Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.     

Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system.

Oxidative demethylation of dimethylnitosamine was studied with both reconstituted and unresolved liver microsomal cytochrome P-450 enzyme systems from rats and hamsters. Proteinase treatment of liver microsomal preparations yielded cytochrome P-450 particulate fractions. Both cytochrome P-450 and NADPH- cytochrome c reductase fractions were required for optimum demethylation activity. Particulate cytochrome P-450 fractions were more effecient than either Triton X-100- or cholatesolubilized preparations of these particles in demethylation activity with rat and hamster liver preparations appear to be due to differences in specificity in their cytochrome P-450 fractions.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

Inhibition of protein synthesis: a basis for tunicamycin-induced decrease in rat liver cytochrome P-450

Tunicamycin caused a dose and time dependent decrease in cytochrome P-450 in rat liver. A dose of 50 micrograms/kg caused a decrease of about 50% in 72 hours. A similar decrease in the activities of rat liver microsomal aniline hydroxylase, aminopyrine N-demethylase and ethoxycoumarin O-deethylase were also seen after the tunicamycin treatment. Tunicamycin also suppressed food and water intake but the decrease in cytochrome P-450 was not related to these effects. NADPH cytochrome c reductase was not markedly decreased by tunicamycin. A decrease in cytochrome P-450 was also observed in cultured rat hepatocytes treated with tunicamycin. It decreased incorporation of [35S]-methionine into total proteins as well as into various cytochrome P-450 isozymes of rat hepatocytes. This indicates that a decrease in protein synthesis may be responsible for the tunicamycin-induced decrease in cytochrome P-450 and drug metabolism.     

STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES : IV. Some Observations on the Properties of Ribosomes and Polysomes from Rat Liver and Hepatomas

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min'' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.     

Effect of mesitylene on ethanol metabolism in rat liver microsomes.

Increased catalase activity was observed in the liver microsomal fraction of ethanol-treated rats (10% v/v aqueous ethanol solution per os for 5 weeks). In contrast, cytochrome P-450 concentration and specific activity of NADPH-cytochrome c reductase remained at the same level as in the liver of control rats (drinking water). The ratio of microsomal H2O2-generation to catalase activity was lower in the "ethanol" group than in the control one. This phenomenon seems to be related to the increased contribution of the "peroxidatic" reaction (increased rate of ethanol oxidation). Administration of mesitylene (1,3,5-trimethylbenzene) by gastric tube for 3 days (5 mmoles per kg daily) increased cytochrome P-450 concentration, specific activity of NADPH-cytochrome c reductase and ethanol metabolism.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.     

Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver.

Nuclei isolated from differentiated normal liver, moderately differentiated Hepatoma 5123 and poorly differentiated Novikoff hepatoma have been compared with respect to ATP-dependence of messenger-like RNA release and resistance to lysis (DNA release) in a cell-free system containing homologous cytosol. The release of RNA from the nuclei of liver, Hepatoma 5123D and the Novikoff hepatoma was totally ATP-dependent, partially ATP-dependent and ATP-independent, repectively. The sensitivity of the nuclear RNA transport in the presence of ATP to beryllium nitrate, an inhibitor of a nuclear pore phosphatase, paralleled their ATP-dependence. Although RNA release from the nuclei of both liver and Novikoff hepatoma has an absolute requirement for cytosol proteins, the structural integrity of liver, but not Novikoff hepatoma nuclei in the presence of ATP, is dependent on macromolecules in the cytosol.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Studies on lipid peroxidation in normal and tumour tissues. The Yoshida rat liver tumour.

Reduced rates of lipid peroxidation have been observed in Yoshida hepatoma cells and microsomes when compared with appropriate control tissue (normal rat liver) under the same pro-oxidant conditions. The pro-oxidant conditions used were incubation with NADPH+ADP+iron or ascorbate+iron or exposure to gamma-irradiation. As previously shown with the Novikoff hepatoma, the relative concentrations of alpha-tocopherol and polyunsaturated fatty acids are important in conferring resistance to lipid peroxidation in the Yoshida hepatoma. Furthermore, NADPH-cytochrome c reductase and the NADPH-cytochrome P-450 electron transport chain, which are involved in the initiation and propagation of certain types of lipid peroxidation, are found at very much reduced levels in the Yoshida hepatoma. The relative importance of these aberrations are discussed.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Ring-andN-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system.

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.     

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.     

The NADPH-dependent cytochrome P-450 reduction in liver microsomes of rats of different ages with and without phenobarbital pretreatment.

The activity of NADPH-cytochrome P-450 reductase in liver microsomes of 10- to 60-day-old rats was determined. Neither the half life time of cytochrome P-450 reduction nor the absolute amount of cytochrome P-450 reduced per time unit depend on age. Phenobarbital pretreatment enhances the reduction rate in all age groups. The addition of hexobarbital or ethylmorphine to microsomal suspension accelerates the reduction of cytochrome P-450 in some age groups only. Age differences corresponding to developmental changes in drug-metabolizing activities are not detectable. The NADPH-cytochrome P-450 reductase seems to be not responsible for the age dependence of drug metabolism.     

Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone

The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.     

Effects of phenobarbitone on hepatic microsomal enzyme activity and liver blood flow in spontaneously hypertensive rats.

Hepatic microsomal enzyme activity, liver blood flow and pentobarbitone sleeping time were determined in spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NR) after pretreatment with saline or phenobarbitone. In NR and SHR the increases in total liver blood flow produced by phenobarbitone were sufficient to maintain liver perfusion despite the increase in liver weight and in both strains of rat the increase was entirely due to increased portal venous return. Saline pretreated SHR had shorter pentobarbitone sleeping times than control NR and their livers had greater total cytochrome c reductase activities and total microsomal protein than those of NR but cytochrome P-450 contents were not significantly different. Phenobarbitone significantly shortened sleeping times in both strains but NR still slept longer than SHR. Total microsomal protein, cytochrome P-450 content and cytochrome c reductase activity were increased by phenobarbitone in both SHR and NR but the increases in cytochrome P-450 and cytochrome c reductase were greater in the hypertensive rats.     

7 Alpha-hydroxylation of cholesterol by reconstituted systems from rat liver microsomes

A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-¹⁴C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.     

Comparison of highly purified sheep liver and lung NADPH-cytochrome P-450 reductases by the analysis of kinetic and catalytic properties

1. Reductase was purified to electrophoretic homogeneity from sheep liver and lung microsomes. The specific activity of both enzymes ranged from 55 to 66 mumol cytochrome c reduced/min/mg protein. 2. Liver and lung reductases appeared to have similar kinetic and spectral properties. Km (NADPH) and Km (cytochrome c) values were calculated to be 14.3 +/- 1.23 microM and 22.2 +/- 2.78 microM for liver and 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM for lung reductase, respectively. Kinetic studies showed that cytochrome c can bind the oxidized form of the enzyme as well as its reduced form and both reductases operated through a ping-pong type mechanism. 3. These reductases cannot be distinguished on the basis of monomer molecular weights (Mr 78,000) except that the liver reductase was found to be more susceptible to proteolytic attack. 4. Both reductases supported aniline 4-hydroxylation and ethylmorphine N-demethylation reactions to the same extent in the reconstituted systems. However, sheep lung reductase appeared only 36.5 and 14.8% as effective in catalyzing benzo[a]pyrene reaction as an equivalent amount of reductase from liver in the presence of liver cytochrome P-450 and 3MC-treated rat liver cytochrome P-448, respectively.