Resolution of Multiple Forms of Rabbit Liver Cytochrome P-450

Cytochrome P-450d rapidly N-demethylates benzphetamine which Is in accord with our suggestion that this i s the pheno- barbital -inducible form descri...
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4 Resolution of Multiple Forms of Rabbit Liver Cytochrome P-450

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ERIC F. JOHNSON and URSULA MULLER-EBERHARD Scripps Clinic and Research Foundation, Department of Biochemistry, La Jolla, CA 92037

Recognition of the existence of multiple forms of cytochrome P-450 is an important step in understanding the complexities of drug and carcinogen metabolism. The characterization of individual forms of cytochrome P-450 is expected to contribute to the knowledge of their function in various metabolic pathways. Investigations undertaken in our laboratory have been directed toward resolving and characterizing forms of cytochrome P-450 from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD. Our efforts have resulted in the partial purification of three distinct forms of cytochrome P-450. These forms possess properties which are unlike those of phenobarbital-inducible cytochrome P-450. The forms of cytochrome P-450 induced by TCDD, 3-methyl-cholanthrene, and related inducers are often referred to as cytochrome P-448. These names are derived from differences in the characteristic absorption maximum of the reduced carbonyl cytochrome complex. In addition, the spectra of the cytochromes contrast in the presence of other ligands (1). The induction of cytochrome P-448 is oTten linked to increases of several monooxygenase a c t i v i t i e s . This was extensively examined with inbred mouse strains where a genetic association was found between the induction of a number of monooxygenase a c t i v i t i e s and mouse liver cytochrome P-448 (2). A prototype activity in this respect is aryl hydrocarbon hydroxylase (benzo(a)pyrene 3-hydroxylase). However, this enzyme is not induced by TCDD or related inducers nor by phénobarbital in liver microsomes of adult rabbits (3-6). Likewise, very few correlate monooxygenase a c t i v i t i e s are increased. However, acetanilide hydroxylase (6), 2-acetylaminofluorene N-hydroxylase (6), and 7-ethoxyresorufin" O-deethylase (7) are increased concomitantly with the induction of rabbiT liver cytochrome P-448 by TCDD or 3-methylcholanthrene. The presence of multiple forms of cytochrome P-450 in rabbit liver microsomes following TCDD treatment might be related to these observations. The form comprising a major portion of

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4.

JOHNSON AND MULLER-EBERHARD

Rabbit

Liver

Cytochrome

P-450

73

cytochrome P-450 d i s p l a y s a spectrum s i m i l a r t o that observed w i t h microsomal p r e p a r a t i o n s f o l l o w i n g TCDD treatment. T h i s cytochrome f u n c t i o n s i n the c a t a l y s i s o f TCDD-inducible monooxygenase r e a c t i o n s . A d d i t i o n a l forms o f cytochrome P-450 are a c t i v e i n those monooxygenase r e a c t i o n s which are not a s s o c i a t e d w i t h TCDD- o r p h e n o b a r b i t a l - i n d u c t ton and are d i s t i n g u i s h a b l e from the p h e n o b a r b i t a l - i n d u c i b l e cytochrome P-450. We have employed column chromatography t o separate i n d i v i d u a l forms o f cytochrome P-450. P r i o r t o chromatography, l i v e r microsomes from TCDD-treated r a b b i t s were s o l u b i l i z e d w i t h sodium c h o l a t e and f r a c t i o n a t e d by p o l y e t h y l e n e g l y c o l p r e c i p i t a t i o n as d e s c r i b e d by van der Hoeven and Coon (8). T h i s method was developed f o r the i s o l a t i o n o f the p h e n o b a r b i t a l - i n d u c i b l e form o f cytochrome P-450. The cytochromes from TCDD-induced microsomes p r e c i p i t a t e over a broad range o f p o l y e t h y l e n e g l y c o l concentrations. Therefore, f r a c t i o n s containing substantial p o r t i o n s o f the p a r t i a l l y p u r i f i e d cytochromes were pooled before chromatography HydroxylapaTite was adopted as the i n i t i a l s e p a r a t i o n medium. Two p r i n c i p a l f r a c t i o n s o f cytochrome P-450 a r e resolved u s i n g a step-wise c o n c e n t r a t i o n g r a d i e n t o f potassium phosphate b u f f e r , pH 7*4, c o n t a i n i n g g l y c e r o l (20%), EDTA (0.1 mM), and Nonidet P-40 ( O . U ) , a n o n i o n i c detergent. The two p r e p a r a t i o n s a r e r e f e r r e d t o as cytochrome P-450ab and P-450c (JJ). Cytochrome P-450ab represents ea. s i x percent (6%) o f t h e microsomal cytochrome and c o n t a i n s 5.8 nmoles o f cytochrome P-450 per mg o f p r o t e i n . The cytochrome P-450c p r e p a r a t i o n c o n t a i n s oa. eighteen percent (18$) o f the o r i g i n a l cytochrome P-450 pool and 10.0 nmoles per mg p r o t e i n . One major p e p t i d e i s detected in the cytochrome P-450c f r a c t i o n when analyzed by polyacrylamide gel e l e c t r o p h o r e s i s in the presence o f sodium dodecyl s u l f a t e . Whereas, s e v e r a l major peptides are observed i n cytochrome P-450ab

(3).

Immunological d i f f e r e n c e s between the cytochromes were i n v e s t i g a t e d w i t h antiserum produced a g a i n s t the major peptide i n the cytochrome P-450c p r e p a r a t i o n . The major peptide was separated from contaminants by polyacrylamide g e l e l e c t r o p h o r e s i s in the presence o f sodium d o d e c y l s u l f a t e . A segment o f the g e l which contained the peptide was used t o educe antibody formation i n a goat. The antiserum was examined by double d i f f u s i o n In agarose, and a s i n g l e p r e c i p i t i n l i n e Is seen when i t r e a c t s w i t h p u r i f i e d cytochrome P-450c o r w i t h sodium c h o l a t e s o l ubt 11 zed, l i v e r microsomes from untreated r a b b i t s o r r a b b i t s t r e a t e d w i t h TCDD o r p h e n o b a r b i t a l . The antiserum t o cytochrome P-450c does not c r o s s - r e a c t w i t h cytochrome P-450ab (Si). In a d d i t i o n , the antibody was t e s t e d f o r i t s a b i T l t y t o i n h i b i t microsomal monooxygenase a c t i v i t i e s . For use In these experiments, an immunoglobulin f r a c t i o n was i s o l a t e d from the goat serum by DEAE-cellulose chromatography (10). Experiments recorded i n Figure 1 d e p i c t the i n h i b i t i o n o f microsomal

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CONCEPTS

acetan H i d e h y d r o x y l a t i o n achieved by a d d i t i o n o f i n c r e a s i n g amounts o f anti-cytochrome P-450c immunoglobulin. A maximum i n h i b i t i o n o f ca. 80% i s observed. Immunoglobulin p u r i f i e d i n the same manner from nonimmune goat serum served as a n e g a t i v e c o n t r o l . N e i t h e r antibody i n t e r f e r e d w i t h the e x t r a c t i o n o r q u a n t i t a t i o n o f the r e a c t i o n products. Thus, antibody t o the major f r a c t i o n o f cytochrome P-450 (form c) i n h i b i t s an a c t i v i t y which i s induced by TCDD. Comparable amounts o f antibody do not i n h i b i t four monooxygenase a c t i v i t i e s which are not a s s o c i a t e d w i t h TCDD-induct Ion. These a c t i v i t i e s are a r y l hydrocarbon hydroxylase (benzo(a)pyrene h y d r o x y l a s e ) , amlnopyrlne N-demethyl a s e , 7-ethoxycoumarin O-deethylase, and coumarin hydroxylase

(2)Two s p e c t r a l p r o p e r t i e s o f cytochrome P-450c agree w i t h t h e concept t h a t t h i s form i s the TCDD-inducible cytochrome P-448. The Soret maximum o f the CO complex o f the reduced cytochrome Is 447 nm, s l i g h t l y lower than the v a l u e o f 449 nm observed f o r cytochrome P-450ab. In the presence o f jv-octylamine the o x i d i z e d cytochromes e x h i b i t d i f f e r e n t s p e c t r a , as shown i n F i g u r e 2. The spectrum o f cytochrome P-450c i s s i m i l a r t o the n-octylamine d i f f e r e n c e s p e c t r a observed f o r l i v e r microsomes f o l l o w i n g i n d u c t i o n by TCDD o r r e l a t e d inducers. The spectrum o f c y t o chrome P-450ab resembles t h a t o f uninduced microsomes (9). By t h r e e c r i t e r i a , cytochrome P-450ab and P-450c appear t o be d i f f e r e n t forms o f cytochrome P-450. They can be chromatog r a p h i c a l l y r e s o l v e d , a r e immunologically d i s c r e t e , and a r e s p e c t r a l l y d i s t i n g u i s h a b l e . Moreover, the s p e c i f i c i n h i b i t i o n o f a c e t a n i l l d e h y d r o x y l a t i o n by antI-cytochrome P-450c suggests that these cytochromes c a t a l y z e d i f f e r e n t monooxygenase r e a c t i o n s However, p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s experiments i n d i c a t e d cytochrome P-450ab might be composed o f more than one form o f the cytochrome. To f u r t h e r r e s o l v e t h i s p r e p a r a t i o n , DEAE-cellulose was employed. Two f r a c t i o n s were resolved and are r e f e r r e d t o as cytochrome P-450a and P-450b (9). Anti-cytochrome P-450c shows no immunological c r o s s - r e a c t i v i t 7 w i t h e i t h e r o f these forms. DEAE-cellulose i s a l s o used t o p u r i f y cytochrome P-450c t o near homogeneity (9). The i s o l a t i o n scheme i s o u t l i n e d i n F i g u r e 3. For comparison, cytochrome P-450 was p u r i f i e d from phenobarbital t r e a t e d r a b b i t s . The m a j o r i t y o f the p h e n o b a r b i t a l - i n d u c i b l e cytochrome P-450 was i n the f r a c t i o n corresponding t o cytochrome P-450a. Anti-cytochrome P-450c d i d not c r o s s - r e a c t w i t h t h i s cytochrome e i t h e r . These c o n d i t i o n s may not be optimal f o r p u r i f i c a t i o n o f t h i s form, but i t was obtained w i t h a content o f 11.0 nmoles per mg p r o t e i n and a y i e l d o f eighteen percent (18%). I t Is presumably the cytochrome P-450 LhL described by van der Hoeven et al (11). However, a d i r e c t comparison has not yet been made, and we w i l l denote t h i s form as cytochrome P-450d. The four cytochrome p r e p a r a t i o n s are summarized In Table I.

JOHNSON AND MULLER-EBERHARD

Rabbit Liver

Cytochrome

Cytochrome P450 Cytochrome P450

P-450

AB

c

T 0.02 O.D. i I—N~

350nm\

400nm/

Figure 2.

1 450nm

500nm

n-Octylamine difference spectra

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DRUG M E T A B O L I S M CONCEPTS

Table I Summary o f Cytochrome P-450 P u r i f i c a t i o n * Cytochrome P-450

Content

2

M o l e c u l a r Weight

Vleld(S)

a

9.7

48,000

b

12.0

60,000

2

c

17.7

54,500

5

d

11.0

51,000

16

3

2

51,000

1

V a l u e s a r e presented f o r t y p i c a l 2

preparations

nmoles o f cytochrome P - 4 5 0 per mg p r o t e i n based on t o t a l microsomal cytochrome P - 4 5 0 The f o u r forms o f cytochrome P-450 appear t o c o n s i s t o f peptides o f d i f f e r e n t molecular weight. These have been d e t e r mined by polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence o f sodium dodecyl s u l f a t e . R e s u l t s o f these experiments a r e shown in F i g u r e 4 and a r e presented i n Table 1. With the e x c e p t i o n o f cytochrome P - 4 5 0 c , the cytochromes seem t o be a s s o c i a t e d w i t h a s i n g l e major e l e c t r o p h o r e t i c band. Cytochrome P - 4 5 0 a comprises two p r i n c i p l e p e p t i d e s ; one o f these corresponds t o the phenob a r b i t a l - i n d u c i b l e form. The f o u r cytochromes c a t a l y z e d i f f e r e n t monooxygenase r e a c t i o n s when r e c o n s t i t u t e d w i t h NADPH cytochrome P-450 reduct a s e . For these experiments, h i g h l y p u r i f i e d reductase Is r o u t i n e l y i s o l a t e d from r a b b i t l i v e r microsomes. We use a s l i g h t m o d i f i c a t i o n o f t h e a f f i n i t y chromatographic procedure d e s c r i b e d by Yasukochi and Masters (12). These reductase p r e p a r a t i o n s c a t a l y z e the r e d u c t i o n o f "55-55 nmoles o f cytochrome c/mln/mg p r o t e i n and are obtained w i t h 38fc t o 55% o v e r a l l y i e l d s . In a d d i t i o n , the p h o s p h o l i p i d d l l a u r o y l - L - a - l e c t t h l n i s included i n the assay mixture. The a c t i v i t y o f t h e i n d i v i d u a l cytochrome P - 4 5 0 p r e p a r a t i o n s w i t h a v a r i e t y o f s u b s t r a t e s i s shown i n Table I I . When r e c o n s t i t u t e d , the f o u r cytochromes show c l e a r d i f f e r e n c e s i n t h e i r metabolism o f t h e s u b s t r a t e s we have t e s t e d thus f a r . Cytochrome P-450d r a p i d l y N-demethylates benzphetamine which Is i n accord w i t h our suggestion t h a t t h i s i s the phenob a r b i t a l - i n d u c i b l e form described by van der Hoeven et al (11)* In a d d i t i o n , cytochrome P - 4 5 0 c a c t i v e l y hydroxylates a c e t a n i l i d e . T h i s was expected as t h i s form o f t h e cytochrome and t h i s

4.

JOHNSON AND MULLER-EBERHARD

Rabbit Liver Cytochrome

P-450

77

Microsomes

Sodium c h o l a t e

solubilization

Polyethylene glycol

fractionation

H y d r o x y l a p a t i t e column chromatography

Cytochrome P-450ab

Cytochrome P-*»50c

DEAEcellulose column chromatography

Cytochroie P-450a (P-450d) Figure 3.

DEAEeellulose column chromatography

Cytochrome P-450b

Cytochrome P - 4 5 0 c

Schematic of the resolution of multiple forms of cytochrome P-450

Figure 4. Polyacrylamide gel electrophoresis of four forms of cytochrome P-450 (8 fig) in the presence of sodium dodecylsulfate

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CONCEPTS

TABLE II RECONSTITUTED ENZYME ACTIVITIES Cytochrome P-450a

P-450b

P-450c

P-450d

12

4.0

2.0

51

0.42

4.1

0.03

0.03

7 ethoxyresorufIn

0.04

0.4

0.4

0.003

Acetanl1Ide

1.2

1.3

6.1

1.1

Substrate Benzphetamlne Benzo(a)pyrene _

A c t i v i t i e s a r e expressed as moles o f product formed per mole o f cytochrome P-450. NADPH cytochrome P-450 reductase was present In excess t o ensure t h a t cytochrome P-450 was t h e r a t e l i m i t i n g component. Reactions were performed In a t o t a l volume of 1 ml c o n t a i n i n g 0.05 M Hepes b u f f e r , pH 7.4 and d i l a u r o y l - L a - l e c l t h l n (30 ug). M g C l (15 umoles) i s present when benzphetamlne and benzo(a)pyrene a r e the s u b s t r a t e s t e s t e d . Products of each s u b s t r a t e were q u a n t i t a t e d as described by the f o l l o w i n g authors: benzphetamine as HC0, T. Nash (1953) Biochem. J . 416-421; benzo(a)pyrene as 3*hydroxybenzo(a)pyrene, D. Nebert and H. Gelboin Tl968) J . B i o l . Chem. 24^, 3242-6249; 7-ethoxyr e s o r u f l n as r e s o r u f i n , M. Burke and R. Mayer (1974) Drug Metab. Dispos. 2, 245-253; a c e t a n i l i d e as hydroxyacetani1ide, K. K r i s c h and H, Staudinger (1961), Biochem. Z. 334, 312-327. Reactions were i n i t i a t e d by a d d i t i o n o f NADPH ( o T T y m o l e s ) . 2

a c t i v i t y a r e both induced by TCDD, and the microsomal enzyme i s i n h i b i t e d by anti-cytochrome P-450c. Benzo(a)pyrene i s metabolized r a p i d l y by cytochrome P-450b. The turnoveF value observed w i t h t h i s cytocrome (9) i s comparable t o that reported f o r a h i g h l y p u r i f i e d p r e p a r a t i o n o f r a t l i v e r cytochrome P-448 (J£). Both the p h e n o b a r b i t a l - and the TCDDi n d u c l b l e forms (d and c) a r e much l e s s a c t i v e w i t h t h i s s u b s t r a t e as assayed by formation o f f l u o r e s c e n t phenols. Thus, r a b b i t l i v e r does appear t o c o n t a i n an a c t i v e a r y l hydrocarbon hydroxylase ( $ ) . The r e s u l t s we have presented a r e c o n s i s t e n t w i t h t h e presence o f m u l t i p l e forms o f cytochrome P-450 i n l i v e r microsomes o f r a b b i t s t r e a t e d w i t h TCDD. The major form, cytochrome P-450c has been p u r i f i e d t o near homogeneity and has s p e c t r a l p r o p e r t i e s a s c r i b e d t o cytochrome P-448. However, I t does not a c t i v e l y hydroxylate benzo(a)pyrene when r e c o n s t i t u t e d w i t h

4. JOHNSON A N D MULLER-EBERHARD

Rabbit

Liver

Cytochrome

P-450

79

NADPH-cytochrome P-450 reductase. T h i s form o f the cytochrome i s Immunologically d i s t i n g u i s h a b l e from the o t h e r forms we have described. A r y l hydrocarbon hydroxylase (benzo(a)pyrene hydroxylase) was r e c o n s t i t u t e d w i t h cytochrome P-450b."" T h i s second form o f the cytochrome has been e x t e n s i v e l y p u r i f i e d and appears t o be composed o f a p e p t i d e o f higher molecular weight than e i t h e r cytochrome P-450c o r cytochrome P-450d. The t h i r d form, cytochrome P-450a, may be a mixture o f s e v e r a l cytochromes. Although i t i s i s o l a t e d i n a manner s i m i l a r t o the i s o l a t i o n o f the p h e n o b a r b i t a l - i n d u c i b l e cytochrome, form d, i t metabolizes benzphetamine a t a slower r a t e . None o f t h e s u b s t r a t e s t e s t e d thus f a r i s r a p i d l y metabolized by t h i s c y t o chrome. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s experiments show t h a t t h i s p r e p a r a t i o n c o n s i s t s o f s e v e r a l peptides w i t h d i f f e r e n t m o b i l i t i e s . Two prominent bands are observed. One band comigrates w i t h cytochrome P-450d, and the presence o f t h i s c y t o chrome i n the p r e p a r a t i o n may account f o r the benzphetamine Ndemethylase a c t i v i t y observed. The o t h e r major band has a f a s t e r m o b i l i t y than those o f the o t h e r t h r e e cytochrome preparations. Several i n v e s t i g a t o r s have d e s c r i b e d m u l t i p l e forms o f cytochrome P-450. These i n c l u d e two h i g h l y p u r i f i e d forms i s o l a t e d from r a t l i v e r microsomes (12) and s e v e r a l forms i s o l a t e d from mouse l i v e r microsomes"Tl4). Several forms have a l s o beenresolved from uninduced (15) and phenobarbital-induced (16) r a b b i t l i v e r microsomes. The number o f d i s c r e t e forms o f cytochrome P-450 present In r a b b i t l i v e r microsomes i s u n c e r t a i n , and the correspondence between p r e p a r a t i o n s i s o l a t e d i n v a r i o u s l a b o r a t o r i e s i s not yet c l e a r . In order t o begin a c l a r i f i c a t i o n o f t h i s s i t u a t i o n , a comparison was made between cytochrome P-450 LM^ i s o l a t e d from G-naphthaflavone induced r a b b i t 1iver microsomes (16), cytochrome P-448 i s o l a t e d from 3-methylcholanthrene induced r a b b i t l i v e r microsomes 0 7 ) > cytochrome P-450c. TCDD, B-naphthaf l a v o n e , and 3-methylcholanthrene are r e l a t e d inducers o f c y t o chrome P-450. These t h r e e cytochrome P-450 p r e p a r a t i o n s react s t r o n g l y w i t h antibody prepared a g a i n s t cytochrome P-450 LM^ o r P-450c. They a l s o e x h i b i t i d e n t i c a l m o b i l i t i e s i n p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s experiments. These experiments were performed i n Dr. M.J. Coon's and our l a b o r a t o r i e s . The work we have d e s c r i b e d i n d i c a t e s that benzphetamine N-demethylase, benzo(a)pyrene 3-hydroxylase, and a c e t a n i l i d e hydroxylase are each a s s o c i a t e d w i t h d i s t i n c t forms o f cytochrome P-450. The demonstration o f s u b s t r a t e s p e c i f i c i t i e s f o r m u l t i p l e forms o f cytochrome P-450 i s an important aspect o f t h i s i n v e s t i g a t i o n and i n d i c a t e s each form may f u n c t i o n i n d i f f e r e n t metab o l i c pathways. In a d d i t i o n , i t may be p o s s i b l e t o use these o r o t h e r assays t o detect i n d i v i d u a l cytochromes i n t i s s u e preparations. a

n

d

o

u

r

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DRUG M E T A B O L I S M C O N C E P T S

This research was supported by National Institutes of Health Grants HD-04445 and CA-17735 (Dr. U. Muller-Eberhard) and by a California Division, American Cancer Society Junior Fellowship Number J-301 (Dr. E . F . Johnson). We would like to thank Dr. Betty Sue Siler Masters for supplying a preprint of her work, and Dr. Russell Prough for providing samples of 7-ethoxyresorufIn and resoruftn. We would also like to express our appreciation to K. Cox, G. Schwab, and M. Zounes for their help in this investi­ gation. Literature Cited: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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