Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450_LM^red$\text{→}\text{O}{}_2$Complex I→Complex II→P-450_LM^ox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome $\text{b}{}_5$ is added. The latter protein does not appear to function as an electron carrier in this process.     

Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism

Two forms of cytochrome P-450 were purified to apparent homogeneity from several different preparations of human liver microsomes. One form, designated P-450DB, had relatively high catalytic activity towards the drugs debrisoquine, sparteine, bufuralol (both the (+)- and (-)-isomers), encainide, and propranolol and appears to be the enzyme involved in the polymorphic distribution of oxidative activities towards these substrates in humans. The other form, designated P-450PA, had relatively high phenacetin O-deethylase activity and appears to be involved in the variation of this activity among humans. Polyclonal antibodies raised to the two enzymes were specific for the antigens as judged by immunoelectrophoresis and immuno-inhibition studies. The two enzymes and their activities were distinguished by chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, immuno-inhibition studies, and steady-state kinetic assays. Immunochemical studies suggest that each form represents only a small fraction of the total cytochrome P-450 in human liver microsomes. These biochemical studies provide a basis for better understanding the mechanisms which underlie genetic polymorphisms involving P-450 cytochromes in humans.     

Immunoquantification of epoxide hydrolase and cytochrome P-450 isozymes in fetal and adult human liver microsomes

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16 beta-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16 alpha increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus.     

Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase

Rates of individual steps in the removal of alkyl groups from O6-methyl (Me) and -benzyl (Bz) guanine in oligonucleotides by human O6-alkylguanine DNA alkyltransferase (AGT) were estimated using rapid reaction kinetic methods. The overall reaction yields hyperbolic plots of rate versus AGT concentration for O6-MeG but linear plots for the O6-BzG reaction, which is approximately 100-fold faster. The binding of AGT and DNA (double-stranded 30-mer/36-mer complex) appears to be diffusion-limited. The rate of dissociation of the complex is approximately 25-fold slower (approximately 1 s(-1)) for DNA containing O6-MeG or O6-BzG than unmodified DNA. The fluorescent dC-analog 6-methylpyrrolo[2,3-d]pyrimidine-2(3H) one deoxyribonucleoside (pyrrolo dC), which pairs with G, was positioned opposite G, O6-MeG, or O6-BzG and used as a probe of the rate of base flipping. A rapid increase of fluorescence (k approximately 200 s(-1)) was observed with O6-MeG and O6-BzG and AGT but not with a Gly mutation at Arg128, which has been implicated in base flipping with crystal structures. Only weak and slower fluorescence changes were observed with G:pyrrolo dC or T:2-aminopurine pairs. These rate estimates were used in a kinetic model in which AGT binds and scans DNA rapidly, flips O6-alkylG residues, transfers the alkyl group in a chemical step that is rate-limiting in the case of O6-MeG but not O6-BzG, and releases the dealkylated DNA. The results explain the overall patterns of rates of alkyl group removal versus AGT concentration and the effects of the mutations, as well as the greater affinity of AGT for DNA with O6-alkylG lesions.     

Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G

The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of human cytochrome P450 enzymes.

Many biochemical approaches have been applied to the human cytochrome P450 enzymes, and more than 20 different gene products have been characterized with regard to their properties and catalytic specificities. The complement of the various cytochrome P450 enzymes in a given individual varies markedly, and dramatic differences may be seen in drug metabolism, pharmacological response, and susceptibility to toxic effects. An understanding of the nature of the individual cytochrome P450 enzymes and their regulation should be useful in determining the most suitable animal models, ascertaining risk from chemicals, and in avoiding undesirable drug interactions.     

Purification and characterization of the rat liver microsomal cytochrome P-450 involved in the 4-hydroxylation of debrisoquine, a prototype for genetic variation in oxidative drug metabolism

Genetic polymorphism in oxidative drug metabolism is perhaps best exemplified in the case of debrisoquine 4-hydroxylase activity, where the incidence of deficient metabolism ranges from 1% to 30% in various populations and this defect is also linked to an impaired ability to metabolize a number of other drugs effectively. Sprague-Dawley (SD) rats possess this activity, but females of the DA strain do not, although total cytochrome P-450 (P-450) levels are similar. We have purified, by using debrisoquine 4-hydroxylase activity as an assay, a minor P-450 to electrophoretic homogeneity from male SD rats and designate this as P-450UT-H. P-450UT-H differs from eight other purified rat liver P-450s as judged by peptide mapping and immunochemical analysis and thus appears to be isozymic with these other P-450s. P-450UT-H exhibited considerably more debrisoquine 4-hydroxylase activity than any of the other purified P-450s and, on a total P-450 basis, more than total microsomal P-450. Antibodies raised against P-450UT-H specifically recognized P-450UT-H and inhibited more than 90% of the debrisoquine hydroxylase activity present in SD rat liver microsomes. The level of P-450UT-H in SD rat liver microsomes accounted for less than 10% of the total P-450, as judged by immunochemical quantitation. These assays also indicated that the level of P-450UT-H in female DA rat liver microsomes is only about 5% of that in male or female SD rat liver microsomes, consonant with the view that deficiency of this form of P-450 is responsible for the defective debrisoquine 4-hydroxylase activity in the former animals.(ABSTRACT TRUNCATED AT 250 WORDS)