Klotz Alan V.

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Klotz
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Alan V.
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  • Thesis
    Purification and characterization of the hepatic microsomal monooxygenase system from the coastal marine fish Stenotomus chrysops
    (Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 1983-08) Klotz, Alan V.
    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.