Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

ARCHIVES

OF BIOCHEMISTRY

Vol. 277, No. 1, February

AND

BIOPHYSICS

15, pp. 166-180,199O

Reconstitution of Testosterone Oxidation by Purified Rat Cytochrome P45Op (IIIAI)’ Michael Halvorson, Denise and Andrew Parkinsonla2 Department of Pharmacology, University of Kansas Medical Received

September

Greenway,

Delmont

Eberhart,

Fitzgerald,

Toxicology and Therapeutics, Center for Environmental Center, Kansas City, Kansas 66103

Health

and Occupational

Medicine,

21,1989

Cytochrome P45Op (IIIAl) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P45Op, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P45Op (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b6, 200 pg/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 pg/ml). Testosterone oxidation by cytochrome P45Op was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P45Op. Under these experimental conditions, purified cytochrome P45Op converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6@-hydroxytestosterone (51%), 2/3hydroxytestosterone (18%), 15&hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 1Shydroxytestosterone (3%), lj3hydroxytestosterone (3%), lG&hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16&hydroxytestosterone and androstenedione, the ’ This work was supported by Grant GM 37044 from the National Institutes of Health (NIH). M.H. was supported by Training Grant ES 07079 from NIH, and A.P. is the recipient of NIH Research Career Development Award ES 00166. ’ To whom correspondence should be addressed. 166

Kathleen

conversion of testosterone to each of these metabolites was inhibited >85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P45Op. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P45Op was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycinor erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P45Op were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P45Op. 0 1990

Academic

Press,

Inc.

The purification of cytochrome P45Op (IIIAl) from rat liver microsomes was first reported in 1980 by Elshourbagy and Guzelian ( 1).3 In this and subsequent immunochemical studies, Guzelian and co-workers (15) and Guengerich et al. (11) identified cytochrome P45Op as the principal P450 enzyme inducible by steroidal agents, such as pregnenolone-16a-carbonitrile and dexamethasone. Similar purification and immunochemical studies by several investigators revealed that cytochrome P45Op is a developmentally regulated enzyme 3 The nomenclature system of Ryan et al. (2, 3) and Arlotto et al. (4,5) is used throughout the manuscript. Cytochromes P450a, P45Ob, P45Oc, P45Og, P450h and P450m are also known as cytochromes P450 IIAl, IIBl, lA1, IIC13, IICll, and IIA2, respectively (6, 7). Cytochrome P45Op is also known as IIIAl (6, 7), PCNl (8,9), and PCNa (10). A closely related isozyme, IIIA2, is also known as PCN2 (6-9) and is apparently identical to cytochrome P450PCN.a (ll), PB-2a (12), P450 PB-1 (13), P450,., (14), and either PCNb or PCNc (10). 0003.9861/90

$3.00

CopyrIght 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

RECONSTITUTION

OF

PURIFIED

(the levels of which decline postpuhertally in female but not male rats) and is also highly inducible by treatment of rats with macrolide antibiotics (16-20). In addition to inducing cytochrome P45Op, macrolide antibiotics, such as troleandomycin, form a stable, inhibitory complex with cytochrome P45Op (16,17). In contrast to numerous other P450 enzymes purified from rat liver microsomes, cytochrome P45Op exhibits little or no catalytic activity when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine (1,7,11-14). Consequently, the biotransformation reactions catalyzed by cytochrome P45Op have been inferred from the effects of cytochrome P45Op inducers and inhibitors on microsomal reactions, and from the results of antibody-inhibition experiments. These types of experiments indicate that rat cytochrome P45Op plays a major role in the microsomal oxidation of testosterone (19-22) and numerous other substrates, such as troleandomycin (16, 17), erythromycin (18), ethylmorphine (l&23), S-mephenytoin (23), nifedipine (22, 24), 17P-estradiol (19, 20, 24, 25), aldrin (24, 26), cyclosporin (27, 28), digitoxin (29), warfarin (30-32), acetylaminofluorene (32), benzo[a]pyrene (32,33), and aflatoxin (34,35). There is now evidence that cytochrome P45Op is one of several closely related members of the IIIA gene family of P450 enzymes (7-10, 31, 36-38). Although cytochrome P45Op, or IIIAl, is the major steroid- and antibiotic-inducible enzyme, a closely related isozyme, namely IIIAS, appears to be the constitutively expressed, developmentally regulated form of cytochrome P45Op (9). The mRNA encoding cytochrome P450 IIIAB is also inducible by treatment of rats with phenobarbital (9). Funae and Imaoka (13) and Yamazoe et al. (14) have purified from untreated or phenobarbital-treated rats a P450p-related protein (designated P450 PB-1 and P450ss.1, respectively) that they believe to be cytochrome P450 IIIA2 (39, 40).4 Both groups of investigators have successfully reconstituted the catalytic activity of this enzyme under conditions that differ significantly from those commonly used for other forms of purified cytochrome P450, which are typically reconstituted with NADPH-cytochrome P450 reductase, dilauroylphosphatidylcholine and, in some cases, cytochrome bg. One of the most important conditions for reconstituting cytochrome P450 PB-1 was the use of phosphatidylcholine, phosphatidylserine and sodium cholate which, compared with phosphatidylcholine alone, stimulated the G/3-hydroxylation of testosterone - 27-fold (from 0.5 to 13.5 nmol/nmol P45O/min) (39). 4 The form of cytochrome P450 PB-1 referred to throughout this paper is the P450p-related protein purified by Funae and Imaoka (13, 39). This protein is not the same as P450 PB-1 purified by Waxman and Walsh (41), which is also known as cytochrome P450 PB-C (ll), P450k (42) and IICG (6, 7).

RAT

CYTOCHROME

P45Op

167

Similarly, one of the most important conditions for reconstituting cytochrome P450,., was the use of a microsomal lipid extract and sodium cholate which, compared with dilauroylphosphatidylcholine, stimulated the O-dealkylation of 7-propoxycoumarin - K&fold (40). Yamazoe et al. (40) reported that, when reconstituted with a microsomal lipid extract and sodium cholate, cytochrome P450G0. the 6P-hydroxylation of testosterone at 10-13 1 catalyzed nmol/nmol P450/min. However, under these same conditions, cytochrome P450 PB-IIb, which appears to be cytochrome P45Op (IIIAl), remained a poor catalyst of testosterone 6P-hydroxylation (40). We have purified cytochrome P45Op from troleandomycin-treated rats, and have attempted to reconstitute its catalytic activity toward testosterone under the conditions described by Yamazoe et al. (40). Although these conditions proved unsatisfactory, these experiments eventually led to the successful reconstitution of testosterone oxidation by purified cytochrome P45Op. Under the reconstitution conditions described in this paper, cytochrome P45Op catalyzed four major pathways of testosterone oxidation (2p-, S/3-, and 15@-hydroxylation and 6-dehydrotestosterone5 formation) at 4.2 to 4.6 times the rate catalyzed by microsomal cytochrome P45Op. Antibody-inhibition experiments established that cytochrome P45Op (or an immunochemically related enzyme) plays a major role in the microsomal oxidation of testosterone to 6-dehydrotestosterone and to lp-, 2p-, 6p-, 15p-, and Whydroxytestosterone. EXPERIMENTAL

PROCEDURES

Chemicals. Sodium cholate, Chaps, octylglucoside, dilauroylpbosphatidylcholine, and DTT were purchased from Calbiochem-Behring (La Jolla, CA); Amberlite XAD-2 beads, Lubrol PX, and Tween 20 were from Sigma Chemical Co. (St. Louis, MO); DE-52 and DE-53 were from Whatman BioSystems (Clifton, NJ); CM-Sepharose, DEAE-Sepharose CL-GB, and DEAE-Sephacel were from Pharmacia Inc. (Piscataway, NJ); Hypatite C (hydroxyapatite) was from Clarkson Chemical Co. (Williamsport, PA), and polyethylene glycol 8000 was from J. T. Baker Chemical Co. (Phillipsburg, NJ). Emulgen 911 was kindly supplied by KAO Corp., Tokyo, Japan (and later by ICI Americas, Wilmington, DE). Troleandomycin was generously provided by Pfizer, Inc. (Brooklyn, NY). All Tris.HCl buffers were prepared at room temperature (23-27”(Z) by addition of HCl to Trizma base. When these buffers were used at 4°C for purification purposes, the pH increased by 0.3 to 0.4 units. Animal treatment and preparation of her microsomes. Female Sprague-Dawley rats (7 weeks old) were purchased from Sasco (Omaha, NE). Rats were housed in polycarbonate cages with corncob bedding, allowed free access to water and Ralston Purina Rodent Chow 5001, and acclimated for at least 1 week to a 12-h diurnal light

’ Abbreviations used: androstenedione, 4-androstene-3,17-dione; Chaps, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propane sulfonate; 6-dehydrotestosterone, 4,6-androstadien-17/3-ol-3-one; octylglucoside, octyl-P-D-glucopyranoside; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; testosterone, 17@hydroxy-4-androstene-3-one. Troleandomycin is also known as triacetyloleandomycin.

168

HALVORSON

cycle. Rats were treated ip once daily for 4 consecutive days with troleandomycin (500 mg/kg), as described previously (21, 29). Liver microsomes were prepared 24 h after the last injection as described by LU and Levin (43), and stored as a suspension in 0.25 M sucrose at 80°C. Cytochrome P45Op was purified Purification of cytochrone P45Op. from liver microsomes from troleandomycin-treated rats by a combination of procedures described by Elshourbagy and Guzelian (I), Wrighton et al., (17) and Halpert (10, 31). Liver microsomes (1.9 g protein) were diluted to 5 mg/ml in 100 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 1 mM EDTA. A 10% solution of sodium cholate was added dropwise to a final concentration of 0.2% to release loosely bound and luminal proteins from the microsomes. After stirring for 20 min at 4”C, the sample was subjected to centrifugation (100,OOOg for 60 min), and the supernatant fraction discarded. The pellet (which contained -80% of the cytochrome P450 but only -55% of the total protein) was resuspended in 50 mM Tris. HCl, pH 7.9, containing 20% glycerol, 5 mM MgCl,, 500 pM DTT and 100 pM EDTA. The resuspended pellet (final volume 270 ml) was solubilized with sodium cholate (56 ml of 10% sodium cholate). A 50% solution of polyethylene glycolSOO0 was added to give a final concentration of 8%, and insoluble material was removed by centrifugation (45,000g for 20 min at 4°C). Additional polyethylene glycol was added to the supernatant fraction to give a final concentration of 15%, and again insoluble material (which included cytochrome P450) was collected by centrifugation (100,OOOg for 60 min at 4°C). The pellet (which contained -50% of the starting cytochrome P450 and -25% of the protein) was resuspended in -50 ml of buffer A (20 mM Tris. HCl, pH 7.9, containing 20% glycerol, 100 pM EDTA, 0.25% sodium cholate, and 0.1% Lubrol PX) and was applied at 4°C to a Whatman DE-52 anion-exchange column (2.5 X 40 cm, -200 ml bed vol) at a flow rate of -1 ml/min. The column packing (250 ml) had previously been conditioned at room temperature by batch treatment with 750 ml of 500 mM (NH&SO,, 4 X 2 liters of water, 2 X 750 ml of 100 mM Tris. HCl, pH 7.9 (after which the pH was adjusted to 7.9), and 3 X 1 liter of 20 mM Tris. HCl, pH 7.9. The column was equilibrated with 5 column vol (-1 liter) of buffer A. After the sample was loaded, the column was washed overnight with 800-1000 ml of buffer A containing 10 mM KCl. The anion-exchange resin was carefully extruded from the column with low pressure Nz gas, and the dark red band (cytochrome P450) at the center of the column was excised. This material was resuspended in -50 ml of buffer A, and applied at 4°C to a Whatman DE-53 anionml bed vol) previously condiexchange column (2.5 X 30 cm, -150 tioned and equilibrated with buffer A as described above for the DE52 column. After the sample was loaded, the column was washed at -0.5 ml/min with a linear gradient of 250 ml of buffer A containing 20 mM KC1 versus an equal volume of buffer A containing 250 mM KCl. The column eluent was monitored at 280 and 405 nm (to detect proteins and hemoproteins, respectively) and a flow-through conductivity meter was used to monitor the salt gradient. Column fractions (5 ml) were collected at the start of the salt gradient. The absolute spectrum (400-500 nm) of heme-containing fractions was recorded, and selected fractions were analyzed by SDS-PAGE. Cytochrome P450 eluted from the DE-53 column as two broad, unresolved peaks (fractions 32-65). Initially, most of the heme-containing fractions were pooled for further purification of cytochrome P45Op. In later experiments, however, only fractions from the first peak (usually 32-35) were pooled for further purification. These fractions contained highly purified cytochrome P45Op, as determined by SDS-PAGE, and displayed an absorbance ratio of A456/A420 3 1.5 (which indicates that most of the cytochrome P45Op was complexed with troleandomycin). The cytochrome P45Op fractions from the DE-53 column were pooled, concentrated (if necessary) to -25 ml by ultrafiltration on an Amicon Diaflo PM-30 membrane, and diluted fivefold with 50 mM Tris HCl, pH 7.9, containing 20% glycerol, 5 mM MgCl,, 500 pM DTT, and 100 pM EDTA. The diluted sample was applied at 1 ml/min to a

ET

AL.

Clarkson Hypatite C column (2.5 X 10 cm), previously conditioned at 4°C with 10 column vol(500 ml) of 20 mM potassium phosphate buffer, pH 7.4, and equilibrated with 2 column vol (100 ml) of buffer B (20 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 500 PM DTT, 100 PM EDTA, and 0.2% Lubrol PX). After the sample was loaded, the column was washed with 150 ml buffer B, followed by a linear gradient of 200 ml buffer B versus an equal volume of the same buffer containing 250 mM potassium phosphate buffer, pH 7.4. The column eluent was monitored as described above, and 5.ml fractions were collected when the sample was loaded. Selected fractions were screened by SDS-PAGE, before pooling those fractions enriched in cytochrome P45Op (usually fractions 85-110). At this stage of the purification procedure, cytochrome P45Op was 295% pure as determined by SDS-PAGE. The source of hydroxyapatite was important because cytochrome P45Op bound tighter to Hypatite C (from Clarkson Chemical Co.) than to Ultrogel (from Pharmacia-LKB Biotechnology). In some cases, the cytochrome P45Op fractions from the Hypatite C column were subjected to CM-Sepharose chromatography at room temperature, as described by Arlotto et al. (4), except that Emulgen 911 was replaced with an equal concentration of Lubrol PX. Although this cation-exchange column removed some minor contaminants, more than half of the cytochrome P450 applied became irreversibly bound to the top of the column. This chromatographic step was subsequently omitted from the purification procedure because minor contaminants in the cytochrome P45Op sample from the Hypatite C column could be removed by anion-exchange chromatography on DEAE-Sepharose CL-6B (or DEAE-Sephacel), which was carried out as described by Halpert (10). The sample from Hypatite C was concentrated by ultrafiltration to 5-10 ml and dialyzed overnight at 4°C against 2 liters of buffer C (5 mM potassium phosphate buffer, pH 7.7, containing 20% glycerol, 0.2% sodium cholate, 0.1% Lubrol PX, and 100 FM EDTA). In the morning, the sample was dialyzed for 2 h against 2 liters of buffer C at room temperature. The dialyzed sample was applied at 0.25 ml/min to a DEAE-Sepharose CL-6B column (1 X 45 cm) previously conditioned at room temperature with 2 column vol (70 ml) of 5 mM potassium phosphate buffer, pH 7.7, and equilibrated with 70 ml of buffer C. Fractions (3 ml) were collected at 4°C. After the sample was loaded (i.e., at fraction 3), the column was washed with 54 ml of buffer C, followed by a linear gradient of 150 ml of buffer C containing 10 mM potassium phosphate versus an equal volume of the same buffer containing 60 mM NaCl. The salt gradient was started at fraction 22, and an increase in the conductivity of the eluent was detected at fractions 32-33. Detergent was removed from purified cytochrome P45Op by a combination of hydroxyapatite chromatography, treatment with amberlite XAD-2 beads, and dialysis. Cytochrome P45Op from the DEAESepharose CL-6B column was concentrated to -5 ml by ultrafiltration, and applied at room temperature to a Hypatite C column (1.5 X 3 cm) previously conditioned and equilibrated with buffer B, as described above. After the sample was loaded, the column was washed with 4 column vol (-20 ml) of detergent-free buffer B, followed by an equal volume of the same buffer containing 0.2% Chaps, followed in turn by 20 ml of detergent-free buffer B. Cytochrome P45Op was eluted from the column with 250 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 500 pM DTT, and 100 pM EDTA, and was treated at room temperature with Amberlite XAD-2 beads (0.1 g wet beads/ml) for 15 min. Although treatment with XAD-2 beads was necessary to remove detergent, some cytochrome P45Op aggregated and was lost by this treatment. The sample was filtered through a fine nylon mesh to remove the XAD-2 beads before being dialyzed overnight at 4°C against 1 liter of 50 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol, 100 pM EDTA, and 100 fiM DTT. The dialyzed sample was concentrated by ultrafiltration on an Amicon Diaflo PM-30 membrane. N-terminal acid sequence

amino acid sequence analysis. of purified cytochrome P45Op

The N-terminal was determined

amino by Ed-

RECONSTITUTION man degradation on a 470A gas-phase systems, Inc.), as previously described

protein (4).

sequencer

OF (Applied

PURIFIED Bio-

Purification of other proteins. Cytochromes P450a, P450b, P45Oc, P45Og, P450h, and P450m, NADPH-cytochrome P450 reductase, and cytochrome b, were purified from rat liver microsomes as described previously (2-4,21,44,45). Extraction of microsomal lipids. A lipid extract from liver microsomes from mature male rats was prepared as described by Folch et al. (46), with modifications described by St. John and Bell (47). Briefly, 50 ml of liver microsomes (10 mg protein/ml in 100 mM potassium phosphate buffer, pH 7.4) was mixed with 83 ml of methanol, followed by 166 ml of chloroform. The aqueous phase was removed, and extracted with 250 ml of chloroform:methanol (2:1, v/v). The combined organic extracts were evaporated in uacuo. The extract was weighed and dissolved in chloroform (5 mg lipid extract per ml). Aliquots (1 ml) were dispensed into screw-cap tubes, evaporated, and stored at -80°C. When needed, the contents of a tube (5 mg lipid extract) were suspended in 0.5 ml of 10 mM EDTA, pH 7.4, and sonicated externally twice for 2 min. It has Dissociation of cytochrome P450p:troleandomycin complex. been shown previously that, in addition to inducing cytochrome P45Op, troleandomycin forms a stable metabolite complex with cytochrome P45Op in uiuo (16,17). This complex, which displays an absorbance maximum at -456 nm, does not bind Oz or carbon monoxide, hence, the cytochrome is catalytically inactive and undetectable by the method of Omura and Sato (48). To dissociate this complex, liver microsomes or purified cytochrome P45Op was treated with potassium ferricyanide (final concentration 100 wM). After a 30-min incubation at room temperature, the sample was dialyzed at 4°C for 2 h against 2 liters of 50 mM potassium phosphate buffer, pH 7.4, containing 20% glycerol and 100 pM EDTA. Testosterone oxidation. Testosterone oxidation was measured at 37°C in l-ml incubation mixtures as described under Results. Metabolites were resolved and quantitated by HPLC as described by Sonderfan et al. (21). A second HPLC system was used to resolve S/J’- and 15ahydroxytestosterone, as described by Arlotto et al. (4). Preparation of antibodies against cytochrome P45Op. Antibody against cytochrome P45Op was raised in four lo-week-old-female New Zealand White rabbits (Small Stock Industries, Pea Ridge, AK), as previously described (4,45). The IgG fraction from high-titer antisera was isolated by fractionation with caprylic acid and ammonium sulfate, essentially as described by McKinney and Parkinson (49). To remove any cross-reacting antibodies, anti-P450p was subjected to absorption chromatography against liver microsomes from mature female rats, liver microsomes from mature female rats treated with 3. methylcholanthrene, and partially purified cytochromes P450b, P450e and P450f (9). These samples were bound either hydrophobically to N-octylamino-Sepharose or covalently to CNBr-activated Sepharose 4B or Affi-Gel 10, essentially as described for the immunoabsorption of antiiP450a (9). Chromatography was carried out at room temperature at a flow rate of 0.1 ml/min. In some cases, the antibody was mixed with the column packing in sealed plastic tubes and gently rotated at room temperature for 2 to 4 h. Column fractions were collected at 4”C, and specificity was determined by Western immunoblot. Western immunoblotting. Liver microsomes (5 pg protein) and purified forms of cytochrome P450 (up to 10 pmol) were analyzed by Western immunoblot essentially as described (9,40). The primary antibody (i.e., anti-P450p) was located with horseradish peroxidase-conjugated, goat anti-rabbit IgG (affinity purified, heavy and light chain specific) and peroxidase-anti-peroxidase complex (both from Organon Teknika, Cappel Division, Durham, NC), as previously described (4). Horseradish peroxidase activity was located with 4-chloro-lnaphthol and H,O*. Other assays. The concentration of microsomal protein was determined by the method of Lowry et al. (50) whereas the protein concentration of purified cytochrome P45Op was determined by the more sen-

RAT

CYTOCHROME

169

P45Op

sitive method of Bradford (51). In both cases, bovine serum albumin was used as standard. The concentration of cytochrome P450 in liver microsomes was determined by the method of Omura and Sato (48), from the carbon monoxide difference spectrum of dithionite-reduced microsomes, based on an extinction coefficient of 91 mM-i cm-‘. In some cases, 5 pM methyl viologen (paraquat) was added to accelerate formation of the ferrous cytochrome P450-CO complex, as described by Koop et al. (52). The concentration of heme was determined from the difference spectrum (A557-A575 nm) between oxidized and reduced pyridine hemochromogen, based on an extinction coefficient of 32.4 mM-’ cm-‘, as described by Falk (53). For reasons outlined under Results, the concentration of purified cytochrome P45Op was routinely determined from its absolute spectrum, based on an extinction coefficient of 125 mM-’ cm-l at 423 nm. Spectra were recorded on a DW2C spectrophotometer (SLM-Aminco, Urbana, IL) calibrated with a holmium oxide crystal (A,,,., = 446.4 nm). SDS-PAGE was carried out according to the method of Laemmli (54), with minor modifications (44).

RESULTS

Purification

of Cytochrome

P45Op

Cytochrome P45Op was purified from troleandomytin-treated female rats by a combination of several chromatographic techniques, based largely on previously published procedures (1, 10, 17). The final stages of the purification procedure included anion-exchange chromatography on DEAE-Sepharose CL-6B or DEAESephacel. This latter column was used by Halpert to resolve cytochrome P45Op (termed PCNa by Halpert) from other members of the P450 III gene family, namely PCNb and PCNc. If only the earliest fractions from the initial Whatman DE-53 column were pooled, the sample eventually applied to the DEAE-Sepharose CL-6B column contained negligible amounts of PCNb, PCNc, and cytochrome P450a, all of which eluted late in the NaCl gradient (20-50 mM) (results not shown). Cytochrome P45Op eluted from DEAE-Sephacel as a single peak early in the NaCl gradient (7-10 mM). In contrast, cytochrome P45Op eluted from DEAE-Sepharose CL-6B as two peaks, as shown in Fig. 1 (upper panel). Peak I eluted at 7-10 mM NaCl, whereas peak II eluted at 15-19 mM. The electrophoretic mobility of the protein in peak I was indistinguishable from that in peak II, as shown in Fig. 2. These two proteins were also indistinguishable with polyclonal antibody against cytochrome P45Op (results not shown). On the basis of their absolute spectra, peak I contained cytochrome P45Op that was not complexed with troleandomycin (x,,, - 423 nm), whereas peak II contained cytochrome P45Op complexed with troleandomycin (X,,, - 456 nm), as shown in Fig. 3. To determine whether the proteins in peaks I and II were identical, the fractions from peak II were pooled, treated with potassium ferricyanide to dissociate the troleandomycin complex, and subjected again to DEAE-Sepharose CL-6B chromatography. As shown in Fig. 1 (bottom), the ferricyanide-treated sample eluted at 7-10 mM NaCl, indicating that decomplexa-

170

HALVORSON

0.15

-I

BEFORE

ET

AL.

A

bFe(CNlr;

PEAK (FRACTION

(FRACTION

II 70)

55)

5 f E 8 9

AFTER

0.08-

KaFe(CN)s

/d ,-25

.d .r

-20 l5

./

-10

2 0 3 2

-5

8

0.04

O-

I

I

I

I

I

I

I

50

55

60

65

70

75

g

‘0

FRACTION

tion shifted peak II to peak I. These results indicate that the free and troleandomycin-complexed form of cytochrome P45Op can be resolved by anion-exchange chromatography on DEAE-Sepharose CL-6B (but not on DEAE-Sephacel).

LS

1

1

410

I

4io WAVELENGTH

FIG. 1. DEAE-Sepharose CL-GB chromatography of cytochrome P45Op before and after dissociation of the complex with troleandomycin. Cytochrome P45Op from troleandomycin-treated rats was purified by anion-exchange and hydroxyapatite chromatography as described under Experimental Procedures, and subjected to DEAESepharose CL-GB chromatography as described by Halpert (10). Cytochrome P45Op was resolved into two peaks, as shown in the upper panel. The second peak, which was complexed with troleandomycin (Fig. 3), was treated with potassium ferricyanide and again subjected to DEAE-Sepharose CL-6B chromatography. The dashed line represents part of the NaCl gradient (O-60 mM) applied to the column.

PEAKI

I

4

PEAK

II

%On

FIG. 2. SDS-Polyacrylamide gel electrophoresis of cytochrome P45Op in peaks I and II separated by DEAE-Sepharose CL-6B chromatography. Cytochrome P45Op from troleandomycin-treated rats was purified by anion-exchange and hydroxyapatite chromatography as described under Experimental Procedures, and resolved into two peaks by DEAE-Sepharose CL-6B chromatography (Fig. 1). An aliquot of the sample applied to the DEAE-Sepharose CL-6B column (designated LS for loading sample), and aliquots of the fractions containing peak I (52-59) or peak II (66-74) were subjected to SDSPAGE, as described by Laemmli (54), with minor modifications (44). Proteins were stained with Coomassie blue R-250.

I

460

I

4io

5 10

(nm)

FIG. 3. Absolute spectra of cytochrome P45Op in peaks I and II separated by DEAE-Sepharose CL-6B chromatography. Cytochrome P45Op from troleandomycin-treated rats was purified by anion-exchange and hydroxyapatite chromatography as described under Experimental Procedures, and resolved into two peaks by DEAE-Sepharose CL-6B chromatography (Fig. 1). The absolute spectrum of fractions 55 and 70, from peaks I and II, respectively, was recorded with an SLM-Aminco DW-2C spectrophotometer. The dashed line is an uncorrected baseline recorded with buffer in both the sample and reference cuvettes.

Prior to anion-exchange chromatography on DEAESepharose CL-GB, care was taken to bring cytochrome P45Op to room temperature (this was achieved by a 2-h dialysis at room temperature as described under Experimental Procedures). This was important because temperature influenced the chromatographic behavior of cytochrome P45Op. At 4°C cytochrome P45Op did not bind to the DEAE-Sepharose CL-6B column, but was largely recovered in the void volume (results not shown). On one occasion, when the room temperature exceeded 3O”C, some cytochrome P45Op became irreversibly bound to the top of the column. As shown in Fig. 4, purified cytochrome P45Op was electrophoretically homogeneous by SDS-PAGE, and comigrated with cytochrome P450h (M, 51,000). The specific content of four preparations of cytochrome P45Op ranged from 14 to 16 nmoljmg protein. Over the first 30 residues, the N-terminal amino acid sequence of cytochrome P45Op was identical to that reported for cytochrome P450 PCNl and PCNa (IIIAl), which differs from the N-terminal amino acid sequence of cytochrome P450 PCNB, PCNb, and PCNc at residues 18 (Val -W Ile), 24 (Gly -+ Arg), 25 (Phe -W Leu), 28 (Arg + His), and 29 (Thr + Arg) (9, 10). N-terminal amino acid sequence analysis of purified cytochrome P45Op did not reveal the presence of contaminating proteins.

RECONSTITUTION

OF

PURIFIED

gel electrophoresis of purified cytoFIG. 4. SDS-Polyacrylamide chrome P45Op. Lanes 1 and 2 contained liver microsomes (5 pg protein) from untreated and troleandomycin-treated female rats, respectively. Lanes 3 and 4 contained 10 and 4 pmol of purified cytochrome P45Op, respectively. Lane 5 contained a mixture (4-6 pmol of each) of cytochromes P45Op (&f, 47,000), P45Og (M, 50,000), P450h (M, 51,000), P450h (M, 52,000) and P45Oc (M, 56,000). Proteins were stained with Coomassie blue R-250.

Spectral Properties The spectral properties of the decomplexed form of purified cytochrome P45Op are summarized in Table I. There was a large discrepancy between the concentration of cytochrome P45Op estimated from the absolute spectrum (2 nmol/ml) and the concentration estimated from the CO-difference spectrum of the dithionite-reduced hemoprotein (0.74 nmol/ml). The former estimate was based on an extinction coefficient of 125 mM-’ cm-l, which was determined from the absolute spectra of purified cytochromes P45Ob and P45Oc. The concentration of heme was determined by the reduced pyridine hemochromogen assay to be 2.06 nmol/ml, which established that the concentration of cytochrome P45Op estimated from the absolute spectrum was essentially correct and that the CO-difference spectrum grossly underestimated the concentration of cytochrome P45Op. Previous studies have shown that the concentration of cytochromes P450b and P45Oc are underestimated in the absence of 0.5% sodium cholate and 0.2% Emulgen 911 (2, 55). In the presence of these detergents, the ap-

RAT

CYTOCHROME

P45Op

171

parent concentration of cytochrome P45Op determined from the CO-difference spectrum increased from 0.74 to 1.24 nmol/ml, although under these conditions cytochrome P45Op was unstable as indicated by its progressive conversion to cytochrome P420. Addition of methyl viologen, which accelerates formation of the ferrous cytochrome P450-CO complex (52), decreased the formation of cytochrome P420 and increased the apparent concentration of cytochrome P45Op to 1.38 nmol/ml. Several detergents were tested for their effects on the CO-difference spectrum of ferrous cytochrome P45Op (in the presence of methyl viologen). As shown in Table I, addition of Emulgen 911, Lubrol PX, or octylglucoside increased the apparent concentration of cytochrome P45Op to 1.9-2.0 nmol/ml, which agreed with the values determined from the absolute spectrum and the reduced pyridine hemochromogen assay. A fourth nonionic detergent, Tween 20, was less effective at increasing the apparent concentration of cytochrome P45Op. In contrast to the other nonionic detergents tested, octylglucoside converted cytochrome P45Op to P42Op. The reason why a combination of Emulgen 911 and sodium cholate destabilized cytochrome P45Op and led to an underestimation of cytochrome P45Op concentration whereas Emulgen 911 alone did not is unknown. Similarly, we are unable to explain why the CO-complex of ferrous cytochrome P45Op absorbed maximally at 452 nm in the absence of detergent or in the presence of sodium cholate and Chaps, but absorbed maximally at 450 nm in the presence of nonionic detergent. Testosterone Oxidation by Purified Cytochrome P45Op Purified cytochrome P45Op was reconstituted with cytochrome b5 and NADPH-cytochrome P450 reductase, an extract of microsomal lipid and sodium cholate, essentially as described by Yamazoe et al. for cytochrome P450G8., (40). Under these conditions, cytochrome P45Op was a relatively poor catalyst of testosterone oxidation, as shown in Table II for testosterone G/3-hydroxylation (the major oxidation product formed). However, under these reconstitution conditions, testosterone oxidation was completely dependent on cytochrome P45Op and NADPH-cytochrome P450 reductase was stimulated by cytochrome b5, microsomal lipid and, to a lesser extent, dilauroylphosphatidylcholine but was unaffected by the presence of sodium cholate. Product formation was also dependent on incubation time. The effect of varying the concentration of several components of the reconstitution system on testosterone G/3-hydroxylation by cytochrome P45Op is shown in Table III. In this experiment, the catalytic activity of cytochrome P45Op was considerably less than that observed previously (see Table II). It was subsequently discovered that the catalytic activity of cytochrome P45Op was influenced by the order of addition and the

172

HALVORSON TABLE Spectral

Properties

of Purified

ET

AL.

I Rat Cytochrome

P45Op

(nm)

Cytochrome (nmol/ml)

423 452 450 450 452 452 450 450 450 450 557

2.00 0.74 1.24 1.38 0.89 0.89 1.93 1.74 1.97 2.00 2.06

Absorbance Additions

Spectrum” Absolute Difference Difference Difference Difference Difference Difference Difference Difference Difference Reduced

pyridine

None None 0.2% Emulgen 911 and 0.5% sodium Above + 5 FM methyl viologen (MV) 0.5% sodium cholate + MV 0.5% Chaps + MV 0.5% octylglucoside + MV 0.2% Tween 20 + MV 0.2% Lubrol PX + MV 0.2% Emulgen 911 + MV hemochromogenb

cholate

maximum

P450

(5% P420) (21% P420) (13% P420) (<5% P420) (<5% P420) (27% P420) (<5% P420) (<5% P420) (<5% P420)

’ All samples were diluted in 50 rnM potassium phosphate, pH 7.4, containing 20% glycerol and 100 pM EDTA. The absolute spectrum was determined with purified cytochrome P45Op in the sample cuvette, and buffer only in the reference cuvette. The concentration of cytochrome spectra, cytochrome P450 was calculated from the absorbance at 423 nm, based on an extinction coefficient of 125 mM-’ cm -r. For difference P45Op was diluted in buffer containing detergent and 5 pM methyl viologen (MV) as indicated. The sample and reference cuvettes both contained dithionite-reduced cytochrome P45Op, and carbon monoxide was bubbled through the sample cuvette. The concentrations of cytochromes P450 cm-l for P420. The and P420 were determined as described (48), based on an extinction coefficient of 91 mM-’ cm-l for P450, and 100 mM-’ percentage contribution of cytochrome P420 to total cytochrome P450 is shown in parentheses. b Cytochrome P45Op (1 ml) was mixed with 0.5 ml pyridine and 1 ml NaOH (0.25 N), and divided between two l-ml cuvettes. After a baseline was recorded (500-650 nm), the reference cuvette was treated with 5 ~1 of 5 mM potassium ferricyanide, whereas the sample cuvette was treated with a few grains of sodium dithionite. The concentration of heme was determined from the absorbance difference between 557 and 575 nm, based on an extinction coefficient of 32.4 mM-’ cm-’ (53).

volume in which the enzymes and lipid were initially combined. In subsequent experiments cytochrome P45Op, NADPH-cytochrome P450 reductase, cytochrome b5, and microsomal lipid were mixed in the order listed in a combined volume of 100 ~1. Despite the low catalytic activity, the results in Table III indicated that testosterone oxidation by cytochrome P45Op was stimulated when the concentration of certain components (particularly microsomal lipid) was increased above that used in Table II. When cytochrome P45Op (50 pmol) was incubated for 20 min with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 pg lipid, 500 nmol testosterone, 5 pmol MgCl,, 1 pmol EDTA, 200 pmol potassium phosphate, pH 7.4, and an NADPH-generating system, the rate of testosterone Go-hydroxylation was 624 pmol/nmol P450p/min, which exceeded the turnover rates shown in Table II. Effects of Detergent The effects of selected detergents on testosterone 6/S hydroxylation by cytochrome P45Op are shown in Table IV. In the absence of detergent, the rate of testosterone Go-hydroxylation was 400 pmol/nmol P450p/min, not 624 as previously determined. This difference was subsequently found to be due to the order of addition of potassium phosphate buffer. The highest rate of testosterone

oxidation by cytochrome P45Op was observed when the buffer was added last. The most striking result shown in Table IV is the marked stimulatory effect (sixfold) of Emulgen 911 on testosterone 6&hydroxylation. To a lesser extent, Chaps and Lubrol PX also stimulated testosterone oxidation by cytochrome P45Op. At the concentrations tested, sodium cholate and octylglucoside had little effect on testosterone oxidation by cytochrome P45Op, whereas Tween 20 was slightly inhibitory. Liver cytosol, which stimulates some microsomal cytochrome P450 reactions, also inhibited testosterone oxidation by cytochrome P45Op (results not shown). Testosterone 6fi-hydroxylation by cytochrome P45Op increased as the concentration of Emulgen 911 was increased to 50 pug/ml, as shown in Fig. 5. The nonionic detergent was inhibitory above 60 pg/ml, and testosterone G&hydroxylation was undetectable when cytochrome P45Op was reconstituted in the presence of 200 pg/ml Emulgen 911. In the absence of Emulgen 911, the rate of testosterone 6P-hydroxylation was -600 pmol/ nmol P450p/min. This value is higher than the corresponding value in Table IV because the potassium phosphate buffer was the last component added before the NADPH-generating system. Effects of pH and Buffer In the presence 6/?-hydroxylation

Concentration

of 50 pg/ml Emulgen 911, testosterone by purified cytochrome P45Op was

RECONSTITUTION TABLE Reconstitution

Incubation

of Testosterone Rat Cytochrome

OF

PURIFIED

II 6fl-Hydroxylation P45Op: Part

by Purified 1

Testosterone Go-hydroxylation (pmol formed per incubation)

conditions

Complete system’ - cytochrome P45Op - reductase - cytochrome b, - microsomal lipid - lipid + DLPC’ - sodium cholate 20.min incubation

250’ 0 0 70 60 170 260 470

u In the complete system, the l-ml incubation mixtures contained 50 pmol P45Op, 100 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b5, 50 pg of microsomal lipid extract, 200 fig sodium cholate, 100 pmol potassium phosphate buffer, pH 7.4, 3 pmol MgCl,, 1 pmol EDTA, and 250 nmol testosterone (dissolved in 20 ~1 of methanol). The first four components were added in a final volume of 100 ~1, followed by 100 ~1 of 0.2% sodium cholate. The last four components were premixed and added in 750 ~1 of water. Reactions were initiated with 50 ~1 of NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose 6-phosphate and 1 rmol NADP), and terminated after a lo-min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21). b Turnover value is 500 pmol metabolite formed/nmol P45Op/min. ’ The microsomal lipid extract was replaced with 50 pg dilauroylphosphatidylcholine (DLPC).

RAT

CYTOCHROME

pmol/ml, but was directly proportional to cytochrome P45Op concentration only up to 25 pmol/ml. A departure from linearity was clearly evident at 50 pmol/ml of cytochrome P45Op, which corresponded to the concentration of cytochrome b5. Effects of NADPH-Cytochrome

Requirement

for Cytochrome

b, and Lipid

The requirement for cytochrome b5 and lipid for testosterone oxidation by cytochrome P45Op was reevaluated in reconstitution systems containing 50 pg/ml Emulgen 911. As previously determined in the absence of nonionic detergent (Table III), testosterone Go-hydroxylation by cytochrome P45Op required the presence of an equimolar amount of cytochrome b5 and 200 pg/ml microsomal lipid, as shown in Table V. In the presence of Emulgen 911, testosterone oxidation by cytochrome P45Op was undetectable in the absence of lipid, or in the presence of 200 pg/ml dilauroylphosphatidylcholine. Effects of Cytochrome

P45Op

The relationship between 6P-hydroxytestosterone formation and cytochrome P45Op concentration is shown in Fig. 7. Product formation increased as the concentration of cytochrome P45Op was increased up to 200

P450 Reductase

The rate of testosterone Go-hydroxylation by cytochrome P45Op (50 pmol/ml) increased as the concentration of cytochrome P450 reductase was increased up to 250 pmol/ml, as shown in Fig. 8. Above 300 pmol/ml, NADPH-cytochrome P450 reductase progressively inhibited testosterone oxidation by cytochrome P45Op. Over the same concentration range, NADPH-cytochrome P450 reductase does not inhibit testosterone oxidation by cytochromes P450a, P450b, or P450h (44),

TABLE Reconstitution

Incubation

stimulated by high concentrations of potassium phosphate buffer, as shown in Fig. 6. At 200 mM potassium phosphate, testosterone G/3-hydroxylation by purified cytochrome P45Op was optimum at pH -7.25 (Fig. 6). In subsequent experiments, cytochrome P45Op was incubated with 200 mM potassium phosphate, pH 7.25.

173

P45Op

of Testosterone Rat Cytochrome

III G/3-Hydroxylation P45Op: PART

conditions

by Purified 2

Testosterone Go-hydroxylation (pmol formed incubation)

Complete system” Ratio of reductase to P45Op 2:l 4:l 8:l Ratio of b5 to P45Op 0.5:1 1:l 2:l 4:l Microsomal lipid

180b 180 210 210 110 180 170 140 180 260 410

50 rg 100 I%

200 I% Testosterone 250 nmol 500 nmol 1000 nmol Magnesium chloride

180 260 270

3mM

180 200 200

5mM 10 mM

Potassium phosphate 100 mM, pH 7.4 200 mM, pH 7.4 a The complete system was the same to Table I, except sodium cholate was nents were added in a final volume of terminated after 20 min. Testosterone quantitated by HPLC (21). ’ Turnover value is 180 pmol metabolite

per

180 210 as that described in footnote a omitted, the first four compo350 ~1, and incubations were metabolites were resolved and formed/nmol

P450p/min.

174

HALVORSON TABLE

Effects

of Detergents

Incubation

IV

on Testosterone Rat Cytochrome

G&Hydroxylation P45Op

by Purified

Testosterone G&hydroxylation (pmol formed incubation)

conditions

per

400b 420 780 (twofold) 380 260 520 2450 (sixfold)

Complete system” + 200 pg sodium cholate + 200 pg Chaps + 200 pg octylglucoside + 20 pg Tween 20 t 20 gg Lubrol PX + 20 pg Emulgen 911

’ In the complete system, the l-ml incubation mixtures contained 50 pmol P45Op, 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b,, 200 pg of microsomal lipid extract, 200 pmol potassium phosphate buffer (pH 7.4), 5 pmol MgClz, 1 amol EDTA, and 500 nmol testosterone (dissolved in 40 pl methanol). The first four components were added in a final volume of 100 ~1, followed by 200 ~1 of 1 M potassium phosphate buffer (pH 7.4) and 100 ~1 of detergent. The last three components were premixed and added in 550 ~1 water. Reactions were initiated with 50 ~1 of NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 prnol glucose 6-phosphate and 1 rmol NADP), and terminated after a 20.min incubation at 37-C. Testosterone metabolites were resolved and quantitated by HPLC

ET

AL.

nor metabolites were 18-hydroxytestosterone (3%), l/I-hydroxytestosterone (3%), 16/3-hydroxytestosterone (2%), and androstenedione (2%). For comparative purposes, cytochromes P450a, P450b, P450h, and P450m were reconstituted under the same conditions that supported testosterone oxidation by cytochrome P45Op, and the results are shown in Table VI. Under the latter conditions, the rate of testosterone oxidation catalyzed by cytochromes P450a, P450b, P450h, and P450m was 63%, 9%, 44%, and 24%, respectively, compared with the rate catalyzed by these enzymes in the presence of dilauroylphosphatidylcholine. The results in Table VI indicate that the reconstitution conditions described in this study supported testosterone oxidation by cytochrome P45Op at rates that are comparable to those catalyzed by other purified forms of rat liver cytochrome P450. Antibody Against Cytochrome P45Op Polyclonal antibody against cytochrome P45Op was raised in rabbits and subjected to immunoadsorption chromatography, as described under Experimental Procedures. This antibody was used to probe a Western blot of liver microsomes from untreated and troleandomycin-

(21). * Turnover

value

is 400 pmol

metabolite

formed/nmol

hence, the reason for the inhibitory P45Op is not clear.

P450p/min.

effect on cytochrome

Effects of Incubation pime The time course of 6@-hydroxytestosterone formation by cytochrome P45Op is shown in Fig. 9. Two concentrations of cytochrome P45Op were examined: 50 pmol/ml was chosen because all previous experiments were carried out with this concentration of cytochrome P45Op, and 25 pmol/ml was chosen because product formation is not directly proportional to cytochrome P45Op above this concentration (as shown in Fig. 7). At either concentration, 6P-hydroxytestosterone continued to accumulate during the 40-min incubation, but product formation was strictly proportional to time during only the first 5 min. Catalytic Turnover and Comparison with Other P450 Enzymes Under the reconstitution conditions described in Table VI, purified cytochrome P45Op converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P45Op/min. The four major metabolites were 6@-hydroxytestosterone (51%), 2/3hydroxytestosterone (18%), 15P-hydroxytestosterone (ll%), and 6-dehydrotestosterone (10%). The four mi-

0

I 25

I 50 EMULGEN

I 75

100

200

911 &g/ml)

FIG. 5. Effects of the nonionic detergent Emulgen 911 on testosterone 6P-hydroxylation by purified cytochrome P45Op. Each l-ml incubation mixture contained 50 pmol P45Op, 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b5, 200 ag microsomal lipid extract, O-200 ag Emulgen 911, 5 Gmol MgC12, 1 pmol EDTA, 500 nmol testosterone (dissolved in 40 pl methanol), and 200 rmol potassium phosphate, pH 7.4. The first four components were added in a final volume of 100 ~1, followed by 100 pl of O-0.2% Emulgen 911. The next three components were premixed and added in 550 ~1 water, followed by 200 ~11 M potassium phosphate buffer (pH 7.4). Reactions were initiated with 50 al NADPH-generating system (1 Unit glucose6-phosphate dehydrogenase, 5 Fmol glucose B-phosphate, and 1 pmol NADP) and terminated after a 20-min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21).

RECONSTITUTION

:

OF

PURIFIED

RAT

CYTOCHROME

175

P45Op

tosterone 6a- and 7cu-hydroxylation), cytochrome P450h (testosterone 2~, 16a-, and 17-oxidation to androstenedione), or cytochrome P450m (testosterone 15a-hydroxylation). The effects of anti-P450p on testosterone 16Phydroxylation depended on the source of liver microsomes. The low rate of testosterone 16P-hydroxylation catalyzed by liver microsomes from mature male rats was inhibited by anti-P450p, as shown in Fig. 11. In contrast, anti-P450p had little effect (~5%) on the high rate of testosterone 16P-hydroxylation catalyzed by liver microsomes from phenobarbital-induced rats, which is catalyzed by cytochrome P450b (21, 56). However, the effects of anti-P450p on the 16&hydroxylation of testosterone catalyzed by liver microsomes from untreated rats were variable (lo-40%), which was largely due to difficulties in measuring this minor pathway of testosterone oxidation. Overall, these results indicate that anti-P450p does not inhibit the catalytic activity of cytochromes P450a, P450b, P450h or P450m, and that cytochrome P45Op (or an immunochemically related protein) is the enzyme in liver microsomes from untreated rats largely responsible for converting testosterone to

111 50

100

CONCENTRATION

6.8

200

(mM)

7.25

7.4

7.7

PH

FIG. 6. Effects of buffer concentration and pH on testosterone 6@hydroxylation by purified cytochrome P45Op. Each l-ml incubation mixture contained 50 pm01 P45Op, 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome bB, 200 fig microsomal lipid extract, 50 pg Emulgen 911, 5 Fmol MgC&, 1 pmol EDTA, 500 nmol testosterone (dissolved in 40 ~1 methanol), and 50-200 pmol potassium phosphate, pH 6.8 to 7.7. The first four components were added in a final volume of 100 ~1, followed by 100 ~1 of 0.05% Emulgen 911. The next three components were premixed and added in 550 ~1 water, followed by 200 ~10.25 to 1 M potassium phosphate buffer, pH 7.4 (left) or 200 ~1 of 1 M potassium phosphate buffer, pH 6.8 to 7.7 (right). Reactions were initiated with 50 ~1 NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose 6-phosphate, and 1 Fmol NADP) and terminated after a 20-min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21).

TABLE

Incubation

treated rats, and various purified rat P450 enzymes. As shown in Fig. 10, the antibody recognized cytochrome P45Op, but not cytochromes P450a, P450b, P45Oc, P45Og or P450h. However, the antibody recognized two proteins in liver microsomes from troleandomycintreated rats, one of which was electrophoretically distinct from purified cytochrome P45Op. Effects of Anti-P450p on Microsomal Testosterone Oxidation Testosterone oxidation by liver microsomes from untreated immature and mature male and female rats, and from mature male rats treated with pregnenolone-16acarbonitrile, dexamethasone, or phenobarbital was examined in the presence and absence of various concentrations of anti-P450p. In all cases, antibody against cytochrome P45Op caused a concentration-dependent inhibition of the microsomal oxidation of testosterone to 6-dehydrotestosterone and to l/3-, a/3-, 6p-, 15/3-, and 18-hydroxytestosterone. The degree of inhibition of these pathways of testosterone oxidation exceeded 85%, as indicated in Fig. 11. Anti-P450p did not inhibit (~10%) the catalytic activity of cytochrome P450a (tes-

V

Reconstitution of Testosterone G/3-Hydroxylation by Purified Cytochrome P45Op in the Presence of Emulgen 911

conditions

Complete systema Cytochrome b, None 25 pmol(50%) 100 pm01 (200%) Microsomal lipid None 100 /lg (50%) 400 pg (200%) DLPC’

200 A%!

Testosterone G@hydroxylation (pmol formed incubation)

Rat

per

3900* 200 2660 4000 0 4120 3180 0

u In the complete system, the l-ml incubation mixtures contained 50 pmol P45Op, 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b5, 200 pg of microsomal lipid extract, 50 fig of Emulgen 911,5 rmol MgC12, 1 pmol EDTA, and 500 nmol testosterone (dissolved in 40 ~1 of methanol), and 200 pmol potassium phosphate, pH 7.25. The first four components were added in a final volume of 100 ~1, followed by 100 ~1 of 0.05% Emulgen 911. The next three components were premixed and added in 500 ~1 of water, followed by 200 ~1 of 1 M potassium phosphate buffer (pH 7.25). Reactions were initiated with 50 pl of NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose 6-phosphate and 1 pmol NADP), and terminated after a 20-min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21). * Turnover value is 3.9 nmol metabolite formed/nmol P450p/min. ’ The microsomal lipid extract was replaced with 200 fig dilauroylphosphatidylcholine (DLPC).

176

HALVORSON

0p 0

. I I I 50 100 150 CYTOCHROME P-450~ (pmolhncubation)

I 200

FIG.

7. Testosterone Go-hydroxylation by purified cytochrome P45Op as a function of P45Op concentration. Each l-ml incubation mixture contained O-200 pmol P45Op, 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b6, 200 pg microsomal lipid extract, 50 pg Emulgen 911, 5 pmol MgQ, 1 Frnol EDTA, 500 nmol testosterone (dissolved in 40 pl methanol), and 200 bmol potassium phosphate, pH 7.25. The first four components were added in a final volume of 100 ~1, followed by 100 ~1 0.05% Emulgen 911. The next three components were premixed and added in 550 al water, followed by 200 ~1 1 M potassium phosphate buffer (pH 7.25). Reactions were initiated with 50 al NADPH-generating system (1 Unit glucose-6phosphate dehydrogenase, 5 I.rmol glucose 6-phosphate, and 1 amol NADP) and terminated after a 20.min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21). The arrow indicates equimolar amounts of cytochrome P45Op and cytochrome b,.

l/3-, 2p-, 6p-, l@?-, and 18-hydroxytestosterone dehydrotestosterone.

ET

AL.

components was found to influence the catalytic activity of cytochrome P45Op. It was also important to premix cytochrome P45Op, NADPH-cytochrome P450 reductase, cytochrome b5, and microsomal lipid in a relatively small volume (i.e., 100 pl), presumably to allow formation of a catalytically active complex. However, preincubating this mixture at various temperatures for up to 30 min did not further enhance the rate of testosterone oxidation by cytochrome P45Op (results not shown). The ability of Emulgen 911 to stimulate testosterone oxidation by cytochrome P45Op was unexpected because this nonionic detergent has been shown previously to inhibit testosterone oxidation by purified cytochromes P450a, P450b, and P450h (55). Cytochrome P450b is particularly sensitive to the inhibitory effects of Emulgen 911, so it was not surprising that testosterone oxidation by this enzyme declined to the greatest extent when cytochromes P450a, P450b, P450h, and P450m were reconstituted under the same conditions as cytochrome P45Op (see Table VI). In addition to its stimulatory effect on testosterone oxidation, Emulgen 911 markedly enhanced the binding of CO to dithionite-reduced cytochrome P45Op (Table I). In the absence of this and cer-

and 6-

DISCUSSION

In the present study, we have established conditions to reconstitute testosterone oxidation by purified cytochrome P45Op, and have determined that this enzyme converts testosterone to four major metabolites (Zp-, So-, and 15P-hydroxytestosterone and B-dehydrotestosterone) and four minor metabolites (lp-, 16p-, and 18hydroxytestosterone and androstenedione). The rates of formation of all eight metabolites varied in unison as the conditions used to reconstitute cytochrome P45Op were varied. However, for convenience only data for testosterone G/3-hydroxylation are shown in the figures and tables. Testosterone oxidation by cytochrome P45Op was stimulated by cytochrome b5and the nonionic detergent, Emulgen 911, and was dependent on high concentrations of a microsomal lipid extract (which could not be substituted with dilauroylphosphatidylcholine). Testosterone oxidation by cytochrome P45Op was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these

100 0 200 300 NADPH-CYTOCHROME P-450 (pmolhncubation)

FIG.

400 500 REDUCTASE

8. Testosterone Go-hydroxylation by purified cytochrome P45Op as a function of NADPH-cytochrome P450 reductase concentration. Each l-ml incubation mixture contained 50 pmol P45Op, O500 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b5, 200 pg microsomal lipid extract, 50 pg Emulgen 911,5 pmol MgCl*, 1 pmol EDTA, 500 nmol testosterone (dissolved in 40 al methanol), and 200 pmol potassium phosphate, pH 7.25. The first four components were added in a final volume of 100 ~1, followed by 100 ~1 of 0.05% Emulgen 911. The next three components were premixed and added in 550 ~1 water, followed by 200 of 1 M potassium phosphate buffer (pH 7.25). Reactions were initiatedwith 50 ~1 NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 wmol glucose 6-phosphate and 1 rmol NADP) and terminated after a 20-min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21). The arrow indicates the concentration of cytochrome P45Op and cytochrome b5.

RECONSTITUTION

1.0

OF

0

50

pm01 P-450p

0

25

pmol

PURIFIED

P-450~

0.5

O-0 0

10

20 TIME

(min)

FIG. 9. Time course of testosterone 6P-hydroxylation by purified cytochrome P45Op. Each l-ml incubation mixture contained 25 or 50 pmol P45Op (open and closed circles, respectively), 250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome bg, 200 pg microsomal lipid extract, 50 pg Emulgen 911, 5 pmol MgCl*, 1 pmol EDTA, 500 nmol testosterone (dissolved in 40 pl methanol), and 200 wmol potassium phosphate, pH 7.25. The first four components were added in a final volume of 100 pl, followed by 100 ~1 of 0.05% Emulgen 911. The next three components were premixed and added in 550 ~1 water, followed by 200 ~1 1 M potassium phosphate buffer (pH 7.25). Reactions were initiated with 50 al NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose 6-phosphate, and 1 Kmol NADP), and terminated after a l- to 40.min incubation at 37°C. Testosterone metabolites were resolved and quantitated by HPLC (21).

tain other nonionic detergents, the concentration of cytochrome P45Op was grossly underestimated when determined by the method of Omura and Sato (48). Over the concentration range shown in Fig. 5, Emulgen 911 inhibited all pathways of testosterone oxidation by liver microsomes from troleandomycin-treated rats (results not shown). This indicates that Emulgen 911 stimulates testosterone oxidation by purified cytochrome P45Op, but not by microsomal cytochrome P45Op. The rate of testosterone 2/3-, 6/?-, and 15P-hydroxylation by microsomal cytochrome P45Op was estimated previously to be 0.73, 2.0, and 0.48 nmol/nmol P45Op/ min, respectively (21). These estimates were based on the increase in testosterone 2p-, 6p-, and 15P-hydroxylation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycinor erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). As shown in Table VI, purified cytochrome P45Op catalyzed the 2/3-, 6p-, and 15P-hydroxylation of testosterone at 3.3, 9.2, and 2.0 nmol/ nmol P450p/min, which is 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P45Op. We previously reported that partially purified cytochrome P45Op, when reconstituted with NADPH-cyto-

RAT

CYTOCHROME

P45Op

177

chrome P450 reductase and dilauroylphosphatidylcholine, catalyzed the 6P-hydroxylation of testosterone at 3.1 nmol/nmol P450p/min (21). The results of the present study indicate this value is erroneously high for two possible reasons. First, the concentration of cytochrome P45Op was estimated from the CO-difference spectrum of the dithionite-reduced hemoprotein. As indicated in Table I, this method underestimates the concentration of cytochrome P45Op by a factor of 2 to 3. Second, the purified protein may have been stimulated by residual contamination with Emulgen 911, which was used throughout the original purification procedure. In the present study, Lubrol PX was used instead of Emulgen 911, and additional steps were taken to remove detergent during the final steps of the purification procedure. We previously acknowledged that our original preparation of cytochrome P45Op (which was purified from induced male rats) was contaminated with lo-15% cytochrome P450h. As shown in Fig. 4, cytochromes P450h and P45Op were indistinguishable by SDS-PAGE. It is also possible that the purified protein was a mixture of cytochromes P45Op (IIIAl) and P4506,., (IIIAB). A comparison between our results and those reported by Yamazoe et al. (40) indicates that purified cytochrome P45Op and cytochrome P45060.1 differ markedly in terms of the conditions required to reconstitute their catalytic activity toward testosterone.

FIG. 10. Western blot of liver microsomes and purified P450 enzymes probed with antibody against cytochrome P45Op. Lanes 1 and 2 contained liver microsomes (5 fig protein) from untreated and troleandomycin-treated female rats, respectively. Lanes 3 and 4 contained 10 and 4 pmol of purified cytochrome P45Op, respectively. Lane 5 contained a mixture (4-6 pmol of each) of cytochromes P450a (M, 47,000), P45Og (M, 50,000), P45oh (M, 51,000), P450b (M, 52,000), and P45Oc (M, 56,000). After SDS-PAGE, proteins were transferred electrophoretically to Immobilon, which was probed with antibody against cytochrome P45Op as described under Experimental Procedures.

178

HALVORSON

ET

TABLE Testosterone

Oxidation

P45Op P450a P450b P450h P450m

Incubation conditions” A A B A B A B A B

2LY

28

6cu

68

In

0.6

~

3.3

0.5 1.1

9.2 -

16 25

-

--

Rat Cytochrome

P450 Isozymes

Testosterone oxidation metabolite formed/nmol

1P

3.5 8.3

VI

by Purified

(nmol P450

AL.

0.2 0.4 0.2 2.0

0.2 1.9

1501

158

* P450/min) 16cu

2.0

1.1 16 3.9 9.5 1.2 11

~ 0.5

160

A

18

A6

Total

0.4

0.3

0.5

1.4 14 -

1.1 16 2.1 5.0 0.2 1.4

~

1.9 0.6 1.2 ~

0.5 4.6

-

18 17 27 4 46 10 23 5 21

’ A, the l-ml incubation mixtures contained 25-50 pmol P450,250 pmol NADPH-cytochrome P450 reductase, 50 pmol cytochrome b,, 200 pg of microsomal lipid extract, 50 ~g of Emulgen 911,5 Fmol MgC&, 1 wmol EDTA, 500 nmol testosterone (dissolved in 40 ~1 of methanol), and 200 pmol potassium phosphate, pH 7.25. The first four components were added in a final volume of 100 ~1, followed by 100 ~1 of 0.05% Emulgen 911. The next three components were premixed and added in 550 ~1 of water, followed by 200 ~1 of 1 M potassium phosphate buffer (pH 7.25). Reactions were initiated with 50 ~1 of NADPH-generating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose 6-phosphate and 1 rmol NADP), and terminated after a 5- to 20.min incubation at 37°C. B, the l-ml incubation mixtures contained 50 pmol P450, 500 pmol NADPH-cytochrome P450 reductase, 15 fig of dilauroylphosphatidylcholine, 3 prnol MgCl,, 1 rmol EDTA, and 250 nmol testosterone (dissolved in 40 ~1 of methanol), and 50 Fmol potassium phosphate, pH 7.4. The first three components were added in a final volume of 100 ~1, followed by the next four components, which were premixed and added in 850 ~1 of water. Reactions were initiated with 50 ~1 of NADPHgenerating system (1 Unit glucose-6-phosphate dehydrogenase, 5 pmol glucose &phosphate and 1 pmol NADP), and terminated after a 5-min incubation at 37°C. b With the exception of A (androstenedione) and A6 (6-dehydrotestosterone), the abbreviations denote the hydroxytestosterone metabolite formed, e.g., 60 denotes G/3-hydroxytestosterone.

The cytochrome P45Op used in the present study was purified by a procedure that involved anion-exchange chromatography on DEAE-Sepharose CL-6B or DEAESephacel. The latter column has been shown by Halpert to be capable of resolving three members of the P450 III gene family, namely PCNa (P45Op), PCNb, and PCNc. The results of our study established that anion-exchange chromatography on DEAE-Sepharose CL-GB, in contrast to DEAE-Sephacel, is also capable of resolving free cytochrome P45Op from cytochrome P45Op complexed with troleandomycin. Apart from a tertiary amine, which is metabolized and complexed with the heme of cytochrome P45Op, troleandomycin contains no ionizable groups that would be expected to interact directly with DEAE-Sepharose CL-6B (16). Hence, the basis for resolving free and troleandomycin-bound cytochrome P45Op by anion-exchange chromatography remains to be established. We recently reported that increasing the concentration of potassium phosphate up to 200 mM or raising the pH up to 8.0 caused a marked increase in the rate of conversion of testosterone to lp-, 2@-, 6/?-, 15&, and 18-hydroxytestosterone and 6-dehydrotestosterone catalyzed by liver microsomes from untreated or phenobarbitaltreated male rats (57). The results in Fig. 6 indicate that testosterone oxidation by purified cytochrome P45Op also increased when the concentration of potassium

phosphate was increased up to 200 mM. However, the pH optimum of purified cytochrome P45Op was -7.25, not 8.0. This discrepancy was due to differences in the procedure to determine the pH optimum of purified versus microsomal cytochrome P45Op. In the former case, purified cytochrome P45Op was incubated with 200 mM potassium phosphate buffer at various pH values. In the latter case, liver microsomes were incubated at the same pH values, but the concentration of potassium phosphate buffer was only 50 mM. When liver microsomes were incubated with 200 mM potassium phosphate buffer, the pH optimum for those pathways of testosterone oxidation catalyzed by cytochrome P45Op shifted from pH 8.0 to pH 7.25 (results not shown). This shift in pH optimum was observed with liver microsomes from untreated male rats, phenobarbital-treated male rats, and troleandomycin-treated female rats (decomplexed with potassium ferricyanide). Hence, under the same experimental conditions, the effects of pH and ionic strength on testosterone oxidation by cytochrome P45Op were the same for both the purified and the microsomal enzyme. In liver microsomes from untreated or phenobarbitaltreated rats, the same pathways of testosterone oxidation catalyzed by cytochrome P45Op are more likely to be catalyzed by cytochrome P450,,, (9,39,40). The uniformity of the results obtained suggest that testosterone oxidation by cytochromes P45Op and P450,,., is stimu-

RECONSTITUTION

n

WITH

q

ANTIBODY

WITHOUT

OF

PURIFIED

ANTIBODY

r300

18

201 28

6a

68

la

TESTOSTERONE

15a 158 160! 168

A

16

METABOLITE

FIG. 11.

Effects of antibody against cytochrome P45Op on testosterone oxidation catalyzed by liver microsomes from mature male Sprague-Dawley rats. Liver microsomes (0.25 and 0.5 mg protein) were incubated at room temperature with anti-P450p (0.1 to 1.0 mg IgG). The amount of antibody was adjusted to 1.0 mg with an IgG fraction purified from preimmune rabbit antiserum. After 15 min, each sample was incubated with testosterone and NADPH (21). Testosterone metabolites were resolved and quantitated by HPLC as described previously (21). Except for A (androstenedione) and A6 (6. dehydrotestosterone), the abbreviations denote the hydroxytestosterone formed (e.g., 6p denotes 6@-hydroxytestosterone).

lated in both cases by high ionic strength, and that the pH optimum of both enzymes is -8.0 at 50 mM potassium phosphate buffer and -7.25 at 200 mM buffer. Polyclonal antibody against cytochrome P45Op strongly inhibited (>85%) the conversion of testosterone to lo-, 2fl-, 6fl-, 15fl-, and 18-hydroxytestosterone and 6-dehydrotestosterone by rat liver microsomes. These results confirm and extend those of Waxman et al. (19, 22), who reported that antibodies against rat cytochrome P450PCN.E or PB-2a (which are apparently identical to IIIAB) or human cytochrome P45Onr (P450 IIIA4) strongly inhibit the G&hydroxylation of testosterone, androstenedione, and progesterone by rat and human liver microsomes. However, the polyclonal antibodies used in these inhibition experiments recognize both IIIAl and IIIAB, hence, the relative contribution of these closely related enzymes to microsome-catalyzed reactions remains to be established. However, by successfully reconstituting its catalytic activity, we have established that cytochrome P45Op catalyzes the oxidation of testosterone to lb-, 2p-, 6p-, 15@-, 16/3-, and 18hydroxytestosterone and 6-dehydrotestosterone and androstenedione. Therefore, cytochrome P45Op can potentially contribute to each of these pathways of testosterone oxidation catalyzed by liver microsomes. The results of the present study also confirm that cytochrome P45Op can oxidize testosterone to 6-dehydrotestosterone, which was first reported by Nagata et al. (58).

RAT

CYTOCHROME

179

P45Op

Although cytochrome P45Op could be resolved from IIIAB by anion-exchange chromatography on DEAESepharose CL-6B or DEAE-Sephacel, as described by Halpert (lo), we were unable to resolve these enzymes by SDS-PAGE (results not shown). However, antibody against cytochrome P45Op recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-induced female rats (see Fig. 10). Purified cytochrome P45Op was not contaminated with the lower molecular weight form of cytochrome P45Op, although both proteins were clearly inducible by treatment of rats with troleandomycin. The regulation and possible function of these electrophoretically distinct forms of cytochrome P45Op will be described in a later paper. The biotransformation reactions catalyzed by cytochrome P45Op have been inferred from the effects of cytochrome P45Op inducers and inhibitors on reactions catalyzed by liver microsomes. Based on these types of studies, we previously proposed that cytochrome P45Op was the enzyme in rat liver microsomes primarily responsible for converting digitoxin to digitoxigenin bisdigitoxoside (at rates comparable to the 15/3-hydroxylation of testosterone) (29). Under the same conditions described for testosterone oxidation, purified cytochrome P45Op did not convert digitoxin to digitoxigenin bisdigitoxoside, as determined by HPLC (results not shown). Based on the recovery of digitoxigenin bisdigitoxoside from spiked samples, we estimated that the rate of digitoxin oxidation by purified cytochrome P45Op was less than 5 pmol/nmol P450p/min. The inability of purified cytochrome P45Op to catalyze digitoxin oxidation may indicate that the conditions established to reconstitute the catalytic activity of cytochrome P45Op toward testosterone may not be suitable for other substrates. Alternatively, it may indicate that the conversion of digitoxin to digitoxigenin bisdigitoxoside is catalyzed by cytochrome P450 IIIAB or another member of the P450 III gene family. Support for this latter possibility will be described in a later paper. ACKNOWLEDGMENTS We are grateful to Steve Wrighton and Dene Ryan for helpful advise and discussions. We thank Drs. Hamilton and Rouse of the Veterans Administration Hospital (Kansas City, MO) for their help with the amino acid sequence analysis.

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