[58]
CYTOCHROMEP450IIE
595
[58] I n d u c t i o n , P u r i f i c a t i o n , a n d C h a r a c t e r i z a t i o n o f Cytochrome P450IIE B y C H U N G S. WANG, CHRISTOPHER J. PATTEN, HIROYUKI ISHIZAKI,
and JEONG-SOOK H. Woo Introduction Most cytochromes P450 are inducible by, and catalyze the oxidation of, rather large hydrophobic molecules. However, it was discovered that many small water-soluble molecules are inducers and substrates of a specific form of cytochrome P450. With a recommended systematic name of P450IIE1, this enzyme has also been referred to as P450LM3a, l P450ac, 2 and P450j 3by various research groups. Some of the inducers and substrates of hepatic P450IIEI are described in subsequent sections. Because the enzyme is related to the metabolism of many environmental chemicals, the study of P450IIE1 is important to our understanding of drug metabolism, biochemical toxicology, and carcinogenesis. Distribution and Induction of P450IIE1 The genes of P450IIE1 have been cloned and characterized in rats, 4 rabbits, 5 and humans. 6 Similar enzymes, such as the low Km form of N-nitrosodimethylamine (NDMA) demethylase, have been shown to exist in mice, guinea pigs, and hamsters based on immunoinhibition of specific catalytic activity.7 Nevertheless, the final designation of these enzymes as P450IIE1 should await the characterization of their gene sequences. Among all tissues liver has the highest concentration, and kidney ranks second, having about 5-10% of the amount in the liver. In the rat, kidney i D. R. Koop, E. T. Morgan, G. E. Tarr, and M. J. Coon, J. Biol. Chem. 257, 8472 (1982). 2 C. J. Patten, S. M. Ning, A. Y. H. Lu, and C. S. Wang, Arch. Biochem. Biophys. 251, 629 (1986). 3 D. E. Ryan, L. Ramanathan, S. Iida, P. E. Thomas, M. Haniu, J. E. Shively, C. S. Lieber, and W. Levin, J. Biol. Chem. 260, 6385 (1985). 4 B.-J. Song, H. V. Gelboin, S.-S. Park, C. S. Wang, and F. J. Gonzalez, J. Biol. Chem. 261, 16689 (1986). 5 S. C. Khani, P. G. Zaphiropoulos, V. S. Fujita, T. D. Porter, D. R. Koop, and M. J. Coon, Biochemistry 84, 638 (1987). 6 M. Umeno, O. W. McBride, C. S. Wang, H. V. Gelboin, and F. J. Gonzalez, Biochemistry 27, 9006 (1988). 7 C. S. Yang, D. R. Koop, T. Wang, and M. J. Coon, Biochem. Biophys. Res. Commun. 128, 1007 (1985).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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P450IIE1 share the same induction pattern as the hepatic enzyme. 8 Lower quantities of P450IIEI have been observed in rat lung, testis, ovaries, brain, and nasal mucosa. Although P450IIE1 is constitutively expressed, it is inducible 2- to 8-fold by a variety of chemicals and physiological conditions. In many early studies, a low K m form of NDMA demethylase was used effectively as an assay for the induction of this enzyme. Some of the induction conditions are outlined as follows:
Fasting and Diabetes Fasting for 2 or 3 days usually produces a 2- to 3-fold increase in the level of P450IIE1 (measured as NDMA demethylase activity) in rats. In some experiments a 2-fold increase in P450IIE1 can be observed after fasting for 24 hr. 9 Because rats eat mostly at night, the time of initiating the fasting may affect the extent of the induction. In many experiments, animals were fasted overnight or were not eating because of the specific treatment; this partial fasting effect may contribute to the induction of P450IIE1. Diabetes can also increase the P450IIE1 level approximately 2-fold. The diabetic conditions can be produced by treatment with chemicals or spontaneously.l° However, these are difficult models to work with owing to the pathological state of diabetes.
Acetone Acetone, given to rats intragastrically at a dose of 2.5 to 5 ml/kg body weight, increases the level of P450IIE1 3- to 4-fold after 18 to 24 hr." However, a clear dose-response effect has not been established within this dose range. Repeated administration of acetone for several days does not seem to produce additional induction. In order to avoid irritation to the gastrointestinal tract, acetone can be given as a 25% solution in two administrations with a 1-hr interval. Following the treatment, the rats may become idle and do not eat for 4-12 hr. This produces a partial fasting effect which may contribute to the induction of P450IIE1. Acetone can also be given by one intraperitoneal injection, or in drinking water (1-5%) for 7 to 10 days. 4'12 2-Propanol, which is readily oxidized to acetone in vivo, produces the same result as acetone. s j._y. Hong, J. Pan, Z. Dong, S. M. Ning, and C. S. Yang, Cancer Res. 47, 5948 (1987). 9 y. y . Tu and C. S. Yang, Cancer Res. 43, 623 (1983). 10 Z. Dong, J. Hong, Q. Ma, D. Li, J. Bullock, F. J. Gonzalez, S. S. Park, H. V. Gelboin, and C. S. Yang, Arch. Biochem. Biophys. 7,63, 29 (1988). II y. y. Tu, R. Peng, Z.-F. Chang, and C. S. Yang, Chem.-Biol. Interact. 44, 247 (1983). 12D. R. Koop, B. L. Crump, G. D. Nordblom, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 82, 4065 (1985).
[58l
CYTOCHROME P450IIE
597
Ethanol Ethanol given to rats as 15% (v/v) solution in drinking water for 3 days produces a 5-fold induction of NDMA demethylase activity (P450IIE1 level). 13 This treatment may cause a decrease in food consumption and result in partial fasting, a possible contributing factor to the induction. A 10% solution of ethanol in drinking water can also be used for the induction, but the extent of induction may be lower. Prolonged drinking of 10 or 15% ethanol (up to 6 days) does not seem to enhance the induction of P450IIEI in rats. Rabbit P450IIE1 is also inducible by 10% ethanol in drinking water. Ethanol can also be put in a liquid diet for 4 to 7 weeks according to Lieber and DeCarli. TM
Pyrazole, lsoniazid, Pyridine, and Related Compounds Four daily doses of pyrazole (200 mg/kg x 4) in saline, 152 daily doses ofpyrazole or 4-methylpyrazole (300 mg/kg × 2), 44 daily doses of pyridine (100 mg/kg × 4), 16 or 0.1% isoniazid 17in drinking water for 10 days cause a 3- to 5-fold induction in P450IIE1. These treatments can retard growth and may cause some toxicity. 15 Imidazole is an effective inducer of P450IIE1 in rabbits 12 but not in rats. 4
Other Chemicals P450IIE 1 is also induced by dimethyl sulfoxide (2 ml/kg twice daily for 4 days), trichloroethylene (one dose of 11 nmol/kg), and diethyl ether (inhalation). Commonly used P450 inducers such as Aroclor, phenobarbital, 3-methylcholanthrene, and pregnenolone 16~-carbonitrile decrease the amount of P450IIE1 in rat liver microsomes) 7 In most studies, Sprague-Dawley, Long Evans, and Wistar rats have been used; a clear strain difference on induction has not been observed. Immature male rats (body weight 50-100 g), which have been used in many studies, usually have 2 to 3 times higher constitutive P450IIE 1 levels than adult rats. 1~R. Peng, Y. Y. Tu, and C. S. Yang, Carcinogenesis (London) 3, 1457 (1982). 14 C. S. Lieber and L. M. DeCarli, Alcohol.: Clin. Exp. Res. 10, 550 (1986). is y. y. Tu, J. Sonnenberg, K. F. Lewis, and C. S. Yang, Biochem. Biophys. Res. Commun. 103, 905 (1981). 16 S. G. Kim, D. E. Williams, E. G. Schuetz, P. S. Guzelian, and R. F. Novak, J. Pharmacol. Exp, Ther. 246, 1175 (1988). ~7p. E. Thomas, S. Bandiera, S. L. Maines, D. E. Ryan, and W. Levin, Biochemistry 26, 2280 (1987).
ISOLATION PROCEDURES
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[58]
TABLE I PURIFICATION OF P450IIE1 FROMHEPATICMICROSOMESOF ACETONE-TREATEDRATS P450
Step Solubilized microsomes Lauric acid-AH-Sepharose
Total P450 (nmol)
Specific content (nmol P450/mg)
Specific activity (nmol/min/nmol)
yield
900 400
I. 1 6.2
-2.5
100.0
106
12.5
7.9
11.7
64
12.8
9.2
5.0
30.5
13.9
10.5
3.4
5.2
16.5
14.0
0.6
(%)
44.0
4B column, peak 2
Carboxymethyl-Sepharose CL-6B column, fraction 3A PBE-94 ion-exchange col-
umn; hydroxyapatite column Antiepoxide hydrolase immunoaffinity column Hydroxyapatite column, 65 mM potassium phosphate eluent; hydroxyapatite column
Purification of Cytochrome P450IIE1
P450IIE1 from Acetone-Induced Rat Liver Microsomes Acetone-induced microsomes are a convenient source for the isolation of P450IIE1. A summary of the purification procedure used in our laboratory 2 is shown in Table I and the steps are described as follows. Solubilization of Microsomes. Acetone-induced rat liver microsomes (700-900 nmol P450) are diluted to 3 mg protein/ml with buffer A (50 mM sodium phosphate, pH 7.25, and 25% glycerol). The mixture is solubilized by the dropwise addition of a 10% solution of Emulgen 911 (E911) to a final concentration of 0.2%. Stirring is continued under N 2 for an additional 30 min, and then the mixture is centrifuged at 100,000 g for 60min at 4 °. Lauric Acid-AH-Sepharose 4B Column. The supernatant is applied to a lauric acid-AH-Sepharose 4B column (2.5 x 25 cm) prepared by the method of Gibson and Schenkman. ~8 The column is preequilibrated with 300 ml of buffer A. The hemoproteins show as a dense red band at the top of the column. The column is washed with 500 ml of buffer B [50 mM sodium phosphate, pH 7.25, 25% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.4% cholate], and followed by 400 ml of buffer B containing 18 G. G. Gibson and J. B. Schenkman, J. Biol. Chem. 253, 5957 (1978).
[58]
CYTOCHROMEP450IIE
599
0.25 M NaC1. The major P450 band is subsequently eluted with 350 ml of buffer B containing 0.7% E911. Most of the P450IIE1 is eluted in this fraction (designated as peak 2) in a volume of about 50 ml. The remaining proteins, including NADPH-cytochrome-P450 reductase and cytochrome b s , are eluted from the column with 300 ml of buffer B containing 1.0 M NaC1 and 1.0% E911 (peak 3). The peak 3 fraction can be used as the starting material in the purification of these enzymes. The column is regenerated by washing with 8 liters of 10 mM potassium phosphate (pH 7.25). Carboxymethyl-Sepharose CL-6B Column. The peak 2 fraction is concentrated on a PM30 membrane to approximately 15 ml (Amicon Co., Danvers, MA), and dialyzed for 24 hr against 3 liters (three changes) of buffer C (5 mM sodium phosphate, pH 6.5,and 25% glycerol). At the end of dialysis, the sample should reach the pH of buffer C (pH 6.5). The sample is diluted to 4 nmol P450/ml with buffer C, and applied to a carboxymethyl-Sepharose CL-6B column (2.5 x 18 cm) preequilibrated with buffer C. The column is then washed with 50 ml of buffer C and eluted stepwise with (a) 200 ml of 30 mM sodium phosphate, pH 6.8, containing 25% glycerol, 0.1 mM EDTA, 0.1 mM DTT, and 0.5% E911 and (b) 250 ml of the same buffer containing 45 mM sodium phosphate, pH 6.7. A subsequent increase in the sodium phosphate concentration to 90 mM, pH 6.6, elutes a broad peak. The first 60 ml of the peak contains 100-120 nmol of P450. This fraction, designated Fraction 3A, is pooled and used for further purification. PBE-94 Ion-Exchange Column and Hydroxyapatite Column. Fraction 3A is concentrated as above and dialyzed overnight against buffer D (25 mM Tris-HCl, pH 7.5, 20% glycerol, and 0.1% E911). The sample is then applied to a Pharmacia (Piscataway, N J) PBE-94 ion-exchange column (0.7 × 20 cm) preequilibrated with buffer D. The P450IIE1 fraction is recovered in the flow through; lower molecular weight P450 isozymes are absorbed on the column and is eluted with 1 M NaC1 added to the buffer. The P450IIE 1 fraction is then applied directly to a hydroxyapatite column (0.9 x 3 cm) preequilibrated with buffer E (5 mM potassium phosphate, pH 7.4, and 20% glycerol). The column is washed with 50 ml of buffer E and eluted with 500 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.2% E911. This step serves to concentrate the protein and to adjust E911 to the desired concentration for the next step of purification. Antiepoxide Hydrolase Immunoaffinity Column. Epoxide hydrolase, a major contaminant at this stage, is removed with a column of epoxide hydrolase antibodies [immunoglobulin G (IgG)] coupled to Sepharose 4B. The column (1.5 x 10 cm) is equilibrated with buffer F (20 mM potassium phosphate, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.2% E911, and 0.1 M KC1). The sample is dialyzed overnight against 2 liters of buffer F without
600
ISOLATION PROCEDURES
[58]
KC1. After dialysis, KCI is added to the sample to a final concentration of 0. I M. The sample is applied to the column at a rate of 10 ml/hr and recycled through the column for 3 hr. The resulting P450 fraction is dialyzed overnight against 2 liters of buffer G (5 mM potassium phosphate, pH 7.4, 20% glycerol, and 0.1 mM EDTA). Hydroxyapatite Columns. The dialyzed sample is loaded onto a hydroxyapatite column (1.5 × 3.5 cm, DNA grade, Bio-Rad, Richmond, CA) preequilibrated with buffer G. The sample is shown as a dense band at the top of the column. After washing with 30 ml of buffer G, the column is washed stepwise with 75 ml each of 10, 50, 65, and 500 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5% E911, and 0.2% sodium cholate. The 65 mM potassium phosphate eluent contains P450IIE1 with the highest purity. This fraction is pooled, dialyzed overnight against 2 liters of buffer H (5 mM potassium phosphate, pH 7.4, 20% glycerol, and 0. I mM DTT), and applied onto a second hydroxyapatite column (0.9 x 1 cm), preequilibrated with buffer H for the removal of E911. The column is washed with the buffer H until the A276 of the eluent is essentially zero. The enzyme is then eluted with 500 mM potassium phosphate (pH 7.4) containing 20% glycerol, 0.1 mM DTT, and 0.2% cholate. The eluted enzyme is dialyzed overnight against 4 liters of 100 mM potassium phosphate (pH 7.4) containing 20% glycerol and 0.2 mM DTT. The final preparation contains 16.0-16.5 nmol P450/mg protein and is stable when stored at - 7 0 °. Other Methods for Purification o f P45OIIE1
P450IIE1 was first purified from liver microsomes of ethanol-treated rabbits by Koop et aL l and of isoniazid-treated rats by Ryan et al. 3 P450IIE 1 has been purified from different sources including human liver microsomes. 19
Assay
Methods
N-Nitrosodimethylamine Demethylase Assay
The NDMA demethylase assay has been extensively used in our laboratories and by others. At low substrate concentrations, P450IIE1 is the only known P450 isozyme showing substantial activity. This low K mform 19S. A. Wrighton, P. E. Thomas, D. T. Molowa, M. Haniu, J. E. Shively, S. L. Maines, P. B. Watkins, G. Parker, G. Mendez-Picon, W. Levin, and P. S. Guzelian, Biochemistry 25, 6731 (1986).
[58]
CYTOCHROME P450IIE
601
of NDMA demethylase can be assayed colorimetrically 9 or radiometrically.20 Colorimetric Assay. The colorimetric assay is based on the measurement of formaldehyde formed by the Nash reaction. The assay mixture in a total volume of 0.5 ml usually contains 50 mM Tris-HCl (pH 7.4), 10 mM MgC12, 150 mM KCI, an NADPH-generating system (0.4 mM NADP + , 10 mM glucose 6-phosphate, and 0.2 units glucose-6-phosphate dehydrogenase), liver microsomes (0.1-1 mg protein), and 0.04-4 mM'NDMA. The components are preincubated in glass test tubes (12 x 75 mm) for 2 min in a 37° shaking water bath prior to initiating the reaction by the addition of NDMA or the NADPH-generating system. The reaction is carried out in duplicate for 10 or 20 min. Blanks are prepared by omitting either NDMA or the NADPH-generating system. For standard, formaldehyde is added, at around the midpoint of the incubation, to the assay mixture in the absence of either NDMA or the NADPH-generating system. The reaction is terminated by the addition of 0.05 ml each of 25% ZnSO4 and saturated Ba(OH)2. The mixture is centrifuged, and 0.35 ml of supernatant is mixed with 0.15 ml of a concentrated Nash reagent (5 g ammonium sulfate and 0.1 ml of acetylacetone in 6 ml of 3% acetic acid, made fresh). After the mixture is incubated at 50° for 30 min, absorbance at 412 nm is measured. Radiornetric Assay. The assay mixture with a total volume of 0.1 ml is similar to the colorimetric assay except lower amounts of microsomal protein (0.01-0.1 mg) and [14C]NDMA (0.01-1 mM) can be used. For standard, 14C-labeled formaldehyde is added to the assay mixture in the absence of [1#C]NDMA. The reaction is carried out at 37° for 5 or 10 min in a shaking water bath in duplicate using 12 × 75 mm glass test tubes. The reaction is terminated by the addition of 0.06 ml of 1 M sodium acetate (pH 4.5), and the tubes are placed on ice, followed by the addition of 0.03 ml of 0.1 M formaldehyde and 0.06 ml of 0.4 M dimedone. Blanks are prepared by the immediate addition of the sodium acetate, formaldehyde, and dimedone to unincubated samples that contained all the incubation components. For each substrate concentration, both blanks and samples are prepared. After the termination of the reaction, the tubes are covered with aluminum foil, placed in a 50° water bath for 15 min, and cooled. To each tube is added 0.2 ml water and I ml hexane. The tubes are vortexmixed, centrifuged, and 0.75 ml of the hexane layer is removed. The hexane is reextracted with 1.5 ml water, and 0.5 ml of the hexane layer is added to 4.5 ml of Betafluor (National Diagnostics, Somerville, NJ) for determination of radioactivity. For this assay, it is important that the 20 J.-S. H. YOO, S. M. Ning, C. J. Patten, and C. S. Yang, Cancer Res. 47, 992 (1987).
602
ISOLATION PROCEDURES
[58]
[14C]NDMA is free from contamination by [14C]formaldehyde. If the background of the assay is too high, [14C]NDMA can be purified by a Dowex-1 bisulfite column. In comparison to other assay conditions, this system uses a higher ionic strength, which increases the activity, and does not include semicarbazide, which is an inhibitor. Previous studies used semicarbazide for trapping the formaldehyde produced, but we found it is not needed. The present assays are more sensitive than many previous ones. Early studies have used a substrate concentration of 4 mM NDMA for the assay of the low K~ form of NDMA demethylase. Although these assays generally reflect the activity of P450IIE1, much lower substrate concentrations (40-200/xM) are recommended to avoid interference by other P450 isozymes. Previous work indicated a low Km value of 60-80 /zM for this enzyme activity. 9 However, with improved assay conditions, such as lower microsomal protein levels and substrate concentrations, and the elimination of possible inhibitors, a K mvalue of 15-20/zMis obtained in our laboratory. Many common solvents such as ethanol, acetone, dimethyl sulfoxide, diethyl ether, benzene, and glycerol are competitive inhibitors. The reconstituted NDMA demethylase system with purified P450IIE1 displays a Km of 3 mM, and the value is decreased to 0.35 mM with the addition of cytochrome bs. 2 Part of the discrepancy in the Km values between the reconstituted system and the microsomes may be due to the presence of glycerol in the former system.
p-Nitrophenol Hydroxylase, Aniline Hydroxylase, and Other Activities Koop 21reported that the conversion ofp-nitrophenol to 4-nitrocatechol is specifically catalyzed by P450IIE1. This assay has been used in several laboratories. The aniline hydroxylase assay has been used widely as a "type II" substrate in microsomal drug metabolism studies. Several laboratories have shown that aniline is a good substrate for P450IIE1.2-4 Other substrates of P450IIE1 include acetone, alcohols, enflurane, ethers, chloroform, carbon tetrachloride, benzene, alkanes, acetaminophen, and azoxymethane. They have not been used as routine assay substrates for P450IIE1.
Immunochemical Assays Antibodies against rat, rabbit, and human P450IIE 1 have been prepared and have been used for the quantitation of P450IIE1 by Western blots 4'8'1° or enzyme-linked immunosorbent assay (ELISA) 17 in many laboratories. 2I D. R. Koop, Mol. Pharmacol. 29, 399 (1986).
[59]
NASAL FORMS OF CYTOCHROME P450
603
Structure and Properties
Spectral Properties The purified P450IIE1 exists in either a high spin state or a mixture of high and low spin states showing a major peak at 394 nm or a broad peak in the 394 to 412 nm region) -3 The ferrous form of P450IIE1 displays an absorption maximum at 412 nm. The ferrous P450-CO spectrum shows an absorption maximum at 451 nm.
Structure Partial amino acid sequences for rat and rabbit P450IIE1 have been reported; these two proteins have 13 identical residues in the first 19 amino acids at the N terminus) Based on the gene sequence, it was shown that the rat and human enzymes both contain 493 amino acids with calculated molecular weights of 56,634 and 56,916, respectively. The rabbit enzyme contains 492 amino acids with a calculated molecular weight of 56,820. The human enzyme shares 75% nucleotide and 78% amino acid similarities to the rat enzyme. Only one gene in the P450IIE subfamily was observed in the rat and human. However, two genes, P450IIE1 and P450IIE2, have been found in the rabbit. The gene product of P450IIE2 is catalytically rather inactive. Acknowledgments Supported by National Institutes of Health Grants CA37037 and ES03938.
[59] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f C y t o c h r o m e s P 4 5 0 in R a b b i t N a s a l M i c r o s o m e s By XINXIN DING a n d MINOR J. COON
Introduction The nasal mucosa is rich in P450 cytochromes and other biotransformation enzymes, with the specific content of P450 in nasal microsomes being the highest among all rabbit extrahepatic tissues examined) A major portion of the microsomal P450 in rabbit olfactory mucosa is made up of I X. Ding, D. R. Koop, B. L. Crump, and M. J. Coon, Mol. Pharmacol. 30, 370 (1986).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
CYTOCHROMEP450IIE
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[58] I n d u c t i o n , P u r i f i c a t i o n , a n d C h a r a c t e r i z a t i o n o f Cytochrome P450IIE B y C H U N G S. WANG, CHRISTOPHER J. PATTEN, HIROYUKI ISHIZAKI,
and JEONG-SOOK H. Woo Introduction Most cytochromes P450 are inducible by, and catalyze the oxidation of, rather large hydrophobic molecules. However, it was discovered that many small water-soluble molecules are inducers and substrates of a specific form of cytochrome P450. With a recommended systematic name of P450IIE1, this enzyme has also been referred to as P450LM3a, l P450ac, 2 and P450j 3by various research groups. Some of the inducers and substrates of hepatic P450IIEI are described in subsequent sections. Because the enzyme is related to the metabolism of many environmental chemicals, the study of P450IIE1 is important to our understanding of drug metabolism, biochemical toxicology, and carcinogenesis. Distribution and Induction of P450IIE1 The genes of P450IIE1 have been cloned and characterized in rats, 4 rabbits, 5 and humans. 6 Similar enzymes, such as the low Km form of N-nitrosodimethylamine (NDMA) demethylase, have been shown to exist in mice, guinea pigs, and hamsters based on immunoinhibition of specific catalytic activity.7 Nevertheless, the final designation of these enzymes as P450IIE1 should await the characterization of their gene sequences. Among all tissues liver has the highest concentration, and kidney ranks second, having about 5-10% of the amount in the liver. In the rat, kidney i D. R. Koop, E. T. Morgan, G. E. Tarr, and M. J. Coon, J. Biol. Chem. 257, 8472 (1982). 2 C. J. Patten, S. M. Ning, A. Y. H. Lu, and C. S. Wang, Arch. Biochem. Biophys. 251, 629 (1986). 3 D. E. Ryan, L. Ramanathan, S. Iida, P. E. Thomas, M. Haniu, J. E. Shively, C. S. Lieber, and W. Levin, J. Biol. Chem. 260, 6385 (1985). 4 B.-J. Song, H. V. Gelboin, S.-S. Park, C. S. Wang, and F. J. Gonzalez, J. Biol. Chem. 261, 16689 (1986). 5 S. C. Khani, P. G. Zaphiropoulos, V. S. Fujita, T. D. Porter, D. R. Koop, and M. J. Coon, Biochemistry 84, 638 (1987). 6 M. Umeno, O. W. McBride, C. S. Wang, H. V. Gelboin, and F. J. Gonzalez, Biochemistry 27, 9006 (1988). 7 C. S. Yang, D. R. Koop, T. Wang, and M. J. Coon, Biochem. Biophys. Res. Commun. 128, 1007 (1985).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ISOLATION PROCEDURES
[58]
P450IIE1 share the same induction pattern as the hepatic enzyme. 8 Lower quantities of P450IIEI have been observed in rat lung, testis, ovaries, brain, and nasal mucosa. Although P450IIE1 is constitutively expressed, it is inducible 2- to 8-fold by a variety of chemicals and physiological conditions. In many early studies, a low K m form of NDMA demethylase was used effectively as an assay for the induction of this enzyme. Some of the induction conditions are outlined as follows:
Fasting and Diabetes Fasting for 2 or 3 days usually produces a 2- to 3-fold increase in the level of P450IIE1 (measured as NDMA demethylase activity) in rats. In some experiments a 2-fold increase in P450IIE1 can be observed after fasting for 24 hr. 9 Because rats eat mostly at night, the time of initiating the fasting may affect the extent of the induction. In many experiments, animals were fasted overnight or were not eating because of the specific treatment; this partial fasting effect may contribute to the induction of P450IIE1. Diabetes can also increase the P450IIE1 level approximately 2-fold. The diabetic conditions can be produced by treatment with chemicals or spontaneously.l° However, these are difficult models to work with owing to the pathological state of diabetes.
Acetone Acetone, given to rats intragastrically at a dose of 2.5 to 5 ml/kg body weight, increases the level of P450IIE1 3- to 4-fold after 18 to 24 hr." However, a clear dose-response effect has not been established within this dose range. Repeated administration of acetone for several days does not seem to produce additional induction. In order to avoid irritation to the gastrointestinal tract, acetone can be given as a 25% solution in two administrations with a 1-hr interval. Following the treatment, the rats may become idle and do not eat for 4-12 hr. This produces a partial fasting effect which may contribute to the induction of P450IIE1. Acetone can also be given by one intraperitoneal injection, or in drinking water (1-5%) for 7 to 10 days. 4'12 2-Propanol, which is readily oxidized to acetone in vivo, produces the same result as acetone. s j._y. Hong, J. Pan, Z. Dong, S. M. Ning, and C. S. Yang, Cancer Res. 47, 5948 (1987). 9 y. y . Tu and C. S. Yang, Cancer Res. 43, 623 (1983). 10 Z. Dong, J. Hong, Q. Ma, D. Li, J. Bullock, F. J. Gonzalez, S. S. Park, H. V. Gelboin, and C. S. Yang, Arch. Biochem. Biophys. 7,63, 29 (1988). II y. y. Tu, R. Peng, Z.-F. Chang, and C. S. Yang, Chem.-Biol. Interact. 44, 247 (1983). 12D. R. Koop, B. L. Crump, G. D. Nordblom, and M. J. Coon, Proc. Natl. Acad. Sci. U.S.A. 82, 4065 (1985).
[58l
CYTOCHROME P450IIE
597
Ethanol Ethanol given to rats as 15% (v/v) solution in drinking water for 3 days produces a 5-fold induction of NDMA demethylase activity (P450IIE1 level). 13 This treatment may cause a decrease in food consumption and result in partial fasting, a possible contributing factor to the induction. A 10% solution of ethanol in drinking water can also be used for the induction, but the extent of induction may be lower. Prolonged drinking of 10 or 15% ethanol (up to 6 days) does not seem to enhance the induction of P450IIEI in rats. Rabbit P450IIE1 is also inducible by 10% ethanol in drinking water. Ethanol can also be put in a liquid diet for 4 to 7 weeks according to Lieber and DeCarli. TM
Pyrazole, lsoniazid, Pyridine, and Related Compounds Four daily doses of pyrazole (200 mg/kg x 4) in saline, 152 daily doses ofpyrazole or 4-methylpyrazole (300 mg/kg × 2), 44 daily doses of pyridine (100 mg/kg × 4), 16 or 0.1% isoniazid 17in drinking water for 10 days cause a 3- to 5-fold induction in P450IIE1. These treatments can retard growth and may cause some toxicity. 15 Imidazole is an effective inducer of P450IIE1 in rabbits 12 but not in rats. 4
Other Chemicals P450IIE 1 is also induced by dimethyl sulfoxide (2 ml/kg twice daily for 4 days), trichloroethylene (one dose of 11 nmol/kg), and diethyl ether (inhalation). Commonly used P450 inducers such as Aroclor, phenobarbital, 3-methylcholanthrene, and pregnenolone 16~-carbonitrile decrease the amount of P450IIE1 in rat liver microsomes) 7 In most studies, Sprague-Dawley, Long Evans, and Wistar rats have been used; a clear strain difference on induction has not been observed. Immature male rats (body weight 50-100 g), which have been used in many studies, usually have 2 to 3 times higher constitutive P450IIE 1 levels than adult rats. 1~R. Peng, Y. Y. Tu, and C. S. Yang, Carcinogenesis (London) 3, 1457 (1982). 14 C. S. Lieber and L. M. DeCarli, Alcohol.: Clin. Exp. Res. 10, 550 (1986). is y. y. Tu, J. Sonnenberg, K. F. Lewis, and C. S. Yang, Biochem. Biophys. Res. Commun. 103, 905 (1981). 16 S. G. Kim, D. E. Williams, E. G. Schuetz, P. S. Guzelian, and R. F. Novak, J. Pharmacol. Exp, Ther. 246, 1175 (1988). ~7p. E. Thomas, S. Bandiera, S. L. Maines, D. E. Ryan, and W. Levin, Biochemistry 26, 2280 (1987).
ISOLATION PROCEDURES
598
[58]
TABLE I PURIFICATION OF P450IIE1 FROMHEPATICMICROSOMESOF ACETONE-TREATEDRATS P450
Step Solubilized microsomes Lauric acid-AH-Sepharose
Total P450 (nmol)
Specific content (nmol P450/mg)
Specific activity (nmol/min/nmol)
yield
900 400
I. 1 6.2
-2.5
100.0
106
12.5
7.9
11.7
64
12.8
9.2
5.0
30.5
13.9
10.5
3.4
5.2
16.5
14.0
0.6
(%)
44.0
4B column, peak 2
Carboxymethyl-Sepharose CL-6B column, fraction 3A PBE-94 ion-exchange col-
umn; hydroxyapatite column Antiepoxide hydrolase immunoaffinity column Hydroxyapatite column, 65 mM potassium phosphate eluent; hydroxyapatite column
Purification of Cytochrome P450IIE1
P450IIE1 from Acetone-Induced Rat Liver Microsomes Acetone-induced microsomes are a convenient source for the isolation of P450IIE1. A summary of the purification procedure used in our laboratory 2 is shown in Table I and the steps are described as follows. Solubilization of Microsomes. Acetone-induced rat liver microsomes (700-900 nmol P450) are diluted to 3 mg protein/ml with buffer A (50 mM sodium phosphate, pH 7.25, and 25% glycerol). The mixture is solubilized by the dropwise addition of a 10% solution of Emulgen 911 (E911) to a final concentration of 0.2%. Stirring is continued under N 2 for an additional 30 min, and then the mixture is centrifuged at 100,000 g for 60min at 4 °. Lauric Acid-AH-Sepharose 4B Column. The supernatant is applied to a lauric acid-AH-Sepharose 4B column (2.5 x 25 cm) prepared by the method of Gibson and Schenkman. ~8 The column is preequilibrated with 300 ml of buffer A. The hemoproteins show as a dense red band at the top of the column. The column is washed with 500 ml of buffer B [50 mM sodium phosphate, pH 7.25, 25% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.4% cholate], and followed by 400 ml of buffer B containing 18 G. G. Gibson and J. B. Schenkman, J. Biol. Chem. 253, 5957 (1978).
[58]
CYTOCHROMEP450IIE
599
0.25 M NaC1. The major P450 band is subsequently eluted with 350 ml of buffer B containing 0.7% E911. Most of the P450IIE1 is eluted in this fraction (designated as peak 2) in a volume of about 50 ml. The remaining proteins, including NADPH-cytochrome-P450 reductase and cytochrome b s , are eluted from the column with 300 ml of buffer B containing 1.0 M NaC1 and 1.0% E911 (peak 3). The peak 3 fraction can be used as the starting material in the purification of these enzymes. The column is regenerated by washing with 8 liters of 10 mM potassium phosphate (pH 7.25). Carboxymethyl-Sepharose CL-6B Column. The peak 2 fraction is concentrated on a PM30 membrane to approximately 15 ml (Amicon Co., Danvers, MA), and dialyzed for 24 hr against 3 liters (three changes) of buffer C (5 mM sodium phosphate, pH 6.5,and 25% glycerol). At the end of dialysis, the sample should reach the pH of buffer C (pH 6.5). The sample is diluted to 4 nmol P450/ml with buffer C, and applied to a carboxymethyl-Sepharose CL-6B column (2.5 x 18 cm) preequilibrated with buffer C. The column is then washed with 50 ml of buffer C and eluted stepwise with (a) 200 ml of 30 mM sodium phosphate, pH 6.8, containing 25% glycerol, 0.1 mM EDTA, 0.1 mM DTT, and 0.5% E911 and (b) 250 ml of the same buffer containing 45 mM sodium phosphate, pH 6.7. A subsequent increase in the sodium phosphate concentration to 90 mM, pH 6.6, elutes a broad peak. The first 60 ml of the peak contains 100-120 nmol of P450. This fraction, designated Fraction 3A, is pooled and used for further purification. PBE-94 Ion-Exchange Column and Hydroxyapatite Column. Fraction 3A is concentrated as above and dialyzed overnight against buffer D (25 mM Tris-HCl, pH 7.5, 20% glycerol, and 0.1% E911). The sample is then applied to a Pharmacia (Piscataway, N J) PBE-94 ion-exchange column (0.7 × 20 cm) preequilibrated with buffer D. The P450IIE1 fraction is recovered in the flow through; lower molecular weight P450 isozymes are absorbed on the column and is eluted with 1 M NaC1 added to the buffer. The P450IIE 1 fraction is then applied directly to a hydroxyapatite column (0.9 x 3 cm) preequilibrated with buffer E (5 mM potassium phosphate, pH 7.4, and 20% glycerol). The column is washed with 50 ml of buffer E and eluted with 500 mM potassium phosphate, pH 7.4, containing 20% glycerol and 0.2% E911. This step serves to concentrate the protein and to adjust E911 to the desired concentration for the next step of purification. Antiepoxide Hydrolase Immunoaffinity Column. Epoxide hydrolase, a major contaminant at this stage, is removed with a column of epoxide hydrolase antibodies [immunoglobulin G (IgG)] coupled to Sepharose 4B. The column (1.5 x 10 cm) is equilibrated with buffer F (20 mM potassium phosphate, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.2% E911, and 0.1 M KC1). The sample is dialyzed overnight against 2 liters of buffer F without
600
ISOLATION PROCEDURES
[58]
KC1. After dialysis, KCI is added to the sample to a final concentration of 0. I M. The sample is applied to the column at a rate of 10 ml/hr and recycled through the column for 3 hr. The resulting P450 fraction is dialyzed overnight against 2 liters of buffer G (5 mM potassium phosphate, pH 7.4, 20% glycerol, and 0.1 mM EDTA). Hydroxyapatite Columns. The dialyzed sample is loaded onto a hydroxyapatite column (1.5 × 3.5 cm, DNA grade, Bio-Rad, Richmond, CA) preequilibrated with buffer G. The sample is shown as a dense band at the top of the column. After washing with 30 ml of buffer G, the column is washed stepwise with 75 ml each of 10, 50, 65, and 500 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5% E911, and 0.2% sodium cholate. The 65 mM potassium phosphate eluent contains P450IIE1 with the highest purity. This fraction is pooled, dialyzed overnight against 2 liters of buffer H (5 mM potassium phosphate, pH 7.4, 20% glycerol, and 0. I mM DTT), and applied onto a second hydroxyapatite column (0.9 x 1 cm), preequilibrated with buffer H for the removal of E911. The column is washed with the buffer H until the A276 of the eluent is essentially zero. The enzyme is then eluted with 500 mM potassium phosphate (pH 7.4) containing 20% glycerol, 0.1 mM DTT, and 0.2% cholate. The eluted enzyme is dialyzed overnight against 4 liters of 100 mM potassium phosphate (pH 7.4) containing 20% glycerol and 0.2 mM DTT. The final preparation contains 16.0-16.5 nmol P450/mg protein and is stable when stored at - 7 0 °. Other Methods for Purification o f P45OIIE1
P450IIE1 was first purified from liver microsomes of ethanol-treated rabbits by Koop et aL l and of isoniazid-treated rats by Ryan et al. 3 P450IIE 1 has been purified from different sources including human liver microsomes. 19
Assay
Methods
N-Nitrosodimethylamine Demethylase Assay
The NDMA demethylase assay has been extensively used in our laboratories and by others. At low substrate concentrations, P450IIE1 is the only known P450 isozyme showing substantial activity. This low K mform 19S. A. Wrighton, P. E. Thomas, D. T. Molowa, M. Haniu, J. E. Shively, S. L. Maines, P. B. Watkins, G. Parker, G. Mendez-Picon, W. Levin, and P. S. Guzelian, Biochemistry 25, 6731 (1986).
[58]
CYTOCHROME P450IIE
601
of NDMA demethylase can be assayed colorimetrically 9 or radiometrically.20 Colorimetric Assay. The colorimetric assay is based on the measurement of formaldehyde formed by the Nash reaction. The assay mixture in a total volume of 0.5 ml usually contains 50 mM Tris-HCl (pH 7.4), 10 mM MgC12, 150 mM KCI, an NADPH-generating system (0.4 mM NADP + , 10 mM glucose 6-phosphate, and 0.2 units glucose-6-phosphate dehydrogenase), liver microsomes (0.1-1 mg protein), and 0.04-4 mM'NDMA. The components are preincubated in glass test tubes (12 x 75 mm) for 2 min in a 37° shaking water bath prior to initiating the reaction by the addition of NDMA or the NADPH-generating system. The reaction is carried out in duplicate for 10 or 20 min. Blanks are prepared by omitting either NDMA or the NADPH-generating system. For standard, formaldehyde is added, at around the midpoint of the incubation, to the assay mixture in the absence of either NDMA or the NADPH-generating system. The reaction is terminated by the addition of 0.05 ml each of 25% ZnSO4 and saturated Ba(OH)2. The mixture is centrifuged, and 0.35 ml of supernatant is mixed with 0.15 ml of a concentrated Nash reagent (5 g ammonium sulfate and 0.1 ml of acetylacetone in 6 ml of 3% acetic acid, made fresh). After the mixture is incubated at 50° for 30 min, absorbance at 412 nm is measured. Radiornetric Assay. The assay mixture with a total volume of 0.1 ml is similar to the colorimetric assay except lower amounts of microsomal protein (0.01-0.1 mg) and [14C]NDMA (0.01-1 mM) can be used. For standard, 14C-labeled formaldehyde is added to the assay mixture in the absence of [1#C]NDMA. The reaction is carried out at 37° for 5 or 10 min in a shaking water bath in duplicate using 12 × 75 mm glass test tubes. The reaction is terminated by the addition of 0.06 ml of 1 M sodium acetate (pH 4.5), and the tubes are placed on ice, followed by the addition of 0.03 ml of 0.1 M formaldehyde and 0.06 ml of 0.4 M dimedone. Blanks are prepared by the immediate addition of the sodium acetate, formaldehyde, and dimedone to unincubated samples that contained all the incubation components. For each substrate concentration, both blanks and samples are prepared. After the termination of the reaction, the tubes are covered with aluminum foil, placed in a 50° water bath for 15 min, and cooled. To each tube is added 0.2 ml water and I ml hexane. The tubes are vortexmixed, centrifuged, and 0.75 ml of the hexane layer is removed. The hexane is reextracted with 1.5 ml water, and 0.5 ml of the hexane layer is added to 4.5 ml of Betafluor (National Diagnostics, Somerville, NJ) for determination of radioactivity. For this assay, it is important that the 20 J.-S. H. YOO, S. M. Ning, C. J. Patten, and C. S. Yang, Cancer Res. 47, 992 (1987).
602
ISOLATION PROCEDURES
[58]
[14C]NDMA is free from contamination by [14C]formaldehyde. If the background of the assay is too high, [14C]NDMA can be purified by a Dowex-1 bisulfite column. In comparison to other assay conditions, this system uses a higher ionic strength, which increases the activity, and does not include semicarbazide, which is an inhibitor. Previous studies used semicarbazide for trapping the formaldehyde produced, but we found it is not needed. The present assays are more sensitive than many previous ones. Early studies have used a substrate concentration of 4 mM NDMA for the assay of the low K~ form of NDMA demethylase. Although these assays generally reflect the activity of P450IIE1, much lower substrate concentrations (40-200/xM) are recommended to avoid interference by other P450 isozymes. Previous work indicated a low Km value of 60-80 /zM for this enzyme activity. 9 However, with improved assay conditions, such as lower microsomal protein levels and substrate concentrations, and the elimination of possible inhibitors, a K mvalue of 15-20/zMis obtained in our laboratory. Many common solvents such as ethanol, acetone, dimethyl sulfoxide, diethyl ether, benzene, and glycerol are competitive inhibitors. The reconstituted NDMA demethylase system with purified P450IIE1 displays a Km of 3 mM, and the value is decreased to 0.35 mM with the addition of cytochrome bs. 2 Part of the discrepancy in the Km values between the reconstituted system and the microsomes may be due to the presence of glycerol in the former system.
p-Nitrophenol Hydroxylase, Aniline Hydroxylase, and Other Activities Koop 21reported that the conversion ofp-nitrophenol to 4-nitrocatechol is specifically catalyzed by P450IIE1. This assay has been used in several laboratories. The aniline hydroxylase assay has been used widely as a "type II" substrate in microsomal drug metabolism studies. Several laboratories have shown that aniline is a good substrate for P450IIE1.2-4 Other substrates of P450IIE1 include acetone, alcohols, enflurane, ethers, chloroform, carbon tetrachloride, benzene, alkanes, acetaminophen, and azoxymethane. They have not been used as routine assay substrates for P450IIE1.
Immunochemical Assays Antibodies against rat, rabbit, and human P450IIE 1 have been prepared and have been used for the quantitation of P450IIE1 by Western blots 4'8'1° or enzyme-linked immunosorbent assay (ELISA) 17 in many laboratories. 2I D. R. Koop, Mol. Pharmacol. 29, 399 (1986).
[59]
NASAL FORMS OF CYTOCHROME P450
603
Structure and Properties
Spectral Properties The purified P450IIE1 exists in either a high spin state or a mixture of high and low spin states showing a major peak at 394 nm or a broad peak in the 394 to 412 nm region) -3 The ferrous form of P450IIE1 displays an absorption maximum at 412 nm. The ferrous P450-CO spectrum shows an absorption maximum at 451 nm.
Structure Partial amino acid sequences for rat and rabbit P450IIE1 have been reported; these two proteins have 13 identical residues in the first 19 amino acids at the N terminus) Based on the gene sequence, it was shown that the rat and human enzymes both contain 493 amino acids with calculated molecular weights of 56,634 and 56,916, respectively. The rabbit enzyme contains 492 amino acids with a calculated molecular weight of 56,820. The human enzyme shares 75% nucleotide and 78% amino acid similarities to the rat enzyme. Only one gene in the P450IIE subfamily was observed in the rat and human. However, two genes, P450IIE1 and P450IIE2, have been found in the rabbit. The gene product of P450IIE2 is catalytically rather inactive. Acknowledgments Supported by National Institutes of Health Grants CA37037 and ES03938.
[59] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f C y t o c h r o m e s P 4 5 0 in R a b b i t N a s a l M i c r o s o m e s By XINXIN DING a n d MINOR J. COON
Introduction The nasal mucosa is rich in P450 cytochromes and other biotransformation enzymes, with the specific content of P450 in nasal microsomes being the highest among all rabbit extrahepatic tissues examined) A major portion of the microsomal P450 in rabbit olfactory mucosa is made up of I X. Ding, D. R. Koop, B. L. Crump, and M. J. Coon, Mol. Pharmacol. 30, 370 (1986).
METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.
