A high spin form of cytochrome P-448 highly purified from PCB-treated rats--II. Characteristic requirement of cytochrome b5 for maximum activity

A high spin form of cytochrome P-448 (PCB P-448-H), highly purified from microsomes of PCB-treated rats, catalyzed oxidations of several compounds and required cytochrome b5 for its full activities in all oxidations examined. PCB P-448-H catalyzed the hydroxylation of aniline and O-dealkylations of p-alkoxy derivatives of aniline and nitrobenzene and 7-alkoxy derivatives of coumarin. Among the activities measured, hydroxylation of aniline and O-dealkylation of p-alkoxy derivatives of aniline were catalyzed by PCB P-448-H more efficiently than by PCB P-448-L, which was a low spin form of cytochrome P-448 purified from liver microsomes of PCB-treated rats. In all reactions, PCB P-448-H required cytochrome b5 for maximum activity. Slight requirements were also seen with PCB P-448-L but varied equivocally depending on the substrates. Cytochrome b5 showed its maximum effects on p-propoxyaniline O-depropylation activity at a molar ratio of cytochrome b5 to PCB P-448-H of 1:2. The enhancement by cytochrome b5 was more pronounced when lower concentrations of either the substrate or NADPH-cytochrome P-450 reductase were added to the reconstituted system. Based on these results, we confirm that PCB P-448-H is a unique form of cytochrome P-448 with respect to the requirements for cytochrome b5 and is a good probe to study the mechanisms involved in the enhancement of drug oxidations by cytochrome b5.     

Inhibition by SKF 525-A of 7-ethoxycoumarin O-deethylation in microsomal and the reconstituted monooxygenase systems from PCB-treated rat livers

Addition of diethylaminoethyl 2,2-diphenylvalerate-HCl (SKF 525-A) to the incubation mixture containing liver microsomes or purified cytochrome P-450 (PCB P-450) from PCB (KC-500)-treated rats resulted in non-competitive inhibition of 7-ethoxycoumarin O-deethylation activity whereas the addition to the incubation mixture containing purified cytochrome P-448 (PCB P-448) showed a competitive inhibition. Fortification of PCB-induced microsomes with purified NADPH-cytochrome P-450 reductase enhanced the O-deethylation activity. With the reductase-fortified microsomes, SKF 525-A inhibited the O-deethylation in a competitive manner. Based on these results, we confirmed that SKF 525-A inhibits non-competitively and competitively, depending on the species of cytochrome P-450. Our results also support the view that in microsomes from PCB-treated rats, PCB P-450 rather than PCB P-448 is mainly involved in the O-deethylation reaction, presumably due to the presence of a limited amount of NADPH-cytochrome P-450 reductase in microsomes.     

One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.     

Solubilisation, purification and reconstitution of hepatic microsomal azoreductase activity

Microsomal NADPH-cytochrome c (P-450) reductase and cytochrome P-450 were purified from the livers of phenobarbitone-treated rats. Purified NADPH-cytochrome c (P-450) reductase effected the NADPH-dependent reduction of FMN and FAD under anaerobic conditions in a non-enzymic manner, but was unable to reduce directly the azo dye, amaranth. In the presence of FMN, the purified reductase effected reduction of amaranth through the production of reduced FMN. Incorporation of NADPH-cytochrome c (P-450) reductase into the microsomal fraction increased the azoreductase activity of liver preparations from phenobarbitone-treated rats, but had no effect on azoreductase activity in preparations from control animals. Azoreductase activity was reconstituted into dilauroyl phosphatidylcholine vesicles containing purified cytochrome P-450 and purified NADPH-cytochrome c (P-450) reductase. In the absence of supplementary FMN, amaranth reduction was completely dependent upon all three components, but in the presence of FMN, the omission of any one component failed to abolish completely azoreductase activity.     

One-electron reduction of mitomycin c by rat liver: Role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H₂O₂ from redox cycling of the reduced mitomycin c in the presence of O₂ and the alkylation of ρ-nitrobenzylpyridine (NBP) in the absence of O₂ were taken as parameters.

2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems.

3. Under aerobic conditions the H₂O₂ production in the microsomal systems was dependent on NADPH, O₂ and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A.

4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O₂, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient.

5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450.

6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase.

7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O₂ tension is more likely the haem site of cytochrome P-450.     

Immunohistochemical localization and quantitation of NADPH-cytochrome P-450 reductase in human liver

An antibody raised in a goat against the human liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4.) enzyme has been used to: 1) immunoquantify the level of this enzyme in human liver microsomes, and 2) study the distribution of the reductase across the human liver acinus. Employing the Western blot procedure, anti-human reductase IgG recognized a single band in human liver microsomes which corresponded in molecular weight to the purified reductase. The content of the NADPH-cytochrome P-450 reductase in six normal human livers varied from 87 to 121 pmol/mg of microsomal protein. NADPH-cytochrome P-450 reductase activity of the same microsomes ranged from 107 to 222 nmol of cytochrome c reduced per min per mg of protein. The correlation between reductase content and activity (r = 0.54) was not statistically significant (p greater than 0.1). The total cytochrome P-450 content (cytochrome P-450 and P-420) of the same microsomes varied from 423 to 1413 pmol/mg of microsomal protein. The average ratio of cytochrome P-450 to NADPH-cytochrome P-450 reductase was 7.1:1 +/- 3.1 (mean +/- SD) in the human liver microsomal preparations studied. The reductase was found to be nonuniformly distributed across the human liver acinus. Although all hepatocytes stained positively for NADPH-cytochrome P-450 reductase, the staining intensity was highest in zone 3 and in some cases also in zone 1 hepatocytes. These results show that human liver contains a gross excess of cytochrome P-450 molecules to NADPH-cytochrome P-450 reductase molecules. Furthermore, the differential distribution of the reductase within the human liver acinus may lead to a better understanding of the mechanism underlining site-specific drug hepatotoxicity.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein

Addition of acrolein to rat lung or liver microsomal suspensions resulted in total inactivation of NADPH-cytochrome c reductase and partial conversion of cytochrome P-450 to P-420 in a concentration- and time-dependent fashion. Acrolein also caused total loss of nonprotein sulfhydryl content in both preparations, whereas protein sulfhydryl content was decreased by 40% and 28% in lung and liver preparations, respectively. Maxima of about 60% of the total lung cytochrome P-450 and 50% of the liver cytochrome P-450 in acrolein-treated microsomes did not support the N-demethylation of benzphetamine or ethylmorphine or hydroxylation of aniline because of the total loss of NADPH-cytochrome c reductase. Addition of purified NADPH-cytochrome c reductase to the acrolein-treated lung or liver microsomal suspension largely restored these monooxygenase activities. Addition of glutathione or dithiothreitol to the lung or liver microsomal suspension prior to the addition of acrolein significantly protected cytochrome P-450 from conversion to cytochrome P-420 as well as NADPH-cytochrome c reductase from inactivation. Thus, selective conjugation of acrolein with lung and liver NADPH-cytochrome c reductase but not cytochrome P-450 was responsible for total loss of these lung and liver monooxygenase activities.     

Stimulation of microsomal drug oxidation activities by incorporation into microsomes of purified NADPH-cytochrome c (P-450) reductase.

The effects of addition of purified NADPH-cytochrome c (P-450) reductase on microsomal activities of aniline hydroxylation, p-phenetidine O-deethylation and ethylmorphine and aminopyrine N-demethylations were investigated utilizing microsomes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats. The purified reductase was incorporated into microsomes. The drug oxidation activities were increased by the fortification of microsomes with the reductase while the extent of increase in the activities varied with the substrate and microsomes employed. The most pronounced enhancement was seen in p-phenetidine O-deethylation, followed by aniline hydroxylation and aminopyrine and ethylmorphine N-demethylations. The enhancement was more remarkable in microsomes from rats treated with 3-methylcholanthrene or phenobarbital. alpha-Naphthoflavone inhibited p-phenetidine O-deethylation activity markedly when the reductase was incorporated into microsomes, indicating that a larger amount of a species of cytochrome P-450 sensitive to the inhibitor was capable of participating in the oxidation of this substrate in the presence of the added reductase. One of the two Km values seen with higher concentrations of aniline or aminopyrine was altered by the fortification of microsomes with the purified NADPH cytochrome c (P-450) reductase. From these results, we propose that NADPH-cytochrome c (P-450) reductase transfers electrons to the selected one or two of multiple species of cytochrome P-450 more preferentially depending upon the substrate and the concentration of the substrate in microsomal membranes.     

Enhanced aryl hydrocarbon hydroxylase activity after interaction between solubilized cytochrome P-448 and microsomes low in endogenous cytochrome P-450

The effects of cumene hydroperoxide on microsomal mixed-function oxidase components and enzyme activities were determined. In vitro cumene hydroperoxide treatment decreased cytochrome P-450 content, benzphetamine N-demethylase activity and aryl hydrocarbon hydroxylase activity of hepatic and renal microsomes from adult male and female rats, and of hepatic microsomes from fetal rats. Cumene hydroperoxide-treated microsomes, as well as fetal liver and adult renal microsomes, which are naturally low in cytochrome P-450 and mixed-function oxidase activity, were used to incorporate partially purified hepatic cytochrome P-448 isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-pretreated immature male rats. This resulted in an enhanced rate of benzo[a]pyrene hydroxylation. Aryl hydrocarbon hydroxylase activity was increased 12-, 26-. 31- and 53-fold when 1.0 nmole of partially purified cytochrome P-448 was incubated with fetal liver microsomes, microsomes from kidney cortex of female rats, and cumene hydroperoxide-pretreated hepatic microsomes from female and male rats, respectively. The increased rate of benzo[a]pyrene hydroxylation was linear with cytochrome P-448 over the range 0.25 to 1.0 nmole. Because cumene hydroperoxide-pretreated microsomes from male rat liver and the hepatic and renal microsomes from female rats have a combination of high NADPH-cytochrome c reductase activity and low mixed-function oxidase activity, they are an attractive choice for catalytic studies of the interaction between cytochrome P-448 and microsomes.     

Studies on the properties of highly purified cytochrome P-448 and its dependent activity benzo[a]pyrene hydroxylase, from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae, produces a cytochrome P-450 enzyme with a Soret peak in the reduced-CO difference spectrum at 448 nm. The enzyme purified to homogeneity (88-97% pure on a specific content basis) has a molecular wt. of 55 500 as determined by SDS-PAGE. Amino acid analysis of yeast cytochrome P-448 revealed 407 amino acid residues per molecule with a 43% complement of hydrophobic residues. Although the number of residues is smaller than cytochrome P-448 enzymes from mammalian sources, the percentage of hydrophobic residues is almost identical. Estimation of the haem content of yeast cytochrome P-448 showed that one haem group was present per molecule. Phospholipid was present at very low levels. The molecular wt. of the polypeptide chain plus an estimated 5-6 units of hexose and of hexosamine is in good agreement with the molecular wt. value obtained from SDS-PAGE. A reconstituted system of purified cytochrome P-448, purified NADPH-cytochrome P-450 (c) reductase and phospholipid showed aryl hydrocarbon hydroxylase activity towards benzo[a]pyrene. Both protein components, NADPH and dilauroyl phosphatidylcholine (or emulgen 911) were necessary for full activity. The NADPH requirement could be replaced by cumene hydroperoxide or H2O2 generated in situ from a glucose oxidase system; in each case Vmax is increased, but the apparent affinity for benzo[a]pyrene, as measured by an increased Km, is lowered. The spin state of purified yeast cytochrome P-448 was 94% low spin (22 degrees C) as determined from the temperature-dependent spin-state equilibrium. The addition of benzo[a]pyrene to this enzyme resulted in a change to higher spin state (18% high spin at 22 degrees C). Equilibrium gel filtration analysis of the number of benzo[a]pyrene binding sites per mole of enzyme monomer showed a value of 1 for purified yeast cytochrome P-448 and 6 for this enzyme in microsomal form. The corresponding values for purified and microsomal cytochrome P-450 from phenobarbital-pretreated rats are 1 and 6, respectively. However, purified cytochrome P-448 from beta-naphthoflavone-induced rats gave a value of 6 benzo[a]pyrene binding sites. Type I binding spectra with purified yeast cytochrome P-448 were observed with benzo[a]pyrene, lanosterol, ethylmorphine, dimethylnitrosamine, sodium phenobarbitone and perhydrofluorene. Type II spectral changes were observed with imidazole, aniline and benzphetamine. Cytochrome P-448 from Saccharomyces cerevisiae is identified as a distinct enzyme of the P-450 family. This enzyme however has many properties in common with cytochrome P-448 from mammalian sources.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of azoreduction of dimethylaminoazobenzene by rat liver NADPH-cytochrome P-450 reductase and partially purified cytochrome P-450. Oxygen and carbon monoxide sensitivity and stimulation by FAD and FMN

We have reported that the hepatocarcinogen dimethylaminoazobenzene (DAB) is reduced by rat liver microsomes in an oxygen- and carbon monoxide-insensitive manner and that activity is induced by clofibrate but no other recognized inducers of cytochrome P-450 activity. In the present study we have shown that the reaction proceeds in a partially purified reconstituted cytochrome P-450 system as well as with purified NADPH-cytochrome P-450 reductase alone. In the latter system, activity is totally inhibited in air whereas the former system is active in air as well as in a carbon monoxide atmosphere. Although clofibrate induces both DAB azoreductase and laurate hydroxylase activities, the suicide substrate 10-undecynoic acid blocks the latter but not the former, implying catalysis by distinct enzymes. FAD and FMN stimulate DAB azoreduction 40-50-fold by both NADPH-cytochrome P-450 reductase alone and by the reconstituted cytochrome P-450 system. However, it was shown that these flavins facilitate electron flow to DAB only from reductase and not from cytochrome P-450. The fact that the reconstituted system, which contains NADPH-cytochrome P-450 reductase, is oxygen insensitive suggests that there is an obligatory electron flow through cytochrome P-450 to DAB, bypassing the oxygen-sensitive step.     

Characterization of mixed-function oxidases and purified cytochrome P-450 of livers of house musk shrew, Suncus murinus

Characteristics of mixed-function oxidases in the liver of house musk shrew, Suncus murinus, were studied. The basal level of cytochrome P-450 and the activity of drug-metabolizing enzyme in hepatic microsomes of Suncus murinus is relatively lower than that of rats, while the level of cytochrome b5 and NADPH-cytochrome c reductase is approximately the same as that of rats. The treatment of Suncus murinus with phenobarbital and 3-methylcholanthrene elevated the level of cytochrome P-450 and the activity of drug-metabolizing enzymes, but not the level of cytochrome b5. Two distinct forms of cytochrome P-450 have been purified from hepatic microsomes of 3-methylcholanthrene-treated Suncus murinus. These forms have their absorption maximum at 448.0 nm and 448.5 nm in CO-bound reduced form, and one is in the high-spin state and the other is in the low-spin state. They are different in their molecular weights (53,500 and 55,000) and in their spectral and catalytic properties. Characteristics of these forms were compared with those of the major forms of cytochrome P-450 purified from livers of rats treated with 3-methylcholanthrene.     

Interactions of diethylphenylphosphine with purified, reconstituted mouse liver cytochrome P-450 monooxygenase systems

Purified mouse liver cytochrome P-450 reconstituted with purified NADPH-cytochrome P-450 reductase and phosphatidylcholine metabolized diethylphenylphosphine to diethylphenylphosphine oxide. NADPH was required for the reaction and the amount of oxide formed was time and cytochrome P-450 dependent. Purified phenobarbital-induced cytochrome P-450 produced more oxide per nmole enzyme than any of the purified uninduced cytochrome P-450s. the phosphine oxide was also formed in lesser amounts in incubation mixtures containing only NADPH-cytochrome P-450 reductase and NADPH. Diethylphenylphosphine bound to oxidized purified phenobarbital-induced cytochrome P-450 and uninduced cytochrome P-450 with Ks values of 16 microM and 11-18 microM respectively. Diethylphenylphosphine was also a competitive inhibitor of p-nitroanisole O-demethylation catalyzed by a reconstituted phenobarbital-induced cytochrome P-450-dependent monooxygenase system, with a Ki value of 5 microM. The phosphine oxide produced no observable optical difference spectrum with oxidized phenobarbital-induced cytochrome P-450 and caused no inhibition of p-nitroanisole O-demethylation.     

Effects of cytochrome b5 on cytochrome P-450-catalyzed reactions. Studies with manganese-substituted cytochrome b5

The effects of cytochrome b5 with manganese-protoporphyrin IX substituted for heme were compared with those of native cytochrome b5 and the apoenzyme on the oxygenation of substrates in the reconstituted system containing liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and phosphatidylcholine. Mn-b5, unlike b5, remains essentially fully oxidized in the presence of NADPH and NADPH-cytochrome P-450 reductase under aerobic conditions. The effects of various concentrations of b5 and its derivatives were determined at constant P-450 and reductase concentrations. Cytochrome b5 inhibits benzphetamine demethylation by isozyme 2, the effect increasing up to the highest concentrations tested, and stimulates 7-ethoxycoumarin deethylation by isozyme 2 and acetanilide p-hydroxylation by isozyme 4, the optimal b5:P-450 molar ratio being about 2. In contrast, Mn-b5 inhibits all three reactions and apo-b5 is either inactive or slightly inhibitory. The activities of the three substrates as well as testosterone were determined with P-450 isozymes 2, 3b, 3c, and 4 in the reconstituted system with no additions or with b5 or Mn-b5 present. Cytochrome b5 is stimulatory, inhibitory, or without any effect, the result depending on both the substrate and P-450 isozyme present, whereas Mn-b5 is inhibitory in most instances. Both b5 and its manganese derivative alter the rates of testosterone 6 beta- or 16 alpha-hydroxylation by most of the P-450 cytochromes. The activities are influenced by the molar ratio of reductase to P-450. The Km values of benzphetamine, ethoxycoumarin, and acetanilide are, with one exception, significantly decreased in the presence of b5 or Mn-b5. We conclude that some of the effects of b5 on the oxygenase system are not accounted for by its role as an electron donor to cytochrome P-450.     

Influence of chronic ethanol consumption on hamster liver microsomal O-dealkylase activities and cytochrome b5 content

The effects of two different methods of administering ethanol to hamsters on liver microsomal cytochrome levels and the activities of ethoxyresorufin O-deethylase and p-nitroanisole O-demethylase have been examined. Administration of ethanol in liquid diets resulted in enhanced levels of cytochrome P-450, NADPH-supported aniline hydroxylase (Form I), and both NADPH- and NADH-supported p-nitroanisole O-demethylase. NADH-ferricyanide reductase was also increased. No change in NADPH-cytochrome c reductase or in the NADPH-supported rate of ethoxyresorufin O-deethylase was observed. In contrast, both NADH-supported ethoxyresorufin O-deethylase and cytochrome b5 levels were decreased. Administration of ethanol in the drinking water to chow-fed animals had no effect on total cytochrome P-450 levels; however, the rates of NADPH-supported aniline hydroxylase (Form I) and p-nitroanisole O-demethylase activity were increased. No changes in NADPH-cytochrome c reductase, NADH-ferricyanide reductase, or NADH-supported p-nitroanisole O-demethylase activity were noted. Cytochrome b5 levels were decreased as were both the NADPH- and NADH-supported rates of ethoxyresorufin O-deethylase. These data suggest that chronic consumption of ethanol by hamsters either in liquid diet form or as ethanol-water solutions to chow-fed animals lowers cytochrome b5 levels. When cytochrome b5 levels are lowered and total chromosome P-450 levels remain unchanged, the NADPH-supported rate of microsomal O-dealkylation of ethoxyresorufin is decreased. These data suggest that cytochrome b5 participates in the NADPH-supported microsomal O-dealkylation of ethoxyresorufin.     

Suppression of constitutive cytochrome P-450 gene expression in livers of rats undergoing an acute phase response to endotoxin

Administration of purified bacterial lipopolysaccharide (LPS) to male rats suppressed the constitutive hepatic expression of the male-specific cytochrome P-450 [AH, reduced flavoprotein:oxygen oxidoreductase (RH hydroxylating), E.C.1.14.14.1] isozyme P-450h (P450IIC11) to about 35% of control levels within 24 hr. The mRNA for P-450h was more rapidly and more profoundly suppressed than was the protein, indicating (a) that the decrease in the mRNA was responsible for the suppression of the protein and (b) that other mechanisms work to maintain expression of P-450h apoprotein in the face of repression of its mRNA. Suppression of P-450h expression was maximal at an endotoxin dose of 30-100 micrograms/kg, indicating that P-450 suppression is concomitant with the acute-phase response of hepatic secretory proteins. The female-specific cytochrome P-450 isozyme, P-450i (P450IIC12), was suppressed to 17% of control levels by LPS administration in female rats. Suppression of the P-450i apoprotein by LPS, and recovery of its expression, was more rapid than was suppression of P-450h in males. P-450i protein and mRNA levels were concomitantly suppressed by LPS, indicating that although there is a pretranslational component to the suppression, other mechanisms may also contribute. Calculations based on estimations of the microsomal contents of P-450h and P-450i relative to the total cytochrome P-450 in untreated rat livers indicate that suppression of these forms contributes significantly to the decreases in total microsomal P-450 after LPS treatment. In these studies, hepatic microsomal NADPH-cytochrome c reductase (TPNH2-cytochrome c reductase, E.C.1.6.2.4) activities and content of cytochrome b5 were decreased by LPS administration in both male and female rats. Like its effects on cytochrome P-450 expression, endotoxin suppression of NADPH-cytochrome c reductase activities and cytochrome b5 levels was more rapid in female rats than in males. The production of a local inflammatory response in male rats by subcutaneous injection of turpentine caused effects on cytochrome P-450, P-450h expression, and cytochrome b5 that were similar to those of endotoxin but were less rapidly achieved.     

Circadian changes of cytochrome P-450-dependent monooxygenase system in the rat liver.

Circadian changes in cytochrome P-450 and cytochrome b5 content and activity of NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases have been studied in rat liver microsomes in season autumn. The obtained results indicate, that cytochrome P-450 in 6-month-old animals shows 12 h rhythm, but in older ones 24 h rhythm. NADPH-cytochrome P-450 reductase activity shows 24 h rhythm in oldest animals only. Cytochrome b5 and its reductase has 24 h rhythm in all examined groups of rats.     

The involvement of cytochrome P-488 and P-450 in NADH-dependent O-demethylation of p-nitroanisole in rat liver microsomes.

These studies have shown that addition of p-nitroanisole to a reaction mixture containing rat liver microsomes resulted in an increase the reoxidation rate of NADH-reduced cytochrome b5. Fortification of rat liver microsomes with partially purified cytochrome b5 produces an increase in both NADPH-dependent and NADH-dependent p-nitroanisole O-demethylation activity. Antiserum to cytochrome P-450 isolated from phenobarbital-treated rat liver microsomes inhibited the NADH-dependent O-demethylation activity as well as the NADPH-dependent O-demethylation activity seen in rat liver microsomes. Addition of either purified cytochrome P-450 or cytochrome P-448 to an incubation mixture containing phenobarbital-treated rat liver microsomes enhanced the NADH-dependent p-nitroanisole O-demethylation activity. These results suggest that NADH-dependent and, in part, NADPH-dependent O-demethylations are catalyzed by cytochrome P-448 and cytochrome P-450 receiving electrons from cytochrome b5.     

Different effects of cyanide on the activities of 7-ethoxycoumarin O-deethylation catalyzed by two forms of cytochrome P-450 purified from 3-methylcholanthrene-treated rats

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.