metabolize benzphetamine as well as the phenobarbital- induced cytochrome P-450. Form 3 had the highest preference for benzphetamine, but form 2 had almost the same activity as form 3. N-demethylation of benzphetamine, as determined fluorometrically, was linear up to 5 min of incubation. The concentrations of cytochrome P-450 for maximum activity were 0.05 and 0.1 nmol/ml; 0.5 mg/ml of sodium cholate was also added. Under the same conditions, a purified form of phenobarbital-induced cytochrome P-450 exhibited a turn- over number about 85 nmol/min/nmol.

0-de-ethylation of 7-ethoxycoumarin, as determined fluo- rometrically, was highly preferred by form 5. The rate of 0- de-ethylation of 7-ethoxycoumarin was linear up to 2.5 min of incubation. The concentration of cytochrome P-450 was 6 pmol/ml. The 0-demethylation of p-nitroanisole was pre- ferred by forms 1 and 4. Cytochrome P-450 reductase was added at 0.75 pmol of cytochrome c reduced/min/ml of re- ductase for all reactions.

This report of the isolation and purification of five distinct forms of cytochrome P-450 from liver microsomes of P- naphthoflavone-pretreated rats extends the elegant work of others (3,6,15) who have isolated several forms of cytochrome P-450 from liver microsomes of rats after treatment with

FIG. 5. Ouchterlony immunodiffusion analysis of purified cytochromes P-450. A. the center well contained 10 pI of antiserum to form 1, the outer wells, forms 1-5; B, the center well contained 10 pl of antibody to form 2, the outer wells, forms 1-5; C, the center well contained 10 pI of antibody to form 3, the outer wells, forms 1-5; D, the center well contained 10 pI of antibody to form 4, the outer wells, forms 1-5; E , the center well contained 10 pI of antibody to form 5, the outer wells, forms 1-5. The outer wells all contained 10 p1 of 1.5 p~ solutions of cytochrome P-450, forms 1-5. Well b contained phosphate-buffered saline buffer.

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Resolution of Cytochrome P-450 Multiple Forms 5261

1 2 3 4 5

FIG. 6. Peptide mapping of cytochromes P-450, forms 1-5. Samples treated with Staphylococcus aureus V-8 protease were run on a 15% Laemmli SDS-polyacrylamide gel. The denatured proteins were digested for 30 min (determined from a time course plot) before being applied to the gel. Lanes 1-5 correspond to forms 1-5.

TABLE I1 Drug metabolism by the reconstituted mixed function oxidase with

the multiple purified forms of cytochrome P-450 from p- naphthoflavone microsomes

Substrate Cytochrome P-450

Form 1 Form2 Form3 Form4 Form5

Benzo[a]pyrene 0.04" 0 0.07 0.13 37 Benzphetamine 1.3 23.7 30 6.8 3.8 7-Ethoxycoumarin 0.3 0.2 0.1 4.0 83.6 p-Nitroanisole 31.6 11.3 15.8 51.8 11.3

a Turnover numbers were determined under the conditions of initial velocity (nanomoles/min/nmol) with concentrations of reductase and lipid optimized for each form of cytochrome P-450.

Aroclor 1254, phenobarbital, 3-methylcholanthrene, or P- naphthoflavone. Form 5 was previously purified and described by our laboratory as cytochrome P-446 (17). Some of the values reported in this paper represent refinement in charac- terization at variance with our previous work (17). The turn- over number for ethoxycoumarin (83.6), improved by elimi- nation of a consistent error in assay, agrees much more closely

with the turnover number reported by Ryan et al. for cyto- chrome P-450, (15). The subunit molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis in this paper (56,500) represents a value obtained in comparison with the four other forms of cytochrome P-450 and the standard pro- teins in the same gel. This value is in agreement with the molecular weight obtained by sedimentation equilibrium (55,000). The CO maximum in CO-reduced absolute spectrum remains at 446 nm as previously reported (17).

Our recovery of over 50% of the starting cytochrome P-450 as the combined contents of the individual forms after the resolution step (30% after final purification steps) gives in- creased hope for a more quantitative assessment of the amount and number of forms of cytochrome P-450 present in microsomes. We must, however, define the composition of the small, unstudied sixth peak eluted from the DE53 column before this hope can be realized. It is clear, though, that the five forms purified are distinct forms by all criteria used in this study. If the remaining peak is a unique form, its total content may be small compared to the others, as judged by the relative size of the elution peak from DE53, the load volume of which contained 92% of the starting content of cytochrome P-450.

The relationship of these five forms purified from liver microsomes of P-naphthoflavone-pretreated rats to those forms present in control rats, or rats pretreated with other inducers, is presently under study. It is the aim of these studies to provide a more adequate picture of which forms of cyto- chrome are constitutive and which forms are inducible by various pretreatments in the rat as Coon et al. (38) and Johnson (39) are doing for the rabbit system.

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5262 Resolution of Cytochrome P-450 Multiple Forms

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P P Lau and H W Strobelcharacterization of five forms.

beta-naphthoflavone-pretreated rats. Separation, purification, and Multiple forms of cytochrome P-450 in liver microsomes from

1982, 257:5257-5262.J. Biol. Chem. 

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