Oxidation of substituted N,N-dimethylanilines by cytochrome P-450: estimation of the effective oxidation-reduction potential of cytochrome P-450

Rates of N-demethylation of N,N-dimethylaniline and of eight meta- or para-substituted N,N-dimethylanilines by rat liver cytochrome P-450PB-B (P-450) were determined under conditions where oxidation was supported by iodosylbenzene or NADPH-P-450 reductase. The rates of dimethylaniline oxidation were found to correlate with the substrate oxidation-reduction potential within each series of substrates supported by a particular oxygen activation protocol; the kcat for each substrate studied was approximately 20-fold faster in the iodosylbenzene-supported system relative to the NADPH-P-450 reductase supported system. Since the N-demethylation of amines is believed to proceed via an initial electron-transfer step, a kinetic scheme for P-450 was proposed that enabled evaluation of the data according to theoretical treatments that correlate rates of electron transfer with extrakinetic parameters. In these analyses, the data could be fitted to the Rehm-Weller and Agmon-Levine equations, providing lambda values (for the energetics of enzyme-substrate reorganization) of 22-26 kcal mol-1 and apparent E1/2 (oxidation-reduction potentials) of 1.7-2.0 V (vs saturated calomel) for the oxidized enzyme. The apparent E1/2 for the enzyme is composed of contributions from the intrinsic potential of the active prosthetic core of the enzyme, the Fe = O - porphyrin species, and a coulombic factor that is a function of the charge-separated radical anion/radical cation pair produced upon electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Iodosylbenzene derivatives as oxygen donors in cytochrome P-450 catalyzed steroid hydroxylations.

The mechanism of cytochrome P-450 catalyzed steroid hydroxylations in rat liver microsomes has been investigated by employing derivatives of iodosylbenzene as oxygen donors. The model steroid substrate androstenedione which was hydroxylated in positions 7 alpha, 6 beta, and 16 alpha was used in reactions supported by NADPH, iodosylbenzene, and iodosylbenzene derivatives. Evidence for cytochrome P-450 involvement in iodosylbenzene-sustained androstenedione hydroxylation included inhibition by substrates and modifiers of cytochrome P-450. The most efficient oxygen donors were (diacetoxyiodo)-2-nitrobenzene greater than (diacetoxyiodo)-2-chlorobenzene greater than 2-nitroiodosylbenzene greater than (dinitratoiodo)-2-nitrobenzene greater than (diacetoxyiodo)benzene greater than (diacetoxyiodo)-2-methoxybenzene greater than 4-(diacetoxyiodo)toluene greater than iodosylbenzene. The capacity of the oxidation agents to serve as oxygen donors in cytochrome P-450 dependent steroid hydroxylation is probably dependent upon several factors such as the tendency of iodosyl compounds to associate, which decreases coordination with the heme iron, the presence of bulky substituents in the 2 position (decreases association), and the presence of electron-withdrawing substituents (tends to decrease coordination with the heme iron). The rates of 7 alpha, 6 beta, and i6 alpha hydroxylation of androstenedione catalyzed by (diacetoxyiodo)-2-nitrobenzene were 108-, 130-, and 167-fold higher, respectively, than the rates of the NADPH-supported reactions. These results strongly suggest that the rate-limiting step in NADPH-sustained cytochrome P-450 catalyzed reactions is the rate of reduction of cytochrome P-450.     

Characterization of the enzymatic and nonenzymatic peroxidative degradation of iron porphyrins and cytochrome P-450 heme

Both purified cytochrome P-450 (P-450) and free ferriprotoporphyrin IX are destroyed by NADPH-P-450 reductase in the presence of NADPH and O2. The process appears to be mediated by H2O2 generated by reduction of O2. Six major products were identified from the reaction of H2O2 with ferri-protoporphyrin IX-hematinic acid, methylvinylmaleimide, and four dipyrrolic propentdyopents. The structures of the propentdyopents were elucidated by mass spectrometry and 1H NMR methods. Both free ferriprotoporphyrin IX and P-450 yielded these same products in similar relative ratios. P-450 heme in rat liver microsomes was degraded in the presence of O2 and NADPH and either NaN3 (a catalase inhibitor) or Fe-ADP (which promotes lipid peroxidation); the products were primarily hematinic acid, methylvinylmaleimide, and small quantities of one propentdyopent. Only the two maleimides were detected in the destruction of microsomal P-450 heme by cumene hydroperoxide and iodosylbenzene. On the basis of the reaction of H2O2 with several metal-octaethylethylporphyrin complexes and free octaethylporphyrin, the iron chelated in ferriprotoporphyrin IX is required for degradation by H2O2. Biliverdin is not an intermediate in the formation of maleimides and propentdyopents from heme. Experiments using the tetraethylpropentdyopent produced from ferrioctaethylporphyrin suggest that propentdyopents are not further cleaved to form the maleimides. A mechanism for oxidative heme destruction consistent with these observations is proposed.     

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.     

Enzymatic oxidation of disulfides and thiolsulfinates by both rabbit liver microsomes and a reconstituted system with purified cytochrome P-450.

Both rabbit liver microsomes and reconstituted system with purified cytochrome P-450 and cofactors enzymatically oxidized o-dithiane (1, 2-dithiane), 3-methyl-o-dithiane, thiane and 2-methylthiane to the corresponding mono-oxygenated products; sulfides or disulfides were oxidized to the corresponding sulfoxides or thiosulfinates, while thiosulfinate was oxidized to thiolsulfonate. The reconstituted systems required oxygen and NADPH and were not affected by the catalase which decomposes H2O2, or by 1,4-diazabicyclo-[2,2,2]octane (DABCO), which is a good quencher of singlet oxygen. The differences in the binding of substrates such as sulfides and disulfides with the enzyme system are discussed in connection with differences in the spectra of the substrates in the reconstituted system with pure cytochrome P-450. A correlation was found between the rates of oxidation of the substrates and the rates of oxidation of NADPH.     

Involvement of an Inducible Cytochrome P-450 in Precocene II Oxidation by Streptomyces Griseus

Streptomyces griseus oxidizes the insecticide precocene II to its cis- and trans-dihydrodiols and 3-chromenol after growth on an enriched medium containing soybean flour. Oxidation of precocene II is dependent on the level of cytochrome P-450 in this organism. Extracts of cells grown on media lacking soybean flour were devoid of cytochrome P-450 and could not oxidize precocene II. In an in vitro reconstituted system containing NADPH, spinach ferredoxin reductase, spinach ferredoxin and ammonium sulfate fractions enriched in cytochrome P-450, precocene II was oxidized to its dihydrodiols. An aerial mycelium-negative variant of S. griseus (AMY mutant), that was unable to elicit cytochrome P-450 when grown on soybean flour-enriched medium, failed to oxidize precocene II.     

Hydroxyl-radical production and ethanol oxidation by liver microsomes isolated from ethanol-treated rats.

In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.     

Oxygen radical generation by microsomes: role of iron and implications for alcohol metabolism and toxicity

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by microsomes, a minor pathway of alcohol metabolism, involved generation of hydroxyl radical (.OH). Microsomes oxidized chemical .OH scavengers (KMB, DMSO, t-butyl alcohol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal .OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal .OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize .OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal .OH production, the oxidation of ethanol by .OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 and NADPH-P-450 reductase indicated that the reductase is the critical enzyme locus for interacting with iron and catalyzing production of reactive oxygen species. Microsomes isolated from rats chronically fed ethanol catalyzed oxidation of .OH scavengers, light emission, and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a .OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.     

Oxidation of cycloalkylamines by cytochrome P-450. Mechanism-based inactivation, adduct formation, ring expansion, and nitrone formation

Mechanism-based destruction of cytochrome P-450 (P-450) and P-450 heme is observed during the oxidation of N-cyclopropyl and N-cyclobutyl benzylamines. The slower inactivation by the cyclobutylamines relative to cyclopropylamines is consistent with known relative rates of ring opening of cycloalkyl-substituted aminium radicals. Evidence was found that porphyrin meso adducts of the type reported for horseradish peroxidase and cyclopropanone hydrate (Wiseman, J. S., Nichols, J. S., and Kolpak, M. X. (1982) J. Biol. Chem. 257, 6328-6332) were not formed. Radiolabels from cyclopropylamine substrates were covalently attached to protein but essentially only from the cyclopropyl portion and not the benzylic portion. Neither label appeared to be bound to extractable heme; however, during oxidations with cyclopropylamines, labeled P-450 heme became covalently attached to protein. Oxidation of 1-phenylcyclobutylamine by P-450 yielded 2-phenyl-1-pyrroline and 2-phenylpyrrolidine, and the ring expansion is interpreted as evidence for the existence of aminium radicals based on precedents with monoamine oxidase (Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138). In addition, purified P-450PB-B oxidized N-(1-phenylcyclobutyl)-benzylamine to N-(1-phenyl)cyclobutyl phenyl nitrone, identified using spectral techniques. This transformation involves two sequential oxidations with either a hydroxylamine or benzylidene intermediate. While P-450 oxidized the amine to both compounds, only the hydroxylamine was rapidly oxidized to give the nitrone. The ring expansion and nitrone products are interpreted in the context of aminium radical intermediates involved in the mechanism of P-450-catalyzed amine oxidation.     

Ethanol oxidation by components of rat liver microsomes

A new method was employed for the purification of cytochrome P-450 from rat liver microsomes. The purified cytochrome was essentially free from possible contaminants and the recovery and degree of purification were high. Although 15% of the original P-450 was recovered through the purification procedure used, only 0.8% of the total original microsomal ethanol oxidation activity was associated with this fraction. Addition of this purified fraction to other fractions isolated did not further stimulate ethanol oxidation. The component of rat liver microsomes that was found most efficient in the oxidation of ethanol was the mixture of catalase and NADPH - cytochrome c - reductase. It is concluded that highly purified cytochrome P-450 by itself does not oxidize ethanol to any appreciable degree.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

Biphasic kinetic behavior of rat cytochrome P-4501A1-dependent monooxygenation in recombinant yeast microsomes

Rat cytochrome P-4501A1-dependent monooxygenase activities were examined in detail using recombinant yeast microsomes containing rat cytochrome P-4501A1 and yeast NADPH-P-450 reductase. On 7-ethoxycoumarin, which is one of the most popular substrates of P-4501A1, the relationship between the initial velocity (v) and the substrate concentration ([S]) exhibited non-linear Michaelis-Menten kinetics. Hanes-Woolf plots ([S]/v vs. [S]) clearly showed a biphasic kinetic behavior. Aminopyrine N-demethylation also showed a biphasic kinetics. The regression analyses on the basis of the two-substrate binding model proposed by Korzekwa et al. (Biochemistry 37 (1998) 4137-4147) strongly suggest the presence of the two substrate-binding sites in P-4501A1 molecules for those substrates. An Arrhenius plot with high 7-ethoxycoumarin concentration showed a breakpoint at around 28 degrees C probably due to the change of the rate-limiting step of P-4501A1-dependent 7-ethoxycoumarin O-deethylation. However, the addition of 30% glycerol to the reaction mixture prevented observation of the breakpoint. The methanol used as a solvent of 7-ethoxycoumarin was found to be a non-competitive inhibitor. Based on the inhibition kinetics, the real V(max) value in the absence of methanol was calculated. These results strongly suggest that the recombinant yeast microsomal membrane containing a single P-450 isoform and yeast NADPH-P-450 reductase is quite useful for kinetic studies on P-450-dependent monooxygenation including an exact evaluation of inhibitory effects of organic solvents.     

Factors involved in the regulation of the levels and activities of rat liver cytochromes P-450

Methods have been developed for the purification of eight rat liver microsomal cytochrome P-450 (P-450) isoenzymes. Another rat P-450, responsible for the metabolism of the genetic polymorphism prototype debrisoquine, has also been partially purified from rat liver. Six P-450s have been purified to electrophoretic homogeneity from human liver preparations. The rat and human P-450s can be quantified in crude samples using 'immunoblotting' methods coupled with peroxidase visualization. A study on the effects of a family of polybrominated biphenyl congeners led to the conclusion that the levels of all of the rat P-450s considered above are under some degree of independent regulation. In monolayer culture, different P-450s show different stabilities and levels of several are selectively regulated by various media components. Studies with the eight isolated rat P-450s indicate that the iron spin state, oxidation-reduction potential (Fe3+/Fe2+ couple), and catalytic activity towards substrates are not related to each other. The major function of phospholipid in reconstituted P-450/NADPH-P-450 reductase systems is the facilitation of formation of a complex of the two proteins. Studies on the regioselective hydroxylation of warfarin have been used to develop an order of binding affinity of the different P-450s for NADPH-P-450 reductase.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.     

Electron transport in the membrane of lutoids from the latex of Hevea brasiliensis.

1. An antimycin-insensitive NADH-cytochrome c oxidoreductase (E.C. 1.6.99.3) activity can be demonstrated in the membrane of lutoids isolated from the latex of Hevea brasiliensis. This electron transport system can also use ferricyanide as an electron acceptor, but is unable to oxidize NADPH. 2. Two beta-type cytochromes are present in the membranes. Cytochrome beta563 is partially reduced by NADH and ascorbate, but is not reducible by NADPH. It shows a double peak at 555 and 561 nm at 77 degrees K. A second cytochrome, cytochrome beta561, seems to be reducible by hydrosulfite only. 3. In the reduced state, these cytochromes do not combine with CO. The occurrence of cytochrome P-450 could not be demonstrated. 4. The role of the NADH oxidation system is considered in relation to the biosynthesis of polyisoprene compounds in the latex.     

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.     

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.     

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.