Cytochrome P-450 ligands: metyrapone revisited

Expressing metyrapone interactions with ferrous cytochrome P-450 as ligand saturation by cytochrome, rather than the more conventional cytochrome saturation by ligand, an extinction coefficient of 68.5 +/- 1.8 mM-1 cm-1 for the metyrapone complex of dithionite-reduced rat hepatic microsomal cytochrome P-450 was derived. Utilizing this new extinction coefficient, the increased cytochrome P-450 present after phenobarbital induction was almost exclusively that which is able to both bind to metyrapone and form a metabolic-intermediate complex from norbenzphetamine. However, it was not the only subpopulation present in microsomes that was able to bind metyrapone, nor the only one capable of forming a metabolic intermediate complex from norbenzphetamine. Thus, neither technique alone can be used to quantitate the "phenobarbital-induced form" of cytochrome P-450.     

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.     

Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration.     

Cytochrome P-450 catalyzed redox cycling of orthophenylphenol

o-Phenylphenol was converted to 2,5-dihydroxy biphenyl (phenylhydroquinone) by microsomal P-450. Depending on the cofactor used, microsomal enzymes catalyzed oxidation and/or reduction of phenylhydroquinone. Phenylhydroquinone was oxidized to phenyl 2,5'-p-quinone by cumene hydroperoxide-supported microsomal P-450. Phenyl 2,5'-p-quinone was reduced to phenylhydroquinone by cytochrome P-450 reductase. This study provides direct evidence of cytochrome P-450 catalyzed redox cycling of o-phenylphenol. It is postulated that redox cycling of o-phenylphenol may play a role in o-phenylphenol-caused bladder cancer.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Cytochrome P-450 mediated biotransformation of organic nitrates

The vascular biotransformation of organic nitrates appears to be a prerequisite for their action as vasodilators. In the current study, we assessed the involvement of cytochrome P-450 in the denitration of glyceryl trinitrate and the enantiomers of isoidide dinitrate. Denitration of organic nitrates by the microsomal fraction of rat liver was NADPH dependent and followed apparent first-order kinetics. Under aerobic conditions, the t1/2 of D-isoidide dinitrate was significantly shorter than that of L-isoidide dinitrate (11.9 vs. 14.1 min, p less than or equal to 0.05), which is consistent with the greater potency of the D-enantiomer for vasodilation. Under anaerobic conditions, the denitration of glyceryl trinitrate was very rapid (t1/2 approximately 30 s). Organic nitrate biotransformation was inhibited by carbon monoxide, SKF 525A, and dioxygen. This suggests that the biotransformation of organic nitrates can occur through the direct interaction with the heme moiety of cytochrome P-450. The biotransformation of glyceryl trinitrate was catalyzed preferentially by those isoenzymes induced by phenobarbital. The biotransformation of glyceryl trinitrate was regioselective for 1,3-glyceryl dinitrate formation except in phenobarbital-induced microsomes under aerobic conditions, in which preferential formation of 1,2-glyceryl dinitrate occurred. These data suggest that cytochrome P-450 is involved in the biotransformation of organic nitrates and raises the possibility that vascular cytochrome P-450 may play a role in the mechanism-based biotransformation of organic nitrates, the result of which is vascular smooth muscle relaxation.     

Cytochrome P-450-mediated redox cycling of estrogens

The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro. Depending on the cofactor used, microsomal enzymes catalyzed reductions and/or oxidations of the estrogens: Phenobarbital-induced rat liver microsomes catalyzed the oxidation of DES to 4',4"-diethylstilbestrol quinone (DES quinone) with cumene hydroperoxide as cofactor. The quinone was unstable and spontaneously rearranged to (Z,Z)-dienestrol. DES quinone was reduced to a mixture of E- and Z-isomers of DES by NADPH catalyzed by purified cytochrome P-450 reductase. After rearrangement of the quinone to (Z,Z)-dienestrol, reduction reactions did not proceed. Rat liver microsomes and NADPH catalyzed the conversion of DES to (Z,Z)-dienestrol and (Z)-DES, but DES quinone could not be detected. The reactions described provide direct evidence for microsome-mediated redox cycling of estrogens. Although DES quinone could not be detected in the incubation of DES, microsomes, and NADPH as cofactor, the intermediacy of the quinone is demonstrated by the formation of (Z,Z)-dienestrol, the marker product for oxidation. The quinone could not be detected because it was rapidly reduced to DES and its Z-isomer. Microsome-mediated redox cycling between 2-hydroxyestradiol and the corresponding quinone was also demonstrated. Using cumene hydroperoxide as cofactor, the oxidation to the quinone was favored, while with NADPH as cofactor the reduction to 2-hydroxyestradiol was preferred. It is postulated that microsome-mediated redox cycling of estrogens plays a role in hormonal carcinogenesis.     

Studies on the Later Stage of the Biosynthesis of Pleuromutilin

Mutilin (4) and deoxy analogues 2 and 3 are biosynthetic precursors of pleuromutilin (1) in the later stage of biosynthesis. Precursors 2 and 3 are required for studies on the oxygenation steps in biosynthesis, and were synthesized from readily available 1 via 4 by deoxygenation of the hydroxy groups. Feeding experiments with the ²H-labeled precursors confirmed their microbial conversion into 1.     

Regional Distribution of Cytochrome P-450 in the Rat Brain: Spectral Quantitation and Contribution of P-450b,e and P-450c,d

The cytochrome P-450 (P-450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P-450b,e and P-450c,d to brain P-450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P-450b and P-450c, respectively). P-450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p-chloroamphetamine-coupled Sepharose. The yield of P-450 from whole brain was 90 +/- 19 pmol/g of tissue, which is approximately 1% of the level in liver microsomes from control rats. The amount of P-450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P-450 content, with yields of up to 400 pmol/g whereas the substantia nigra yielded only 16-20 pmol/g. Immunohistochemical studies with anti-P-450b and anti-P-450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti-P-450c. In the cerebellum, both anti-P-450b and anti-P-450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti-P-450b gave weak staining of certain astroglia, but with anti-P-450c, there was intense staining of neuronal somata.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome P-450 in culture forms of Trypanosoma cruzi

Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40-60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. Epimastigotes show induced peptides of mol. wt 56,000, 52,000, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.     

Cytochrome P-450 catalyzed oxidation of 1-piperidinoanthraquinone]

Oxidation of 1-piperidinoantraquinone (1-PA) in microsomal fractions of rat liver was studied. The only product of complete oxidation of 1-PA--(N-antraquinone-1)-delta-aminovaleric acid-was identified using paper and thin-layer chromatography. The participation of cytochrome P-450 in oxidation of 1-PA was demonstrated by sharp inhibition, involving blowing of the microsomes with CO and treatment with sodium deoxycholate. Studies of differential spectra of cytochrome P-450 in the presence of 1-PA are indicative of the first type of binding between 1-PA and cytochrome P-450. The binding constants (Ks) and the kinetic parameters (Km and V) for the above substrate in control microsomes and in those induced by phenobarbital and 3-methyl cholanthrene were determined. The results obtained suggest that cytochrome P-450 is involved in oxidation of a number of heterocyclic compounds resulting in the opening of the ring.