Conversion of arachidonic acid to two novel products by a cytochrome P450-dependent mixed-function oxidase in polymorphonuclear leukocytes

Canine polymorphonuclear leukocytes metabolize [14C] arachidonic acid into 2 unidentified products, separated by thin-layer chromatography and high performance liquid chromatography, and called peak 1 and peak 2. The formation of peak 1 is maximal at 5 minutes and then declines, while the synthesis of peak 2 increases throughout the 30 minute incubation period. The formation of peak 1 and, to a lesser extent, peak 2, was enhanced after dual inhibition of lipoxygenase and cyclo-oxygenase enzymes with BW755C (94 microns) or nafazatrom (37 microns), or after incubation in a calcium-free buffer. In contrast, the formation of these products was inhibited by SKF-525A (50 microns), suggesting a cytochrome P450-dependent mechanism. The presence of cytochrome P450 in neutrophil microsomes was confirmed by measuring aryl hydrocarbon hydroxylase activity and cytochrome P450 content.     

Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceus TS: Assay and characterization of the enzyme system

Microsomal preparations of $\text{Aspergillus ochraceus}$ TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and O₂ and exhibits a pH optimum of 8.0–8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistant with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.     

Metabolism of diphenyloxazole (PPO) by mouse liver microsomes.

2, 5-Diphenyloxazole (PPO) is an inducer and inhibitor of aryl hydrocarbon hydroxylase. We report that PPO is itself metabolized to an alkali-extractable metabolite with intense fluorescence. The fluorescence spectra of excitation and emission indicate peaks at 345 nm and 510 nm, respectively. The reaction is linear with respect to time and enzyme concentration. NADPH is required for activity and the reaction is inhibited by carbon monoxide and 7, 8-benzoflavone but not by SKF-525A or hexobarbital. The intensity of fluorescence produced is similar to that of benzo (a) pyrene. PPO may be a useful model compound in studies of drug metabolism by the mixed function oxidase.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Microsomal 11α-hydroxylation of progesterone in Aspergillus ochraceus: Part I: Characterization of the hydroxylase system

Microsomes (105,000xg sediment) prepared from induced cells of $\text{A.ochraceus}$ was found to hydroxylate progesterone to 11α-hydroxyprogesterone (11α-OHP) in high yields (85–90% in 30 min.) in the presence of NADPH and O₂. The pH optimum for the hydroxylase was found to be 7.7. However, for the isolation of active microsomes grinding of the mycelium should be carried out at pH 8.3. Metyrapone, carbon monoxide, SKF-525A, p-CMB and N-methyl maleimide inhibited the hydroxylase activity indicating the involvement of cytochrome P-450 system. The inhibition of the hydroxylase by cytochrome $\text{c}$ and the presence of high levels of NADPH-cytochrome $\text{c}$ reductase in induced microsomes suggest that the reductase could be one of the components in the hydroxylase system.     

Kinetic studies of inhibition of estradiol 2- and 16 alpha-hydroxylases in rat liver microsomes with various cytochrome P-450 inhibitors

Kinetic studies of inhibition of estradiol 2- and 16 alpha-hydroxylase activities in male rat liver microsomes with cytochrome P-450 inhibitors, alpha-naphthoflavone, DL-aminoglutethimide, SKF-525A and metyrapone, were extensively carried out. All of the inhibitors competitively blocked the two enzyme activities. The former three inhibitors preferentially inhibited the 16 alpha-hydroxylase activity while the reverse result was obtained in the case of metyrapone, and SKF-525A was the most potent inhibitors for the two enzyme among the four inhibitors. The kinetic data, the apparent Ki's for the four inhibitors and Km's for the substrate estradiol in the assays, along with the inhibition results with carbon monooxide suggest that different forms of cytochrome P-450 may be involved in the two hydroxylations. Kinetic parameters of the two hydroxylase activities in female rat liver microsomes were then determined to be an apparent Km of 23.0 and 158 microM and Vmax of 99.0 and 5.65 pmol/min/mg protein for the 2-hydroxylation and the 16 alpha-hydroxylation, respectively. The kinetic data show that the 2-hydroxylation may be quantitatively an exclusive hydroxylative pathway in estrogen metabolism in female.     

Peroxide-supported in-vitro cytochrome P450 activities in Haemonchus contortus.

The potential for cytochrome P450 from Haemonchus contortus to operate in the oxygen-poor intestinal environment was investigated by examining the ability of the cytochrome to act in vitro as a peroxygenase in utilising cumene hydroperoxide for substrate oxidations not requiring molecular oxygen. Peroxygenase and NADPH-supported monooxygenase activities were measured in microsomes prepared from L3 and adult nematodes. Both cumene hydroperoxide- and NADPH-supported ethoxycoumarin O-deethylase and aldrin epoxidase activities were detected in larval microsomes. Adult microsomes showed low levels of cumene hydroperoxide-supported ethoxycoumarin O-deethylase, as well as NADPH- and cumene hydroperoxide-supported aldrin epoxidase activities. The use of inhibitors in ethoxycoumarin O-deethylase assays with larval microsomes indicated that the peroxygenase pathway does not proceed via ferrous cytochrome P450 (no inhibition by carbon monoxide), did not require molecular oxygen, and did not depend on electron flow from cytochrome P450 reductase. Larval activity was inhibited by typical cytochrome P450 inhibitors (piperonyl butoxide, SKF-525A, chloramphenicol, metyrapone, n-octylamine) and was unaffected by the peroxidase inhibitor salicylhydroxamic acid. In contrast, adult microsomal cumene hydroperoxide-supported ethoxycoumarin O-deethylase activity was significantly inhibited by both cytochrome P450 inhibitors and salicylhydroxamic acid. Adult microsomes also contained potassium ferrocyanide peroxidase activity utilising cumene hydroperoxide. This activity showed a similar pattern of inhibition by both cytochrome P450 and peroxidase inhibitors. Whilst the ability of larval H. contortus cytochrome P450 to act as a peroxygenase in vitro was demonstrated, the inhibition results with adult microsomes showing both cytochrome P450 and peroxidase activities require further investigation to clarify the nature of the adult microsomal cumene hydroperoxide-supported O-deethylase activity.     

Anti-P450lpr antiserum inhibits specific monooxygenase activities in LPR house fly microsomes.

Monooxygenase activity in microsomes from the LPR strain of house fly (Musca domestica L.) was inhibited by anti-P450lpr, and antiserum specific for house fly cytochrome P450lpr. Anti-P450lpr did not inhibit house fly cytochrome P450 reductase or rat cytochrome P450 monooxygenase assays, consistent with specific inhibition of P450lpr. Anti-P450lpr inhibited the ability of cytochrome P450 reductase to reduce carbon monoxide treated LPR microsomal cytochrome P450, up to 49% of the total, showing that inhibition of cytochrome P450 reduction is the major mechanism of inhibition. Anti-P450lpr inhibited 98% of methoxyresorufin-O-demethylase activity and all the benzo(a)pyrene hydroxylase activity in LPR microsomes, but none of the pentoxyresorufin-O-dealkylase activity. The antiserum partially inhibited ethoxyresorufin-O-dealkylase and ethoxycoumarin-O-dealkylase activity. These results demonstrate that methoxyresourfin-O-demethylase activity and benzo(a)pyrene hydroxylase activity are characteristic substrates for P450lpr activity in the LPR strain of house fly.     

A soluble Bacillus cereus cytochrome P-450cin system catalyzes 1,4-cineole hydroxylations.

A cytochrome P-450-dependent monooxygenase system that catalyzes the stereospecific hydroxylation of the monoterpene substrate 1,4-cineole was demonstrated in cell-free preparations of Bacillus cereus UI-1477. 1,4-Cineole hydroxylations were catalyzed by a 100,000 x g (1-h)-centrifuging soluble, hexane-inducible enzyme that activated and incorporated molecular oxygen into hydroxylated products; required NADH; was inhibited by SKF-525A, imidazole, metyrapone, and octylamine; and displayed a 452-nm peak in the carbon monoxide difference absorption spectrum. The constant 7:1 ratio of endo/exo alcohol products formed when 1,4-cineole was hydroxylated by normal cells, hexane-induced cells, and cell extracts suggested that a single enzyme designated cytochrome P-450cin was responsible for both reactions.     

The role of cytochrome P-450 in the synthesis of polar metabolites of aldosterone by microsomes of male rat liver

Among the tissues of the male rat studied, the largest quantities of the neutral polar metabolites of aldosterone were synthesized by the hepatic microsomal fraction. The polar metabolites of aldosterone were separated by HPLC into six peaks. Three peaks of non-polar (reduced) metabolites were also synthesized. Synthesis of at least four of the neutral polar metabolites was induced by phenobarbital and inhibited by both CO and SKF-525A. The rates of synthesis of these metabolites, which were linear up to 5 minutes, correlated well with the concentration of cytochrome P-450 in the liver microsomes. Addition of aldosterone to the microsomal fraction caused a pronounced type 1 change in the cytochrome P-450 spectrum. The half maximal spectral change (K_s) for aldosterone was calculated to be 8 μM. These experiments indicate that the neutral polar metabolites of aldosterone are produced by cytochrome P-450 dependent hydroxy lations.     

Inhibition of lipoxygenase enzyme in vitro by the cytochrome P-450 inhibitors metyrapone and SKF-525-A

It has recently been demonstrated that agents which block lipoxygenase enzymes (i.e. nordihydroguaiaretic and eicosatetraynoic acid) also block cytochrome P-450 in some $\text{in vitro}$ preparations. In some cases, therefore, results which were based on these lipoxygenase inhibitors could have been produced by the inhibition of cytochrome P-450. We therefore sought a way to distinguish between effects produced by metabolites of arachidonic acid generated by lipoxygenase enzymes and those produced by metabolites of the cytochrome P-450 system. seemed that a straightforward approach might be to administer drugs that inhibit the cytochrome P-450 system first, and then examine effects of lipoxygenase inhibitors. However, it then became necessary to establish the effect of inhibitors of the cytochrome P-450 system on lipoxygenase enzymes. Initial work was carried out using crystalline soybean lipoxygenase enzyme to avoid the complexities involved in isolating enzymes from biological tissues.Enzyme activity was assessed spectrophotometrically by measuring the increase in absorbance at 240 nm produced by the formation of a conjugated triene (15-HETE) from arachidonic acid. Effects of the two most commonly used cytochrome P-450 antagonists, metyrapone and SKF-525A, were determined by Lineweaver-Burke analysis. One major difficulty was that arachidonic acid is soluble in aqueous media at Ph 8 whereas both SKF-525-A and metyrapone are soluble at pH 7. In order to maintain all components in solution at the same time, experiments were carried out at pH 7.35 in the presence of 5% dimethylsulfoxide. There was no observable difference in the activity of 15-lipoxygenase at pH 9 in the absence of DMSO and that observed at pH 7.35 in the presence of DMSO.Studies were carried out in 2 ml quartz spectrophotometer cuvettes. For each experiment, two cuvettes were prepared containing arachidonic acid, DMSO (0.1 ml), SKF-525-A or metyrapone, and Tris buffer (pH 7.35). The cuvettes were placed in a Beckman DB/G spectrophotometer. One cuvette was used as reference and to the other was added 3ug (0.1 ml) of a solution of crystalline soybean lipoxygenase (Sigma). The change in absorbance at 240 nm was recorded and reflected product formation. Concentrations of arachidonic acid ranged from 10–50 uM, each tube contained either 0–50 uM SKF-525-A or 0–200 uM metyrapone.Results show that both SKF-525-A and metyrapone inhibited soybean lipoxygenase. Lineweaver-Burke analysis indicated that both agents acted in uncompetitive fashion. Of particular importance is that these concentrations of SKF-525-A and metyrapone are similar to those routinely used to block cytochrome P-450. From these results, it appears that experiments in which SKF-525-A and metyrapone were employed may need to be reevaluated. Work is underway using platelet-derived 12-lipoxygenase.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Genetic regulation of coumarin hydroxylase activity in mice. Biochemical characterization of the enzyme from two inbred strains and their F1 hybrid.

Hydroxylation of coumarin to 7-hydroxycoumarin by liver microsomes from control or phenobarbital-pretreated mice is 5- to 10-fold higher in the DBA/2J strain compared to the AKR/J strain, while activities of nine other cytochrome P-450 mediated oxidations show only minor differences. Mixing experiments with whole liver homogenates and subcellular fractionations do not reveal the presence of enzyme activators or inhibitors or competing enzyme reactions in either strain. Comparisons of pH optima (pH 7.6), heat stability at 52 degrees C (6 to 8 min for 50% inactivation), and Km values (0.45 to 0.50 microM coumarin) for coumarin hydroxylase show no significant differences in the two strains of mice or their F1 hybrid. Similarly, only minor differences in inhibition of coumarin hydroxylase by carbon monoxide, SKF-525A, menadione, and several other inhibitors of microsomal mixed function oxidase reactions are observed in the two strains. In contrast to these data, aniline and metyrapone, two compounds which bind to the heme iron of cytochrome P-450 to form ferrihemochromes, show differential and opposite patterns of inhibition of enzyme activity in the DBA/2J and AKR/J mouse strains. This latter observation suggests that a structurally different cytochrome P-450 may hydroxylate coumarin in these two inbred mouse strains.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

The occurrence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in Drosophila melanogaster microsomes, and the importance of this metabolizing capacity for the screening of carcinogenic and mutagenic properties of foreign compounds.

The presence of cytochrome P-450 and aryl hydrocarbon hydroxylase activity in microsomes prepared from three strains of Drosophila melanogaster was investigated.The first microsomal preparations, for which mortar pounding was used to homogenize the insects, indicated the occurrence of cytochrome P-450, as concluded from carbon monoxide difference spectra, exhibiting an absorbance maximum at 452 nm.As the reproducibility of the mortar procedure was irregular, a new homogenization procedure was developed, using glass marbles. This method led to a better reproducibility and an increase in the quality of the carbon monoxide difference spectra.An empirical correction by a computer was used for analysis of the spectra.Aryl hydrocarbon hydroxylase activity, one of the expressions of the mixed function oxidase system mediated by cytochrome P-450, was studied by using benzo(a)pyrene as substrate and measuring enzymically produced 3-hydroxy-benzo(a)pyrene. The observed activity was NADPH dependent and was absent (i) when NADH was given as cofactor, (ii) when the microsomes were destroyed by boiling, and (iii) when they were deteriorated by treatment with deoxycholate.These findings provide the basis for further investigations, by which a better understanding of the metabolizing capacity of Drosophila can make this species even more meaningful for the screening of pre-carcinogens and indirectly acting mutagens.     

Metabolism of prostaglandin A1 by hepatic microsomal monooxygenase P-450 system in the guinea pig and rat.

This study demonstrates the metabolic transformation of prostaglandin A₁ (PGA₁) by guinea pig and rat liver microsomes. The transformation, which required NADPH and oxygen, yielded polar (presumably hydroxylated) products. Incubations with guinea pig liver microsomes yielded one zone of product on tlc, whereas rat liver microsomes produced two discernable metabolic zones. It was demonstrated that PGA₁ metabolism in the guinea pig and the rat was inhibited by the addition of SKF-525A, metyrapone, carbon monoxide and cytochrome $\text{C}$; nicotinamide (10 mM) inhibited only the guinea pig system. These findings indicate that the enzymatic activity responsible for PGA₁ metabolism is composed of a typical cytochrome P-450 monooxygenase system.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes.

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37 degrees C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Catalytic properties of monooxygenases from isolated immunocompetent cells

The kinetic parameters of two monoxygenase (N-demethylase and benzo(a)pyrene hydroxylase) reactions occurring in murine immunocompetent cells were determined. It was shown that the pH optimum for macrophage enzymes lies at 7.4, whereas that for thymocytes and spleen cells at 8.5. The effects of cytochrome P-450 inhibitors (methyrapone, SKF-525A, tilorone) on monooxygenase systems of immunocytes were investigated. Methyrapone and SKF-525A were shown to be effective inhibitors of N-demethylase. The mode of inhibition changes from non-competitive to competitive, depending on the inhibitor concentration. Tilorone, an immunostimulator and inhibitor of liver cytochrome P-450-dependent enzymes, competitively inhibits N-demethylase and benzo(a)pyrene hydroxylase of immunocytes.