Purification and characterization of the hepatic microsomal monooxygenase system from the coastal marine fish Stenotomus chrysops
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https://hdl.handle.net/1912/2761DOI
10.1575/1912/2761Abstract
Three cytochrome P-450 forms were highly purified
(8-12 nmol/mg) from the hepatic microsomes of the untreated
coastal marine fish Stenotomus chrysops (scup) by detergent
solubilization and column chromatography. Scup cytochromes
P-450A, P-450B and P-450E (labeled in order of elution from
the first ion exchange column) had distinct spectroscopic
properties, substrate profiles and minimum molecular weights
on SDS-polyacrylamide gels (52.7, 45.9 and 54.3 K,
respectively). The three purified cytochrome P-450 isozymes
yielded different peptide maps when digested with
a-chymotrypsin or S. aureus V8 protease. An additional
hemoprotein fraction called cytochrome P-450 fraction D was
also resolved and partially purified. This cytochrome P-450
preparation was characterized by a red shift in the
CO-ligated, reduced difference spectrum with a chromophore
near 451 nm. The scup NADPH-cytochrome P-450 reductase was
purified to electrophoretic homogeneity (Mr = 82.6 K), had
a specific activity of 45-60 U/mg with cytochrome c and
contained both FAD and FMN.
Scup cytochrome P-450E (Mr = 54.3 K) is the major
aryl hydrocarbon hydroxylating form in untreated hepatic
microsomes as judged by both its abundance (30-50% of the
total resolved cytochromes P-450) and catalytic activity
with benzo[a]pyrene (turnover number = 0.9 nmol product/nmol
P-450/min). Further, reconstituted cytochrome P-450E was
inhibited 70-80% by 100 uM 7, 8-benzoflavone in catalytic
assays, similar to the 80-90% inhibition of benzo[a]pyrene
hydroxylase in microsomal incubations. Analysis of
benzo[a]pyrene products derived from reconstitutions of
purified cytochrome P-450E revealed that greater than 50% of
the oxidation occurred at benzo-ring positions, like the
product profile observed in incubations with microsomes.
The molecular weight of the purified cytochrome P-450E is
identical to the major microsomal hemoprotein induced by
3-methylcholanthrene pretreatment, suggesting cytochrome
P-450E is the major aromatic hydrocarbon-inducible
cytochrome P-450 form in scup. Rabbit antisera raised
against purified scup cytochrome P-450E reacts in
Ouchterlony diffusion analysis with cytochrome P-450E
antigenic determinants in microsomes but not purified
cytochrome P-450A. Further, the antisera cross-reacts with
an apparent 3-methylcholanthrene-inducible cytochrome P-450
isozyme purified from trout liver (TLM-4a; Williams and
Buhler, Compo Biochem. Physiol. 7SC: 25-32, 1983), giving a
pattern of fusion without visible-spurring in Ouchterlony
analysis. These observations imply common antigenic
determinants for the apparent major 3-methylchoianthrene-inducible
cytochrome P-450 forms in trout and scup.
Monooxygenase reconstitution experiments indicated that
purified scup cytochrome P-450A actively hydroxylated
testosterone at the 6ß position (turnover number = 0.8
nmol/min/nmol cytochrome P-450). Reconstituted cytochrome
P-450B oxidized testosterone at two different sites
tentatively identified as the 2-a and l5-a positions (total
turnover number = 0.07 min-1). Cytochrome P-450 fraction
D produced several metabolites upon reconstitution (sum
turnover number = 0.2 min-1) including two chromatographically
similar to 16a- and 16ß-hydroxytestosterone.
Reconstituted cytochrome P-450E was a poor catalyst of
testosterone hydroxylase but the only detectable product was
6 ß-hydroxytestosterone (turnover number = 0.04 min-1).
However, besides benzo[a]pyrene, reconstituted cytochrome
P-450E was active with 7-ethoxycoumarin, acetanilide and
7,8-benzoflavone as substrates.
Addition of purified scup cytochrome b5 to monooxygenase
reconstitutions had a stimulatory effect on some
catalytic rates. The magnitude of the cytochrome b5
stimulation depended on the P-450 isozyme and the substrate
used in the reconstitution; cytochrome P-450A was generally
influenced by the presence of cytochrome b5. This rate
stimulation was greater than the effect obtained with
purified rabbit liver cytochrome b5. In an extreme
example, cytochrome P-450E was completely inactive in
reconstitutions of 7-ethoxyresorufin O-deethylase (an
activity associated in microsomes with aromatic hydrocarbon
induction) in the presence or absence of rabbit cytochrome
b5 but the addition of scup cytochrome b5 to the
reconstitution led to an observed O-deethylation rate of 7.0
min-1. It is uncertain whether these cytochrome b5
effects are exhibited in microsomes or in vivo but the
stimulation in reconstitutions appears to be important in
the evaluation of catalytic activity with purified isozymes.
Description
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution August 1983
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Suggested Citation
Thesis: Klotz, Alan V., "Purification and characterization of the hepatic microsomal monooxygenase system from the coastal marine fish Stenotomus chrysops", 1983-08, DOI:10.1575/1912/2761, https://hdl.handle.net/1912/2761Related items
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