Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats

Biochimica et Biophysica Acta 926 (1987) 349-358 Elsevier 349 BBA 22845 Purification and characterization of liver microsomal cytochrome P-454) fro...

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Biochimica et Biophysica Acta 926 (1987) 349-358 Elsevier

349

BBA 22845

Purification and characterization of liver microsomal cytochrome P-454) from untreated male rats Yoshihiko Funae and Susumu Imaoka Laboratory of Chemistry, Osaka Ci(v University Medical School, Osaka (Japan) (Received 17 March 1987) (Revised manuscript received 24 August 1987)

Key words: Cytochrome P-450; Constitutive form; NH2-terminal sequence; (Male rat)

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH2-terminal sequence and catalytic activity of them were determined. The NH2-terminal sequences of six forms of cytochrome P-4S0 (designated P4S0 UT-I, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome/-450 isozymes are of different molecular species. The hydrophobicity values of the NH2-terminal sequences of P450 UT-I and P4S0 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54 000, of the six forms of P-4S0. P4S0 UT-2 was active in demethylation of benzphetamine, P450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-I and P450 UT-2 were active in the 2aand 16a-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6fl-, 7or- and ISa-hydroxylation of the same steroid. We believe that P450 UT-I, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P.4SO.

Introduction The hepatic monoxygenase system can metabolize a large number of endogenous and exogenous substrates. This can be accounted for by the participation of multiple forms of cytochrome P-450. The population of cytochrome P450 forms changes quantitatively and qualitatively after treatment of animals with a variety of compounds [1]. Many forms of cytochrome P-450 have been purified and characterized [2,3]. Using an anion-exchange HPLC column, we detected more than ten forms of cytochrome P-450 present

Abbreviation: DLPC, dilauroylphosphatidylcholine. Correspondence: Y. Funae, Laboratory of Chemistry, Osaka City University Medical School, Abeno-ku, Osaka 545, Japan.

in liver microsomes of the untreated male rat [4]. Schenkman et al. [5] have detected 11 forms of cytochrome P-450 from untreated rats. However, it is uncertain how many forms of cytochrome P-450 actually exist and the biological properties have yet to be determined. Although the main function of cytochrome P-450 seems to be metabolism of xenobiotics, cytochrome P-450, in particular, constitutive cytochrome P-450, probably plays a role in homeostasis. Liver microsomal cytochrome P-450 forms metabolize steroids [6-8] and prostaglandins [9,10]. They also form various biologically active lipoxygenase products from arachidonic acid [11]. Several forms of cytochrome P-450 from untreated rat liver microsomes have been purified to homogeneity as indicated by SDS-polyacrylamide gel electrophoresis [12-17]. Since the content of cytochrome P-450 in the liver of untreated rats is

0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

350

low and the protein-chemical properties of different forms of cytochrome P-450 are similar, it is difficult to separate and simultaneously purify many different froms of cytochrome P-450, using conventional chromatography. We have studied the resolution of cytochrome P-450 isozymes using and HPLC technique [4,18,19]. We have used this method for the purification of multiple forms of cytochrome P-450 from rats using preparative DEAE-column [20]. We have further developed the HPLC techniques using a preparative cationexchange column and a hydroxyapatite column. Six different froms of cytochrome P-450 from the livers of untreated male rats were purified. We describe here the NH2-terminal sequence, testosterone hydroxylation activity and other properties. The NH2-terminal amino acids sequence facilitate identification of the cytochrome P-450 isozymes. Materials and Methods

Purification of cytochrome P-450 Preparation of microsomes and fractionation with poly(ethylene glycol). Preparation of liver microsomes and purification of cytochrome P-450 forms were performed as described previously [20]. Hepatic microsomes were prepared from male Sprague-Dawley rats weighing 200-250 g (Nippon Clea, Kyoto, Japan). The microsomal preparation (7 g of microsomal protein) was diluted to 7 mg protein/ml with 0.1 M potassium phosphate buffer (pH 7.4) containing 30% glycerol, 1.0 mM EDTA and 1.0 mM dithiothreitol. A solution of 10% sodium cholate (adjusted to pH 7.5) was added to a final concentration of 3 mg/mg protein. After stirring for 30 rain, a solution of 50% poly(ethylene glycol) 6000 (recrystallized with diethyl ether and acetone) was added and the fraction between 7-15% was obtained. Octylamino-Sepharose 4B chromatograph),. This fraction was suspended in a final concentration of 8 mg protein/ml in 0.1 M potassium phosphate buffer (pH 7.2) containing 20% glycerol, 1 mM EDTA and 0.5 mM dithiothreitol (designated OA buffer). A solution of sodium cholate was added dropwise, with stirring, to a final concentration of 0.7%. This solution containing 1.7 g protein was

loaded on an octylamino-Sepharose 4B column (2.6 x 50 cm) which had been equilibrated with OA buffer/0.5% sodium cholate. OctylaminoSepharose 4B was prepared according to the method of Cuatrecasas [21]. 1,8-Diaminooctane had been coupled to cyanogen bromide-activated Sepharose 4B (Pharmacia, Sweden). After washing with OA buffer containing 0.5% sodium cholate cytochrome P-450 was eluted with OA buffer containing 0.4% sodium cholate and 0.1% Emulgen 911. The eluate (500 ml) from octylamino-Sepharose 4B chromatography was concentrated to 20 ml, using an ultrafiltration membrane (UK-50, Toyo Roshi, Tokyo). During filtration, Emulgen 911 is removed to some extent (one-third) by the UK-50 membrane, which cuts off 50-kDa proteins. Preparative anion-exchange HPLC. The concentrated material was diluted 15-fold with 20 mM Tris-acetate buffer (pH 7.5) containing 20% glycerol and adjusted to 5 mg protein/ml. This sample, containing 300 mg protein was applied to HPLC. The HPLC was equipped with a preparative DEAE-5PW anion-exchange column (2.15 × 15 cm, Toyo Soda, Tokyo) performed with a linear salt gradient made by buffer A (20 mM Tris-acetate (pH 7.5), containing 20% glycerol and 0.4% Emulgen 911) and buffer B (1 M sodium acetate was added to buffer A (pH 7.5)). When a sodium cholate was added to the elution buffers, the separation of different forms of cytochrome P-450 got worse. The most critical points of the conditions of anion-exchange HPLC on the resolution of multiple forms of cytochrome P-450 are the type and concentration of detergent added to the mobile phase. Triton X-100, Lubrol and Emulgen 913 did not give a good resolution. Emulgen 911, with an optimum concentration of 0.4% for the DEAE-5PW column, proved to be the most effective [19]. The elution profile of hemoprotein and protein were simultaneously monitored at 417 and 244 nm, respectively. When the detergent Emulgen 911 is present in the solution, it is impossible to monitor the elution profile of protein at 280 nm. However, proteins can be monitored at 244 nm, the absorption minimum of Emulgen 911. The peaks which absorbed at both 417 and 244 nm were collected, separately. The adsorbed fractions in preparative DEAE-HPLC

351 were further rechromatographed by HPLC using an analytical DEAE-5PW column and the passthrough fraction was further purified by HPLC using cation-exchange column. Preparative cation-exchange HPLC. The passthrough fraction (50-100 ml) from the preparative DEAE-5PW column was concentrated to 10-20 ml using the UK-50 ultrafiltration membrane and diluted 3-fold with 20 mM sodium phosphate buffer (pH 6.5) containing 20% glycerol to a concentration of 1-2 mg/ml, to prevent the need for dialysis. This solution containing 100-150 mg protein was further purified by HPLC equipped with a preparative cation-exchange column (Asahipak EF-502CP, 21.5 x 100 mm, Asahi Chemical, Tokyo). Cytochrome P-450 was eluted at a flow rate of 2.0 ml/min, at room temperature, with a linear salt gradient made by buffer C (20 mM sodium phosphate (pH 6.5) containing 20% glycerol and 0.4% Emulgen 911) and buffer D (1.0 M sodium acetate was added to buffer C (pH 6.7)) (50% buffer D in 180 rain). Hydroxyapatite HPLC. When the purified cytochrome P-450 was not homogeneous, the obtained cytochrome P-450 was further applied on HPLC using a hydroxyapatite column (KB-column Type S, 0.6 × 10 cm, Koken, Tokyo) as described previously [22]. This column was packed with spherical particles of hydroxyapatite. Cytochrome P-450 containing 1-3 mg protein (5 mg protein can be loaded on this column) was loaded on a hydroxyapatite column equilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 20% glycerol, 0.2% sodium cholate and 0.4% Emulgen 911. Cytochrome P-450 was eluted with a linear gradient from 10 to 350 mM phosphate buffer (pH 7.4), containing 20% glycerol, 0.2% sodium cholate and 0.4% Emulgen 911 in 70 min at a flow-rate of 0.5 ml/min, at room temperature. Emulgen 911 was removed by hydroxyapatite chromatography packed in a mini-column with Bio-Gel HT (Bio-Rad, Richmond, CA) as described previously [20]. Purified cytochromes P450 eluted with 0.35 M sodium phosphate buffer (pH 7.4) containing 20% glycerol and 0.05% sodium cholate were stored at - 9 0 ° C.

Metabolic activities of purified cytochrome P-450 Benzphetamine demethylation activity, 7-

ethoxycoumarin deethylation activity and p-nitroanisole hydroxylation were measured as described previously [20]. Aniline hydroxylation activity was measured according to the method of Imai et al. [23]. The reaction mixture, in a final volume of 0.5 ml of 0.1 M potassium phosphate buffer (pH 7.5), contained 30 pmol of cytochrome P-450, 0.3 units of cytochrome P-450 reductase, 5 /~g of DLPC (Sigma, St. Louis, MO), 0.2 /~mol of NADPH (Oriental Yeast, Tokyo) and 1 /~mol of aniline sulfate (Wako Pure Chemicals, Tokyo), was incubated for 20 min at 37 °C with shaking.

Hydroxylation of testosterone Testosterone hydroxidation activity was assayed in a 0.5 ml of reaction mixture containing 30 pmol of purified cytochrome P-450, 0.3 units of cytochrome P-450 reductase, 5/~g of DLPC, 0.2 /~mol of NADPH and 0.5 /~mol of testosterone. This reaction was carried out in 0.1 M potassium phosphate buffer (pH 7.4) and initiated by addition of NADPH. The reaction mixture was incubated at 37°C for 10 min and terminated by addition of ethyl acetate. Testosterone metabolites were extracted twice with 1 ml of ethylacetate. The extracted solution was evaporated under reduced pressure and the resulting residue was dissolved in 100 /~1 of 50% methanol. 50 /~1 of this solution were injected onto HPLC equipped with reverse-phase C18 column (NOVA-PAK, Waters Associates, Milford, MA). The column developed by isocratic elution with methanol/ H 20 (25:75) from 0 to 10 min, followed by a linear gradient to methanol/HzO/CH3CN (64: 30: 6) at 40 min. The flow rate was 0.8 ml/min and the metabolites were monitored at 254 nm. HPLC was performed at 50 ° C. Metabolites were generally quantitated by comparison of their peak areas with those of authentic hydroxylated testosterone, such as 6a-, 6/3-, 15a, 7a-, 16et- and 2ct-hydroxytestosterone. 6a- and 15t~-hydroxytestosterone was provided by Professor D.N. Kirk (Queen Mary University of London, London, U.K.), and 6fl-, 7a-, 16a- and 2a-hydroxytestosterone were kind gifts from Dr. Y. Nakamura (Shionogi Research Laboratory, Osaka). We confirmed that the hydroxylated testosterone by cytochrome P-450 was eluted at the same retention time as that of authentic sample. And we also confirmed that hydroxylated

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testosterones were derived from testosterone by using [14C]testosterone as a substrate. Quantitative analysis of hydroxylated testosterones generated by purified cytochrome P-450 in the reconstituted enzyme system was performed by calculation of the peak area eluted on HPLC, by dataprocessor.

NH,-terminal sequence analysis Purified cytochrome P-450 samples (5 nmol) were dialyzed overnight against distilled water, lyophilized and the resulting samples were dissolved to make a thin film on a glass column. Fine glass beads were poured into the glass column equipped with an LKB 4030 sequencer, and automated Edman degradation was performed using the LKB program 51.6. Phenylthiohydantoin derivatives were identified by HPLC equipped with an C1~ column (4.6 × 250 mm, Irica RP-18, Kyoto) with isocratic elution of solvent (CH3CN / 10 mM acetate buffer (pH 4.5), 42.7 : 57.3) at 35 o C, monitoring at 254 nm. When the amount of cytochrome P-450 obtained was small, the NHz-terminal sequence was analyzed by a gas-phase sequencer (Applied Biosystems Model 470A, U.S.A.).

Other methods Cytochrome P-450 was measured according to the spectral method [24]. Protein concentrations were measured by the method of Lowry et al. [25]. For the measurement of the protein of the eluate on HPLC containing Emulgen, we used the method of Dulley and Grieve [26], with some modification (final concentration of SDS was 1.5%). SDS-polyacrylamide gel electrophoresis of cytochrome P-450 was performed according to the method of Laemmli [27], using a slab separating gel (1.5 mm thick) containing 10% acrylamide. Cytochrome P-450 reductase was purified from rat liver microsomes as described previously [20]. Results

Purification of cytochrome P-450 Different forms of cytochrome P-450 from adult male Sprague-Dawley rats were purified by poly(ethylene glycol) fractionation, octylaminoSepharose chromatography and HPLC, using an-

ion- and cation-exchange columns and a hydroxyapatite column. The data of initial three steps are summarized in Table I. On the fractionation by poly(ethylene glycol), 63.7% of cytochrome P-450 was recovered and the specific content of cytochrome P-450 was over 3-times elevated over levels in microsomes solubilized with sodium cholate. When large amounts of microsomes were used, this step was very effective to remove lipids and to protect the octylaminoSepharose 4B column. Recovery of cytochrome P-450 on the octylamino-Sepharose 4B column was 54%, a value close to that (47%) of the commercially available hexylamino-Sepharose chromatography [20]. We did not intend to separate many different forms of cytochrome P-450 on this hydrophobic affinity chromatography. All of the different forms of cytochrome P-450 which were detectable by HPLC using the DEAE type of column [4] in solubilized microsomes were present in the fraction eluted with 0.1% Emulgen 911 on octylamino-Sepharose 4B chromatography. The specific content of cytochrome P-450 was approx. 3-times elevated with this step. The elution profile of cytochrome P-450 on preparative DEAE-HPLC is shown in Fig. 1. Recovery of cytochrome P-450 on the preparative DEAE-HPLC was 64%. We monitored simultaneously the elution pattern of hemoprotein and protein by measuring the absorbance at 417 and 244 nm, respectively. On the DEAE-HPLC of cytochrome P-450, phenyl-based detergent Emulgen 911 is essential for resolution of different forms of cytochrome P-450 [19]. When the elution curves of cytochrome P-450 on DEAE-HPLC are monitored simultaneously at 417 nm (detection of hemoprotein) and at 244 nm (detection of protein at the presence of Emulgen), it is easy to ascertain the purity of cytochrome P-450 in the peak fractions. Elution profiles of hemoprotein were highly reproducible on three experiments of HPLC chromatography, using a preparative DEAE-5PW column. Seven fractions (pass-through and UT3, 4, 5, 6, 7, 8) were collected manually. Specific content and recovery of these cytochrome P-450 fractions are given in Table I. 48% of cytochrome P-450 loaded on a preparative DEAE-column was eluted in the pass-through fraction and 6.8 and 6.4% of loaded cytochrome P-450 was eluted in

353 TABLE I PURIFICATION OF DIFFERENT FORMS OF CYTOCHROME P-450 IN LIVER MICROSOMES FROM UNTREATED RATS The elution pattern of UT-PT-UT-8 is shown in Fig. 1. The procedures of solubilization with sodium cholate, poly(ethylene glycol) fractionation, octylamino-Sepharose 4B chromatography and HPLC using preparative DEAE-5PW column are described in Materials and Methods. UT-PT, pass-through fraction. Fraction

Microsomes Solubilized with sodium cholate Poly(ethylene glycol) Octylamino-Sepharose 4B Preparative DEAE-5PW UT-PT UT-3 UT-4 UT-5 UT-6 UT-7 UT-8

Protein

Cytochrome P-450

(mg)

total (nmol)

specific content (nmol/mg)

recovery (%)

7 000 8 257 1498 300

3150 2 623 1670 902

0.45 0.32 1.11 3.01

100 63.7 34.4

136 4.71 6.47 10.2 12.4 2.63 0.97

436 19.3 16.9 38.9 36.5 5.3 3.4

the UT-5 and UT-6 fractions, respectively. Content of cytochrome P-450 in UT-7 and UT-8 fractions was very low. The elution peak of UT-8 was also detected on the HPLC of solubilized microsomes from phenobarbital- and 3-methyl-

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Fig. 1. Anion-exchange HPLC of octylamino-Sepharose 4Beluted cytochrome P-450. A fraction eluted with 0.1% Emulgen 911 from an octylamino-Sepharose 4B column was applied on HPLC using a preparative DEAE-5PW column (2.15 × 15 cm). HPLC was performed at flow rate of 2 ml/ml at 20-25 o C, with a linear gradient of buffer A (20 mM Tris-acetate (pH 7.5) containing 20% glycerol and 0.4% Emulgen 911) and buffer B (1 M sodium acetate was added to buffer A (pH 7.5)). Cytochrome P-450 was monitored at 417 nm and proteins were monitored at 244 nm (dotted line). PT, pass-through.

3.20 4.06 2.62 3.82 2.91 2.0 4.0

16.6 0.74 0.64 1.48 1.39 0,20 0,12

cholanthrene-treated rats [4]. HPLC using a preparative DEAE-5PW column could separate different forms of cytochrome P-450 but specific contents of cytochrome P-450 did not increase so much from the specific content of fractions from octylamino-Sepharose 4B chromatography. Therefore, the adsorbed fractions (UT-3-UT-8) were further purified by HPLC using an analytical DEAE-5PW column. Their specific contents were remarkably increased. The pass-through fraction of preparative DEAE-5PW HPLC was further applied on HPLC using a cation-exchange column (Asahipak EF-502CP). The pass-through fraction was separated into two fractions, designated UT-1 and UT-2 (Fig. 2). Recovery of P450 UT-1 and P450 UT-2 was 11 and 29%, respectively, and the specific content of cytochrome P-450 was 8.1 and 14.7 n m o l / m g protein, respectively. The UT-1 fraction obtained by cation-exchange HPLC and the UT-8 fraction obtained by anion-exchange HPLC were further purified by HPLC, using a hydroxyapatite column. The elution pattern of HPLC, using a hydroxyapatite column, of UT-1 is shown in Fig. 3. Emulgen and non-hemoproteins was eluted at the first and at the second peaks detected at 244 nm, respectively. SDS-polyacrylamide gel electrophoresis of the

354

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Fig. 2. Cation-exchange HPLC of pass-through fraction on anion-exchange HPLC. A pass-through fraction on a preparative DEAE-5PW HPLC was further applied on a preparative cation-exchange column (2.15 × 10 cm). HPLC was performed at a flow rate of 2.0 m l / m l at 20-25 o C with a linear gradient of buffer C (20 mM sodium phosphate (pH 6.5) containing 20% glycerol and 0.4% Emulgen 911) and buffer D (1 M sodium acetate was added to buffer C (pH 6.7)). Cytochrome P-450 was monitored at 417 nm and proteins were monitored at 244 nm (dotted line).

purified forms of cytochrome P-450 is shown in Fig. 4. These purified cytochrome P-450 forms were homogeneous on SDS-polyacrylamide gel

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Fig. 3. Hydroxyapatite HPLC of P450 UT-1. P450 UT-I prepared by cation-exchange HPLC was applied on HPLC using a hydroxyapatite column (0.6 x 10 cm). HPLC was performed at a flow rate of 0.5 m l / m i n with a linear gradient of sodium phosphate buffer (pH 7.4) from 10 to 350 raM, containing 20% glycerol, 0.2% sodium cholate and 0.4% Emulgen 911. Cytochrome P-450 was monitored at 417 nm and proteins were monitored at 244 nm (dotted line).

1

2

3

4

5

6

7

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Fig. 4. SDS-polyacrylamide gel electrophoresis of purified cytochrome P-450 forms. Protein standard mixtures consisted of bovine serum albumin (68000), catalase (57000), glutamate dehydrogenase (53000), ovalbumin (45 000) and c~-chymotrypsinogen (25000) (lane 1 and 10). Purified forms of cytochrome P-450 are seen in lane 2 (P450 UT-1), lane 3 (P450 UT-2), lane 4 (P450 UT-3), lane 5 (P450 UT-4), lane 6 (P450 UT-5), lane 7 (P450 UT-6), lane 8 (P450 UT-7) and lane 9 (P450 UT-8).

electrophoresis. The specific content of cytochrome P-450 and absorption maximum and molecular weight of the purified cytochrome P-450 are shown in Table II. Specific contents of purified cytochrome P-450 were over 11 n m o l / m g . P450 UT-8 is unstable, and easily denatured during purification. The apparent molecular weight of cytochrome P-450 from the untreated rats, as calculated from the mobility on SDS-polyacrylamide gel electrophoresis, was 48000-50000, except for P450 UT-8. The molecular weight of P450 UT-8 was higher than other cytochrome P-450 forms we purified. The oxidized absolute spectra of P450 UT-2 and P450 UT-3 had a shoulder peak at 398 and 395 nm, respectively, indicating that these two cytochrome P-450 forms consisted of a mixture of a low- and high-spin state of heme. The CO-reduced absorption maxima were in the range of 448-452 nm. Interestingly, the CO-reduced maximum of P450 UT-4 was as high as 452 nm.

NH2-terminal sequence The NH2-terminal sequences of the purified cytochrome P-450 (UT-1-UT-8) are summarized in Table III. Four forms of cytochrome P-450 and

355 T A B L E 1I SPECIFIC C O N T E N T , A B S O R P T I O N M A X I M U M , SPIN STATE O F H E M E A N D M O L E C U L A R W E I G H T OF P U R I F I E D C Y T O C H R O M E P-450 Absorption m a x i m u m in parentheses is the value of shoulder peak. L and H, low and high, respectively. Cytochrome

Specific content

Absorption m a x i m u m (nm)

P-450 UT-1 UT-2 UT-3 UT-4 UT-5 UT-6 UT-7 UT-8

(nmol/mg) 12.5 14.7 16.0 11.7 14.4 14.6 13.0 12.8

oxidized 417 418, (398) 418, (395) 416 418 418 417 419

reduced 415 416 415 414 414 414 414 418

two forms of cytochrome P-450 purified from phenobarbital- and 3-methylcholanthrene-treated rat liver microsomes, respectively [20] are also listed in Table III. The NH2-terminal sequences of P450 UT-3, P450 UT-7 and P450 UT-8 were determined by gas-phase type sequencer, as the amount of purified cytochromes P-450 was scanty, and all others were determined by solid-phase type sequencer. The NH2-terminal amino acid of

CO-reduced 450 451 451 452 449 449 448 449

Spin state

Molecular

L L+ H L+ H L L L L L

weight 49000 50000 50000 48 000 48000 48 000 49000 54000

P450 UT-1-P450 UT-6 was methionine. The first cycle of the NH2-terminal amino acid of P450 UT-7 and UT-8 was not determined. All the NH2-terminal amino acids of purified cytochrome P-450 start with methionine, except for the 3methylcholanthrene-inducible P450 MC-1 and P450 MC-5. The second cycle of the NH2-terminal amino acid of constitutive purified cytochrome P-450 forms, except for P450 UT-4, was acidic

T A B L E Ili N H 2 - T E R M I N A L S E Q U E N C E OF P U R I F I E D C Y T O C H R O M E P-450 A m i n o acid residues marked X were not identified. Cytochrome P-450 1 Untreated rats UT-1 UT-2 UT-3 UT-4 UT-5 UT-6 UT-7 UT-8

Ret-Asp-GLu-Ser-

5 X -ALa-Pro-

10

15

20

X -Leu-Leu-Pro

Ret-Asp-Pro-Vat-Leu-VaL-Leu-VaL-Leu-Thr-Leu-Ser-Ser-Leu-Leu-Leu-Leu-Ser Ret-Asp-Pro-VaL-Leu-VaL-Leu-VaL-Leu-Thr-Leu-Ser-Ser-Leu-Leu-Leu-Leu-Ser-Leu Het-Leu-Asp-Thr-GLy-Leu-Leu-Leu-VaL-VaL-ILe-Leu-ALa-Ser-LeuX -VaL-Ret-Phe-Leu Ret-Asp-Pro-VaL-VaL-VaL-Leu-Leu-Leu-Ser-Leu-Phe-Phe Ret-Asp-Pro-VaL-vaL-VaL-Leu-Leu-Leu-Ser-Leu-Phe-Phe-Leu-Leu-Phe X -Asn-GLy-Thr-GLy-Leu-Trp-Ser-Ret-ALa-ILe-Phe-Thr-VaL-ILe-Phe-Ite-Leu-Leu-VaL X - X -Pro-Gtu-VaL-ALa-Phe-Pro-Trp-rtu-VaL

Phenobarbital-treated rats a PB-1 Ret-Asp-Leu-VaL- X -ALa-Leu-Thr-Leu-GLu- X - X -Val-Leu PB-2 Re t - A s p - L e u - V a L-Re t - L e u - L e u - V a t - L e u - T h r - L e u - T h r - S e r - L e u - I Le - L e u - L e u PB-4 Re t - G Lu - P r o - S e r - I Le - L e u - L e u - L e u - L e u - A La - L e u - L e u - V a L-G L y - P h e - L e u - L e u - L e u - L e u - V a L PB-5 Re t - G L u - P r o - S e r - I L e - L e u - L e u - L e u - L e u - A La - L e u - L e u - V a L-G L y - P h e - L e u - L e u - L e u - L e u - V a L 3-Methylcholanthrene-treated rats a MC-1 A L a - P h e - S e r - 6 l n - T y r - I L e - S e r - L e u - A l a - P r o - G L u - L e u - L e u - L e u - A La-Th r - A L a - I t e - P h e MC-5 P r o - $ e r - V a L-Ty r - G L y - P h e - P r o - A I a - P h e - T h r - S e r - A La-Th r a

These NH2-terminal sequences have been reported [20].

356

amino acid (aspartic acid) or their amide (asparagine). P450 UT-4 has an unique amino acid sequence, leucine at the second cycle of the NH 2terminal sequence. Phenobarbital-inducible P450 PB-4 and P450 PB-5 has also acidic amino acid with glutamic acid at the second cycle of the NH2-terminal sequence. The third cycle of the NH2-terminal of P450 UT-2, UT-3, UT-5, UT-6, PB-4 and PB-5 was proline. Each purified cytochrome P-450 possesses a high content of hydrophobic amino acid in the NH2-terminal region. Purified cytochromes P-450 (P450 UT-1-P450 UT-8) possess a hydrophobic leader sequence consisting of 22% (2 residues/9 residues), 61% (11/18), 63% (12/19), 63% (12/19), 69% (9/13), 75% (12/16), 48% (11/23) and 33% (3/9) hydrophobic amino acid (leucine, valine, phenylalanine and isoleucine), respectively, in the NH2-terminal region. The clusters of potent hydrophobic amino acid were not present in P450 UT-1, UT-8, PB-1 and MC-5. The NH2-terminal sequences of P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8 were distinct. Although P450 UT-2 and P450 UT-3 eluted at different positions on DEAE-HPLC, the NH2-terminal sequences were identical. The same results were obtained in the case of P450 UT-5 and P450 UT-6. The NH2-terminal sequence of P450 UT-2 and UT-3 showed 63% homologous (10/16 identical residues) to P450 UT-5 and UT-6. The NH2-terminal sequence of P450 UT-2 also showed a high homology to P450 PB-2 (71%, 12/17 identical residues).

Metabolic activities of purified cytochrome P-450 Metabolic activities of benzphetamine demethylation, 7-ethoxycoumarin deethylation, aniline hydroxylation and p-nitroanisole demethylation of eight purified cytochrome P-450 forms were measured (Table IV). P450 UT-2 and P450 UT-3 showed active demethylase activities of benzphetamine. P450 UT-1 and P450 UT-7 also have considerably high catalytic activities. Benzphetamine is a pertinent substrate for the identification of phenobarbital-inducible cytochrome P450. P450 UT-4 was active in deethylation of 7-ethoxycoumarin and demethylation of pnitroanisole. Aniline hydroxylation activity was detected in P450 UT-1, UT-2 and UT-3 but the others were not significant. Considering that the hydroxylation activity of microsomes for aniline hydroxylation from untreated rats was 0.34 nmol/ nmol cytochrome P-450 per min, P450 UT-1, UT-2 and UT-3 showed high specificity for the hydroxylation of aniline. Hydroxylation of testosterone Constitutive forms of cytochrome P-450 are of interest in view of the ability of cytochrome P-450 to metabolize testosterone and play a role in regulating homeostasis of the organism. Authentic hydroxylated testosterones were eluted in the order of 6a-, 6fl-, 15a-, 7a-, 16a-, 2a-hydroxytestosterone and testosterone, on reverse-phase HPLC, and their retention times (min) are 20.7, 23.8, 24.5, 25.5, 28.2 and 31.5, respectively (Fig.

T A B L E IV CATALYTIC

ACTIVITY OF PURIFIED

CYTOCHROME

P-450

M e t a b o l i c a c t i v i t i e s w e r e m e a s u r e d in the r e c o n s t i t u t e d s y s t e m ( d e s c r i b e d in M a t e r i a l s a n d M e t h o d s ) w i t h o u t c y t o c h r o m e b 5 a n d shown with nmol p r o d u c t s / n m o l

c y t o c h r o m e P - 4 5 0 p e r m i n . - , v a l u e s lower t h a n 0.5 n m o l / n m o l

Cytochrome

Benz-

7-Ethoxy-

p-Nitro

P-450

phetamine

coumarin

anisole

Aniline

Testosterone 2 a-OH

UT-I

12.8

0.5

2.2

1.33

4.7

UT-2

29.4

1.4

2.0

1.63

9.5

1.58

10.8

UT-3

39.4

1.7

UT-4

8.7

13.7

3.2

-

UT-5 UT-6

5.0 5.5

0.9 0.7

1.9 1.8

-

UT-7

18.1

0.7

1.7

-

UT-8

1.3

-

1.7

.

cytochrome P-450 per min.

6 a-OH

6fl-OH

-

-

15 a - O H

-

0.6

-

3.2

-

2.2 1.9

.

3.5 -

1.0 .

16 a - O H 7.6

-

-

.

7a-OH

.

.

-

13.7

-

19.7

19.6

0.6

1.4 1.2

0.6 0.5 -

.

357

tivity was detected only in P450 UT-4. P450 UT-5 showed 6/3-, 15a- and weak 16c~-hydroxylase activity. Differences in testosterone hydroxylation between P450 UT-5 and UT-6 were not apparent. P450 UT-7 showed weak 6fl-hydroxylase activity. Distinct testosterone hydroxylase activity was not detected in P450 UT-8. 6a

Discussion

6~ 15~

2¢x

1 I

I

I

1

I

0

10

20

30

40

TIME

(min)

Fig. 5. HPLC profile of testosterone and hydroxytestosterones. Testosterone and authentic hydroxylated testosterones (6a, 6/3, 15a, 7a, 16a and 2 a ) were resolved by H P L C using a reversephase C:8 column at 50 o C. H P L C conditions are described in Materials and Methods. Testosterone and hydroxylated testosterones were detected at 254 nm.

5). Testosterone hydroxylated by cytochrome P450 was identified from the retention time of the authentic sample. The hydroxylase activities are shown in Table IV. Constitutive forms of cytochrome P-450 hydroxylated testosterone, regioselectively and stereoselectively. Hydroxylation of testosterone catalyzed by P450 UT-1, UT-2 and UT-3 resulted in two products of 2a- and 16a-hydroxytestosterone. The amount of 16a-hydroxytestosterone was higher than that of 2a-hydroxytestosterone. P450 UT-4 had a unique regioselective hydroxylase activity at 63-, 7a- and 15a-position of testosterone. 15a-Hydroxylated testosterone was the main product, the content of which was 73% of the total. Distinct 7a-hydroxylase ac-

Different forms of cytochrome P-450 range from cationic to anionic, as determined on ion-exchange chromatography [4]. To purify the many cytochrome P-450 isozymes, with a high reproducibility, anion-exchange HPLC alone is inadequate. We used a preparative anion-exchange and cation-exchange column for HPLC and isolated cytochrome P-450 forms with a high reproducibility. HPLC techniques showed better resolution of cytochrome P-450 isozymes than by conventional chromatography. Cytochrome P-450 forms are numbered in their relative order of elution on anion-exchange chromatography. The NH 2-terminal sequence, spectral properties and catalytic activities of P450 UT-2 and P450 UT-3, and P450 UT-5 and P450 UT-6 are the same, respectively. Only the chromatographic behavior by HPLC using ion-exchange column was distinguishable. Cytochromes P-450h and P450 2c, which correspond to P450 UT-2 or UT-3, are considered to be the same cytochrome P-450 isozyme; however, there seems to be a microheterogeneity, determined from the result of two-dimensional electrophoresis [28]. We compared our purified forms of cytochrome P-450 with cytochrome P-450 forms purified by other researchers from the results of the NH2-terminal sequences and catalytic activities. P450 UT-2 may correspond to cytochromes P450h [29], RLM5 [30], P-450male [31] and P-450 (M-l) [17]. P450 UT-4 may correspond to RLM2 [16] and P-450 (M-2) [17]. P450 UT-5 may correspond to P-450g [29], RLM3 [16] and P-450 (M-3) [17]. To the best of our knowledge, no cytochromes P-450 corresponding to P450 UT-1, P450 UT-7 and P450 UT-8 have heretofore been isolated. The regulation of constitutive and inducible forms of cytochrome P-450 is complicated. Gener-

358

ally, an inducer will lead to new forms of cytochrome P-450, while the constitutive forms of cytochrome P-450 are decreased [32]. On the other hand, immunochemical studies demonstrated that inducible forms of cytochrome P-450, such as phenobarbital or pregnenolone-16a-carbonitrile and isosafrole, were detected in untreated male rats, although the amount was scanty [33]. In the present study, inducible forms of cytochrome P450 were not included in our purified forms of cytochrome P-450 from untreated male rats, judging from the NH2-terminal sequence.

Acknowledgements We thank Drs. H. Ohkawa and T. Sakaki for determination of the NHz-terminal sequence and M. Ohki for expert technical assistance.

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