Search

To elucidate the nature of substrate specificity and intrinsic mechanism of hydroxylation of steroids, in the present work we carried out molecular cloning and heterologous expression of cDNA for three new forms of cytochrome P45017alpha from species of the Bovidae family (sheep, goat, and bison), which catalyze 17alpha-hydroxylation of both progesterone (P4) or pregnenolone (P5) and 17,20-lyase reaction resulting in cleavage of side chain with formation of C(19)-steroids. Recombinant cytochromes P45017alpha were expressed in E. coli as derivatives, containing a six-His tag at the C-terminal sequence that simplifies purification of the cloned heme proteins using metal-affinity chromatography. Highly purified cytochromes P45017alpha were used for determination of enzyme activity and specificity in relation to progesterone, pregnenolone, 17alpha-hydroxyprogesterone, and 17alpha-hydroxypregnenolone with registration of the kinetics of reaction product formation using HPLC. It is shown that each form of cytochrome P45017alpha is characterized by a specific profile of enzyme activity and dependence of 17,20-lyase reaction on the presence of cytochrome b(5) in the reaction mixture. The analysis of the activity of the known forms of cytochrome P45017alpha in view of the data obtained in the present work allows the division of known cytochromes P45017alpha into three main group: group A (pig, hamster, rat), cytochromes P45017alpha catalyze the reaction of 17alpha-hydroxylation of both P4 and P5 steroids and the 17,20-lyase reaction of 17alpha-hydroxyprogesterone and 17alpha-hydroxypregnenolone; group B (human, bovine, sheep, goat, and bison), cytochromes P45017alpha, which have no or have insignificant 17,20-lyase activity in relation to 17alpha-hydroxyprogesterone; group C (guinea pig), cytochrome P45017alpha which either has no or has insignificant 17,20-lyase activity on transformation 17alpha-hydroxypregnenolone to dehydroepiandrosterone.

Cytochrome P450s (P450 or CYP) are the largest family of hemeproteins yet characterized. X-ray crystallographic studies have shown that the heme of the P450 hemeproteins is buried in the interior of the protein molecule. Unexplored are answers to questions concerning the role of heme in the folding of newly synthesized apo-P450s and the factors that influence changes in heme accessibility following modification of the pattern of folding of the holo-P450s. We have carried out the present studies to measure changes in heme accessibility in P450s. This is an initial step to determining whether heme-binding confers structural and functional integrity and stability to a P450 molecule. Recently, we have shown that apo-high molecular weight cytochrome b5 (apo-HMWb5) is an efficient acceptor of heme when added to a preparation of purified recombinant CYP3A4. In the present work we have studied heme binding by apo-HMWb5 when mixed with a number of different hemeproteins (myoglobin, hemoglobin, catalase, CYP4A1, CYP101, and CYP3A4). These hemeproteins differ in the location of the heme (i.e., surface or internal) allowing one to study changes in structure as measured by the process of heme transfer from one protein to another. It was found that heme transfer to apo-HMWb5 occurs relatively rapidly from hemeproteins where the heme is located at or near the surface or when the hemeprotein is denatured. In contrast, heme transfer from P450s to apo-HMWb5 occurs only following modification of the P450 structure with chaotropic agents. An exception is CYP3A4 where a measurable amount of heme is transferred to apo-HMWb5 in the absence of denaturing agents. The preliminary results described here employs apo-HMWb5 as an indicator for assessing changes in heme-availability of P450s as the protein-folding of the molecule is altered.

The antiandrogenic drug, flutamide, is widely used in the treatment of carcinoma of the prostate. The present study examines the metabolism of flutamide by human liver microsomes and purified recombinant human cytochrome P450s (CYP), expressed as fusion proteins. These studies show the principal role of CYP1A2 in the metabolism of flutamide to 2-hydroxyflutamide. A minor metabolite is formed during the metabolism of flutamide by CYP3A4 in the presence of an excess of added purified NADPH-P450 reductase. The metabolism of flutamide is inhibited by low concentrations of alpha-naphthoflavone and ketoconazole. Other substrates of CYP1A2, such as phenacetin, imipramine, caffeine, and estradiol, are also inhibitors of flutamide metabolism by CYP1A2. Of interest is the inhibition of flutamide metabolism by its metabolite, 2-hydroxyflutamide, and the inhibition of the 2- and 4- hydroxylation of estradiol by flutamide. CV1 cells do not metabolize flutamide to 2-hydroxyflutamide. In assays performed using this cell line transfected with the cDNA for the androgen receptor, flutamide is a pure antagonist, and 2-hydroxyflutamide, while a more potent androgen receptor (AR) antagonist, activates the AR at higher concentrations. Stable expression of CYPIA2 in these CV1 cells causes flutamide to exhibit agonistic properties at higher concentrations, a behavior not exhibited by cells stably transfected only with the expression vector encoding the AR. These findings raise the possibility that increased conversion of flutamide to 2-hydroxyflutamide or accumulation of 2-hydroxyflutamide in cells may contribute to the anomalous responses to flutamide that are observed in some advanced prostate cancers.

The metabolism of terfenadine was studied with a cDNA-expressed/purified recombinant fusion protein containing human liver microsomal cytochrome P4503A4 (CYP3A4) linked to rat NADPH-P450 reductase (rF450[mHum3A4/mRatOR]L1) and was compared with that observed in the presence of human liver microsomes and precision-cut human liver tissue slices. In all three cases, [3H]terfenadine was metabolized to at least three major metabolites. LC/MS (electrospray) analysis confirmed that these metabolites were alpha, alpha-diphenyl-4-piperidinomethanol (M5), t-butyl hydroxy terfenadine (M4), and t-butyl carboxy terfenadine (M3), although the level of M5 detected in the presence of fusion protein was greater than that found with microsomes or tissue slices. Two additional metabolites, M1 (microsomes and tissue slices) and M2 (fusion protein), were also detected, but remain uncharacterized. Consumption of parent drug (microsomes: KM = 9.58 +/- 2.79 microM, Vmax = 801 +/- 78.3 pmol/min/nmol CYP; fusion protein: KM = 14.1 +/- 1.13 microM, Vmax = 1670 +/- 170 pmol/min/nmol CYP) and t-butyl hydroxylation to M4 (microsomes: KM = 12.9 +/-3.74 microM, Vmax = 643 +/- 62.5 pmol/min/nmol CYP, ; fusion protein: KM = 30.0 +/- 2.55 microM, Vmax = 1050 +/- 141 pmol/min/nmol CYP) obeyed Michaelis-Menten kinetics over the terfenadine concentration range of 1-200 microM. Ketoconazole, a well-documented CYP3A inhibitor, effectively inhibited terfenadine metabolism in all three models. The conversion of M4 to M3, studied with human liver microsomes and fusion protein, was NADPH-dependent and inhibited by ketoconazole. It is concluded that cDNA-expressed CYP3A4, in the form of a NADPH-P450 reductase-linked fusion protein, may also serve as a model for studying the metabolism of terfenadine in vitro and many other drugs.

Cytochrome P-450BM-3 is a catalytically self-sufficient fatty acid omega-hydroxylase with two domains. Functional and primary structure analyses of the hemo- and flavoprotein domains of cytochrome P-450BM-3 and the corresponding microsomal cytochrome P-450 system have shown that these proteins are highly homologous. Prior attempts to reconstitute the fatty acid hydroxylation function of cytochrome P-450BM-3, utilizing the two domains, obtained either by trypsinolysis or by recombinant methods, were unsuccessful. In this paper, we describe the reconstitution of the fatty acid hydroxylation activity of cytochrome P-450BM-3 utilizing the recombinantly produced flavoprotein domain (Oster, T., Boddupalli, S. S., and Peterson, J. A. (1991) J. Biol. Chem. 266, 22718-22725) and its hemoprotein counterpart. The rate of fatty acid-dependent oxygen consumption was shown to be linear when increasing concentrations of the hemoprotein domain are added to a fixed concentration of the flavoprotein domain and vice versa. The combination of the hemo- and flavoprotein domains in a ratio of 20:1 respectively, in the reaction mixture, results in the transfer of 80% of the reducing equivalents from NADPH for the hydroxylation of palmitate at 25 degrees C. The ratio of the regioisomeric products obtained for lauric, myristic, and palmitic acids was similar to that obtained with the holoenzyme form of cytochrome P-450BM-3. The reconstitution of the fatty acid omega-hydroxylase activity, using the soluble domains of cytochrome P-450BM-3, without added factors such as lipids, may be useful for structure/function comparisons to their eukaryotic counterparts.

Arachidonic acid and many products of the arachidonate cascade serve as substrates for cytochrome P450-mediated metabolism via allylic oxidation, omega hydroxylation, and epoxygenation as well as peroxide rearrangement. Defining the physiological importance of these metabolites is an area of intense research interest. Cytochrome P450-catalyzed reactions play prominent roles in multiplying the structural and functional diversity of the arachidonate metabolic cascade.

Cytochrome P-450BM-3 is a catalytically self-sufficient enzyme which monooxygenates saturated and unsaturated fatty acids, alcohols, and amides. The protein has two domains: one which contains heme and is P-450-like and the other which contains FAD and FMN and is P-450 reductase-like. Both domains are on a single polypeptide chain. Utilizing a plasmid containing the gene encoding P-450BM-3, we have transformed the Escherichia coli strain DH5 alpha. This clone overexpresses P-450BM-3 to make approximately 20% of the soluble protein of this organism under optimal conditions. P-450BM-3 can be purified to homogeneity from the soluble fraction of the protein of these cells with a recovery of 50% making this cell line an excellent source of this important enzyme. Purified preparations of P-450BM-3 hydroxylate palmitic acid at a rate of 1600 mol/min/mol of heme at 25 degrees C. The stoichiometry of NADPH to oxygen utilized was 1 for all conditions; however, the ratio of oxygen or NADPH utilized per molecule of fatty acid substrate metabolized was different for different homologs of saturated fatty acids, when low concentrations (less than 100 microM) of substrate were used. Lauric and myristic acids were metabolized to two hydroxylated products, irrespective of the initial concentration of fatty acid in the reaction mixture, and the ratio of oxygen consumed to fatty acid hydroxylated was 1. High concentrations of palmitic acid (greater than 200 microM) led to the formation of three polar metabolites and a stoichiometry of 1:1 was observed for oxygen and palmitic acid utilization. These results indicate that a single hydroxyl group was inserted into each of these molecules. Lower concentrations (less than 50 microM) of palmitic acid were metabolized to additional polar metabolites, and the ratio of oxygen consumed to fatty acid substrate consumed approximated 3:1. These results can be explained best by a hypothesis that the initial hydroxylated compounds, which accumulate during the oxidation of palmitic acid by P-450BM-3, can be further oxidized by this enzyme to polyhydroxy- or hydroxy-ketone products.

The cytochrome P-450s catalyze the oxidative transformation of a large number of endogenous and exogenous chemicals in plants, insects, and mammals. One consequence of this type of reaction is the generation of highly reactive electrophilic metabolites that can react with intracellular macromolecules. It has been postulated that the initiation reaction for chemical carcinogenesis and/or cellular toxicity involves the metabolism of xenobiotics by P-450s. The naturally occurring steroid dehydroepiandrosterone (DHEA), when administered as a supplement to the diet of rodents, has been reported to have anti-carcinogenic and other chemoprotective activities. A change in the inventory of liver P-450s occurs during treatment of rodents with DHEA, the most pronounced being that involved in the omega-hydroxylation of medium-chain length fatty acids. In addition, changes in the activities of other liver P-450s, viz., P-450IIB1, P-450IIC11, and P-450IIIA, occur as shown by in vitro experiments to assess the P-450-dependent formation of hydroxylated metabolites of testosterone and androstenedione. The effect of feeding rodents a diet supplemented with DHEA mimics some of the changes seen when animals are treated with hypolipidemic drugs (such as ciprofibrate, and other chemicals which are known to be peroxisome proliferators). Studies comparing the enzymatic functions of the heterologous expressed recombinant forms of P-45017 alpha, responsible for the two step conversions of progesterone and pregnenolone to the C19-steroids, androstenedione, and DHEA, respectively, illustrated the marked differences in enzymatic properties between the human and the rat orthologues. The findings serve to demonstrate that DHEA is an obligatory intermediate for the synthesis of androgens in the human, but not in the rodent. The biochemical changes responsible for the anti-carcinogenic properties of DHEA remain to be identified. The studies presented here suggest that DHEA, when administered as a dietary supplement, functions like a xenobiotic, and that its effects may result from alterations in the inventory of cellular P-450s, thereby influencing the balance of metabolic activities associated with the initiation phase of chemical carcinogenesis and/or toxicity.

A 27-yr-old woman with lifelong severe exercise intolerance manifested by muscle fatigue, lactic acidosis, and prominent symptoms of dyspnea and tachycardia induced by trivial exercise was found to have a skeletal muscle respiratory chain defect characterized by low levels of reducible cytochromes a + a3 and b in muscle mitochondria and marked deficiency of cytochrome c oxidase (complex IV) as assessed biochemically and immunologically. Investigation of the pathophysiology of the exercise response in the patient revealed low maximal oxygen uptake (1/3 that of normal sedentary women) in cycle exercise and impaired muscle oxygen extraction as indicated by profoundly low maximal systemic arteriovenous oxygen difference (5.8 ml/dl; controls = 15.4 +/- 1.4, mean +/- SD). The increases in cardiac output and ventilation during exercise, normally closely coupled to muscle metabolic rate, were markedly exaggerated (more than two- to threefold normal) relative to oxygen uptake and carbon dioxide production accounting for prominent tachycardia and dyspnea at low workloads. Symptoms in our patient are similar to those reported in other human skeletal muscle respiratory chain defects involving complexes I and III, and the exaggerated circulatory response resembles that seen during experimental inhibition of the mitochondrial respiratory chain. These results suggest that impaired oxidative phosphorylation in working muscle disrupts the normal regulation of cardiac output and ventilation relative to muscle metabolic rate in exercise.

The addition of the methyl ester of 15-hydroperoxyeicosa-5,8,11,13-tetraenoic acid, an arachidonic acid hydroperoxide, to rat liver microsomes results in the uptake of molecular oxygen and the formation of thiobarbituric acid reactive products, indicative of lipid peroxidation. Addition of the hydroperoxide to boiled microsomes does not lead to lipid peroxidation. Metyrapone and imidazole anti-mycotic agents, inhibitors of cytochrome P-450-dependent reactions, inhibit the initial rates of lipid peroxidation. The antioxidant butylated hydroxyanisole inhibits lipid peroxidation at a sub-stoichiometric concentration relative to the hydroperoxide, suggesting that the reaction is an autocatalytic free-radical process. The results indicate that microsomal cytochrome P-450 may play a role in the transformation of lipid hydroperoxides (such as those produced by the irradiation of membranes) to products of lipid peroxidation.

The present studies illustrate the ability to carry out kinetic measurements of steroid hydroxylation using a cytochrome P-450 expressed in a tissue culture cell system. For these experiments a single species of cDNA, incorporated into a suitable expression vector, has been introduced via transfection. A number of interesting preliminary observations have been made on the function of the cytochrome P-450 associated with adrenocortical microsomes which catalyses the 17-hydroxylation of progesterone and pregnenolone. In confirmation of earlier reports the adrenal 17-OHase possesses both 17-hydroxylase as well as C17,20-lyase activities. However, the latter is only functional with 17-OH pregnenolone and not with 17-OH progesterone as substrate. This result differs from the numerous reports that a lyase activity for both substrates is associated with this P-450. The reason for this difference between a delta 4 and a delta 5 steroid remains unresolved although initial experiments indicate that the 5-alpha reduced progesterone is a suitable substrate for both the 17-OHase as well as lyase reactions. This result suggests an inhibitory effect of the delta 4 double bond preventing the carbon-carbon cleavage of the C-17,20 bond in 17-OH progesterone. Clearly more experiments will be required to resolve this question. Measurements of substrate affinity for the cytochrome P-450 expressed in COS cells appears to be influenced by a permeability barrier to the steroid effecting the transport of the steroid across the cell membrane into the cells. This conclusion is suggested by the presence of a time lag before the onset of metabolism as well as by the discrepancy in the concentration of substrate required to give half-maximal rates of metabolism, cf. the results obtained where the initial concentration of progesterone present in the reaction medium is altered versus those experiments measuring the kinetics of substrate depletion. The presence of such a barrier to the free movement of steroid across the membrane is interesting to contemplate when considering the build up of 17-OH pregnenolone required for the lyase reaction. Most unexpected where the results obtained when a comparable expression vector containing the cDNA for cytochrome b5 was cotransfected with pCD17 alpha 2. Cytochrome b5 has been postulated to be an electron transfer component participating in the cyclic function of some cytochromes P-450 (Hildebrandt and Estabrook, 1971).(ABSTRACT TRUNCATED AT 400 WORDS)

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)

Phenylhydrazine interacted with oxidized and reduced cytochrome P-450 of rat liver microsomes to produce binding difference spectra typical of many nitrogenous compounds. The phenylhydrazine-induced difference spectrum observed with oxidized microsomal cytochrome P-450 was converted, in a time-dependent process, to yield a new spectral intermediate with an absorbance maximum around 480 nm. The time required to form this new phenylhydrazine-induced spectral intermediate was decreased from hours to minutes when either NADPH or NADH was added to the reaction mixture. Phenyldiazene generated by addition of the decarboxylation product of methyl phenyldiazenecarboxylate or by addition of potassium ferricyanide and phenylhydrazine (2:1 molar equivalents) instantly formed the new spectral intermediate. This suggests that phenyldiazene is formed during the NADPH-dependent reaction. The appearance of the new spectral intermediate occurred concomitant with the loss of CO-reactive cytochrome P-450 (less than 90%) and loss of absorbance at 418 nm. The interpretation of the optical spectral changes was supported by a loss of the low spin signals characteristic of oxidized cytochrome P-450 as determined by electron paramagnetic resonance spectroscopy. The loss of CO-reactive cytochrome P-450 apparently resulted from the formation of a binary complex of phenyldiazene and the heme of oxidized cytochrome P-450 giving rise to the 480 nm spectral intermediate. In addition, the diazene-bound heme of cytochrome P-450 apparently was modified irreversibly in the presence of oxygen. The effects observed with phenylhydrazine could be produced to a lesser degree by other hydrazine derivatives. The possible role of phenylhydrazine as a new type of suicide substrate is discussed.

The microsomal electron transport complex, in particular the segment associated with cytochrome P-450 function, is both qualitatively and quantitatively an important contributor to the cellular respiration of tissues such as liver. Although our knowledge is still limited, it is apparent that oxygen plays a pivitol role in dictating the mode of substrate hydroxylation or the generation of hydrogen peroxide. As illustrated in Figure 7, current evidence suggests that hydrogen peroxide is formed by the dismutation of the superoxide anion resulting from the dissociation of oxycytochrome P-450. Of interest are recent studies demonstrating the ability of hydrogen peroxide to initiate a cytochrome P-450 dependent peroxidatic reaction competent for supporting substrate hydroxylation. The fact that the function of cytochrome P-450 is sensitive to changes in oxygen tension establishes its role as an "oxygen sensor" for cellular metabolism.

Optical spectral repetitive scan analysis during the oxidative metabolism of benzo(a)pyrene by liver microsomal suspensions reveals the time-dependent formation of an intermediate(s) of which the visible spectra resemble those of several benzo(a)pyrene phenols. Liver microsomes from 3-methylcholanthrene-treated rats showed a greater rate of formation of the phenols than did microsomes from control animals; the rate of formation catalyzed by liver microsomes from phenobarbital-pretreated rats was intermediate. When 3-hydroxybenzo(a)pyrene was used as a standard for comparison of activity, the rates of formation of phenols were compared when measured by fluorometric, spectrophotometric, or high-pressure liquid chromatographic analytical techniques. An epoxide hydrase inhibitor, 1,1,1-trichloropropene-2,3-oxide, enhanced phenol formation regardless of the source of liver microsomes, and 7,8-benzoflavone inhibited control and 3-methylcholanthrene-induced microsomal metabolism of benzo(a)pyrene, 7,8-Benzoflavone did not effect benzo(a)pyrene metabolism by liver microsomes from phenobarbital-pretreated rats. The effect of inhibitors on the spectrophotometric assay correlates well with the results obtained from benzo(a)pyrene metabolite analysis using high-pressure liquid chromatography.

Four different experimental studies are described which were designed to evaluate the role of oxycytochrome P-450 in the formation of superoxide anions and hydrogen peroxide. The use of lipophilic copper chelates with superoxide dismutase like activity revealed that the primary site of interaction of these agents is related to the inhibition of the flavoprotein. NADPH-cytochrome P-450 reductase. Measurements of the proton assisted nucleophilic displacement of superoxide from oxycytochrome P-450 by high concentrations of sodium azide indicated an increase in the rate of hydrogen peroxide formation concomitant with the inhibition of the N-demethylation of ethylmorphine. Studies on the effect of NADH on the rate of hydrogen peroxide formation during NADPH oxidation by liver microsomes failed to reveal a stimulatory or synergistic effect in a manner analogous to results obtained during the cytochrome P-450 dependent oxidation of substrates such as ethylmorphine. These results suggest that hydrogen peroxide formation may not require the reduction of oxycytochrome P-450 to peroxycytochrome P-450. Measurements of the reduction of succinylated cytochrome c using purified cytochrome P-450 and the flavoprotein, NADPH-cytochrome P-450 reductase, directly demonstrate the formation of superoxide anions. It is concluded that oxycytochrome P-450 may decompose to generate hydrogen peroxide.

Fifteen different secondary and tertiary methyl amines have been examined as substrates for the cytochromes P-450 of rat-liver microsomes to determine the similarities or differences between the NADPH and oxygen-dependent N-demethylation reaction and the reaction occurring in the presence of hydrogen peroxide. No apparent correlation of the rates of formaldehyde formation using the two different conditions of oxidation was observed. The types of cytochromes P-450 were altered by using rat-liver microsomes from animals treated with various inducing agents. No obvious predictable dependence on the animals treated with various inducing agents. No obvious predictable dependence on the type of cytochrome P-450 present was obtained for the hydrogen peroxide-supported peroxidatic reaction. It is concluded that the hydrogen peroxide-dependent N-demethylation reaction occurs by a reaction mechanism distinct from that occurring during the mixed-function oxidase activity of cytochrome P-450 obtained in the presence of NADPH and oxygen.

Lipophilic chelates of divalent copper, possessing superoxide dismutase-like activity, have been proposed to enhance the decay of oxycytochrome P-450 to explain their inhibitory effect on microsomal mixed-function oxidation reactions (Richter, C., Azzi, A., Weser, U., and Wendel, A. (1977) J. Biol. Chem. 252, 5061-5066). The present investigation, however, failed to provide evidence in favor of this hypothesis. In particular, it was found that the reported inhibition of cytochrome P-450-catalyzed hydroxylation reactions by copper-tyrosine is associated with an inhibition rather than a stimulation of the formation of hydrogen peroxide, the product of the dismutation of the superoxide radicals generated as a result of the decay of oxycytochrome P-450. The attenuation of both these reactions was shown to be the consequence of an impaired function of the NADPH-cytochrome P-450 reductase. Additional sites of interaction of copper chelates and the microsomal electron transport system appear to exist since divalent copper was found to undergo reduction reactions with NADPH and NADH as electron donors. These reduction reactions do not involve superoxide radicals and, therefore, are unrelated to the ability of copper chelates to function in a superoxide dismutase-like manner.

Publisher Summary This chapter summarizes the types of results that may be obtained by utilizing a few typical protocols in general use. The chapter also describes a few examples of general applicability to the study of the cytochromes of microsomes, emphasizing those areas where problems are generally encountered. Cytochrome P-450 is best characterized by the presence of an absorbance band at about 450 nm for the CO adduct of the reduced hemoprotein. Much of recent knowledge on the function of cytochrome P-450 is derived from spectrophotometric observations obtained by examining spectral pertubations occurring on addition of various substrates, oxygen, or reduced pyridine nucleotides to microsomal fractions, that is, turbid suspensions. The microsomal fraction of many types of tissues, such as liver, kidney, and the adrenal cortex, contains two hemoproteins, cytochromes b5 and P-450, which can be measured by difference spectrophotometry during the oxidative transformation of a wide variety of substrates by mixed-function oxidation reactions.