5 The Use of Animal Subcellular Fractions to Study Type I Metabolism of Xenobiotics
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R O N A L D W. ESTABROOK, JURGEN and J U L I A N A . P E T E R S O N
1
WERRINGLOER,
Department of Biochemistry, Southwestern Medical School, University of Texas Health Science Center, Dallas, TX 75235
The genius of man has r e s u l t e d i n the synthesis of a vast number of organic compounds during the l a s t century. The quest to seek new drugs, s y n t h e t i c polymers f o r i n d u s t r i a l use, i n s e c t i c i d e s and p e s t i c i d e s — to name but a few areas of chemical development — has provided both b e n e f i c i a l as w e l l as d e t r i m e n t a l r e s u l t s which touch the l i v e s of each of us. The l a s t decade has seen a surge of a c t i v i t y d i r e c t e d toward the e v a l u a t i o n of a wide a r r a y of organic chemicals which have the p o t e n t i a l of n e g a t i v e l y modifying the homeostasis of c e l l s and organs. Nearly every day the popular press i d e n t i f i e s yet another agent which can be shown i n experimental animals or b a c t e r i a l t e s t systems t o cause mutagenesis and c e l l u l a r dysf u n c t i o n or n e o p l a s i a . I t i s now w e l l e s t a b l i s h e d that many of these chemicals are merely precursors of metabolic products which a r e the true causative agents of c e l l u l a r changes. Therefore considerable e f f o r t i s now being expended to i d e n t i f y and c h a r a c t e r i z e the r e q u i s i t e enzyme systems r e s p o n s i b l e f o r the metabolic transformation of such chemicals. The purpose of t h i s p r e s e n t a t i o n i s to review the methodology employed and the r e s u l t s obtained when studying one such enzyme system — an enzyme complex which has a broad s p e c i f i c i t y f o r the o x i d a t i v e a l t e r a t i o n of a v a r i e t y of both n a t u r a l and s y n t h e t i c organic chemicals. C e n t r a l to t h i s enzyme complex i s a f a m i l y of hemeproteins c a l l e d cytochromes P-450. I t i s the p r o p e r t i e s and r e a c t i o n s of t h i s type of hemeprotein and i t s a s s o c i a t e d e l e c t r o n transport c a r r i e r p r o t e i n s which w i l l serve as the primary emphasis of the present r e p o r t .
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Supported in part by a research grant from the United States P u b l i c Health S e r v i c e N a t i o n a l I n s t i t u t e s of Health (GM-16488).
0-8412-0486-l/79/47-097-149$07.75/0 © 1979 American Chemical Society
Paulson et al.; Xenobiotic Metabolism: In Vitro Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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XENOBIOTIC METABOLISM
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The
C h a r a c t e r i z a t i o n of Cytochrome P-450
Cytochrome P-450 i s widely d i s t r i b u t e d i n nature spanning the phylogenetic t r e e from b a c t e r i a to the human organism. I t has been observed i n yeast, f l o w e r i n g p l a n t s , i n s e c t s , rept i l e s , b i r d s and animals (1,2). Indeed, i t i s i n t e r e s t i n g that the f i r s t r e p o r t of cytochrome P-450 occurred as a consequence of studying the r e s p i r a t o r y pigments of the mid-gut of the silkworm (3). The ease of d e t e c t i n g cytochrome P-450, using spectrophotometric methods to assess the c h a r a c t e r i s t i c absorbance band of the carbon monoxide adduct of the reduced hernep r o t e i n , has g r e a t l y f a c i l i t a t e d the q u a n t i t a t i v e e v a l u a t i o n of t h i s c e l l u l a r pigment (4,5_). In organisms other than some s p e c i a l i z e d b a c t e r i a , cytochrome P-450 i s a membrane bound p r o t e i n — a property which u n t i l r e c e n t l y has rendered i t r e f r a c t o r y to i s o l a t i o n and p u r i f i c a t i o n . Methodologies a r e now a v a i l a b l e , however, which permit the s o l u b i l i z a t i o n and s t a b i l i z a t i o n of the hemeprotein p e r m i t t i n g i t s c h a r a c t e r i z a t i o n as a homogeneous p r o t e i n s p e c i e s . I t i s p o s s i b l e to g e n e r a l i z e and i d e n t i f y two b a s i c types of cytochrome P-450 depending on the a s s o c i a t e d e l e c t r o n transport c a r r i e r proteins responsible f o r t r a n s f e r r i n g electrons (reducing e q u i v a l e n t s ) from reduced p y r i d i n e n u c l e o t i d e s to the hemeprotein. As shown i n F i g u r e 1, we have designated these as C l a s s A o r B. The C l a s s A type of cytochrome P-450 i s found a s s o c i a t e d w i t h the mitochrondria of many c e l l types where i t f u n c t i o n s together with an i r o n - s u l f u r p r o t e i n and a f l a v o p r o t e i n (6). The r e a c t i o n s of c h o l e s t e r o l metabolism i n s t e r o i d o g e n i c organs such as the adrenal, t e s t i s , and placenta are c a t a l y z e d by t h i s c l a s s of cytochromes P-450.
ClQSS Reduced Pyridine Nucleotide
h
A
Flavonrntein _Jron-Sulfur ^ Protein ,
a
v
o
ÇlQSS
p
r
o
t
e
m
^ Cytochrome P-450
Β
P y r i ^ N u c l e o t i d e - - Flavoprofin (FMN.FAD) V
* /
Cytochrome bs Figure 1. Two general classes of cytochrome F-450 based on the type of electron carriers functional in the transfer of reducing equivalents from reduced pyridine nucleotides
Paulson et al.; Xenobiotic Metabolism: In Vitro Methods ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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5.
ESTABROOK ET A L .
Type I
Metabolism
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Best c h a r a c t e r i z e d (_7,8) are the r e a c t i o n s f o r the o x i d a t i v e degradation of camphor by the bacterium Pseudomonas putida where t h i s c l a s s of cytochrome P-450 p l a y s a key r o l e i n the i n i t i a t i o n of the metabolism of t h i s complex molecule. The Class Β type of cytochromes P-450 i s g e n e r a l l y assoc i a t e d with the endoplasmic r e t i c u l u m (or i t s membrane equiva l e n t ) where i t f u n c t i o n s i n concert with a f l a v o p r o t e i n which contains both FMN and FAD as p r o s t h e t i c groups (9-11). This c l a s s of cytochrome P-450 i s g e n e r a l l y found a s s o c i a t e d with a second hemeprotein, cytochrome b . Although the r o l e of cytochrome b
