Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P450.

This study has investigated the mechanism of steroid hydroxylation in bovine adrenocortical microsomes and mitochondria by employing NaIO₄, NaClO₂, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. In the microsomal hydroxylating system, progesterone, 17α-hydroxyprogesterone, and androstenedione were found to act as substrates. Progesterone was chosen as the model substrate and was converted mainly to the 21-hydroxylated derivative in the presence of microsomal fractions fortified with hydroxylating agent. Using saturating levels of hydroxylating agent, NaIO₄ was found to be the most effective in promoting progesterone hydroxylation followed by cumene hydroperoxide, t-butyl hydroperoxide, NADPH, NaClO₂, and pregnenolone 17α-hydroperoxide. Evidence for cytochrome P₄₅₀ involvement included a marked inhibition of the activity by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. Steroid hydroxylation was studied in adrenocortical mitochondria that had been previously depleted of endogenous pyridine nucleotides by aging for 1 h at 30 dgC in a phosphate-supplemented medium. Androstenedione was converted to its respective 6β-, 11β-, 16β-, and 19-hydroxylated derivatives when incubated with aged mitochondrial fractions fortified with hydroxylating agent whereas progesterone was hydroxylated in the 1β-, 6β-, and 15β- positions. These hydroxylations were completely abolished by preheating the mitochondria for 5 min at 95 dgC prior to assay, indicating the enzymic nature of the reactions. Deoxycorticosterone and deoxycortisol were effective substrates for NADPH-dependent enzymic 11β-hydroxylation but were extensively degraded nonenzymically to unidentified products in the presence of NaIO₄ and hydroxylating agents other than NADPH and consequently could not be utilized as substrates in these reactions. Using androstenedione as substrate, NaIO₄ was the most effective hydroxylating agent, followed by cumene hydroperoxide, NaClO₂, t-butyl hydroperoxide, and NADPH. These hydroxylations were inhibited by substrates and modifiers of cytochrome P₄₅₀ and by reagents that convert cytochrome P₄₅₀ to cytochrome P₄₂₀. A mechanism for steroid hydroxylation in adrenocortical microsomes and mitochondria is proposed in which the ferryl ion (compound I) of cytochrome P₄₅₀ functions as the common “activated oxygen” species.     

The mechanism of hydroperoxide-dependent reactions with participation of cytochrome P-450

Cytochrome P-450 destruction kinetics by cumene hydroperoxide (CHP) has been studied at 25 degrees C in phosphate buffer, pH 7.25-7.50, in various systems: intact and induced rat or rabbit microsomes, highly purified LM2- and LM2- and LM4-forms of cytochrome P-450 from rabbit liver microsomes. The destruction kinetics is characterized by three phases in all systems. The CHP-influenced cytochrome P-450 destruction is a radical chain process with linear termination of the chains. The acidic phospholipids, phosphatidylserine and phosphatidylinositol and total microsomal phospholipids containing the acidic lipid components activate cytochrome P-450 in the hydroxylation of aniline and naphthalene by CHP. Phosphatidylcholine and sphingomyelin have no effect upon the cytochrome P-450 activity in the type I and II substrates oxidation by CHP. The phase transitions of the microsomal phospholipids influence the interaction of cytochrome P-450 with its reductase, altering the activation energy of type I substrates oxidation. The type II substrate oxidation is not affected by phase transitions in the full microsomal hydroxylating system.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

Benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae. Substrate binding, spectral and kinetic data

Saccharomyces cerevisiae, brewer's yeast, produces a microsomal benzo(a)pyrene hydroxylase when grown at high glucose concentrations of which the haemoprotein, cytochrome P-450 (RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating) EC 1.14.14.1) is a component. We report here kinetic data derived from Lineweaver-Burk plots of benzo(a)pyrene hydroxylation. The Michaelis constant was decreased by growth of the yeast in the presence of benzo(a)pyrene showing the induction of a form of the enzyme more specific for this compound. NADPH or cumene hydroperoxide could be used as cofactors by this enzyme, although with different Km and V values for benzo(a)pyrene. A solubilised and a solubilised, immobilised enzyme preparation were capable of benzo(a)pyrene hydroxylation, using cumene hydroperoxide but not NADPH as the cofactor. Benzo(a)pyrene was found to produce a modified type I spectral change with yeast and rat liver microsomes. The interaction of benzo(a)pyrene with cytochrome P-450 was investigated further by means of an equilibrium gel filtration technique. There appeared to be 20 binding sites per mol ofcytochrome P-450 for benz(a)pyrene, in both yeast and rat liver microsomes.     

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

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

Possible higher valence states of cytochrome P-450 during oxidative reactions

The addition of the organic hydroperoxide, cumene hydroperoxide, to liver microsomes results in the appearance of a transient spectral change associated with cytochrome P-450. In addition, unique electron paramagnetic resonance signals are observed with liver microsomal cytochrome P-450 comparable to signals obtained when peroxides interact with metmyoglobin. It is suggested that higher valence states of cytochrome P-450 may function during the activation of oxygen for the hydroxylation of a variety of xenobiotics.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex: comparison of cholesterol side-chain cleavage P-450 with steroid 11beta-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450.

Adrenocortical mitochondrial cytochrome P-450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11beta-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0 degrees C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11beta-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H2O2, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents. Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

Cytochrome P-450 mediated interaction between hydroperoxide and molecular oxygen. A proposed method for P-450 kinetic studies

Rat liver cytochrome P-450 mediates a novel reaction between equimolar quantities of dissolved oxygen and organic hydroperoxides. The reaction shares some of the properties of both NADPH-O2 dependent hydroxylation and NADPH-O2 independent peroxidase reactions, but does not require either NADPH, phosphatidylcholine, or any substrates other than hydroperoxide and oxygen. It proceeds at a rate approximately 100 times faster than other well known P-450 hydroxylation reactions. Monitoring the rate of O2 consumption in this novel reaction may be a simple and rapid means for studying the kinetics of cytochrome P-450.     

Oxidation of cytochrome b5 by hydroperoxides in rat liver.

1. Spectral changes following the addition of hydroperoxides to isolated hepatocytes and to perfused rat liver were observed. Cytochrome b5 is the major, if not the only, hemoprotein exhibiting redox changes under these conditions; cytochrome b5 is oxidized by added hydroperoxides, e.g. tert-butyl or cumene hydroperoxides. No spectral changes attributable to cytochrome b5 were observed with tert-butanol. 2. The effect is present also when the mitochondrial respiratory chain is inhibited by antimycin A, and it is not observable with isolated mitochondria. On the other hand, the oxidation of cytochrome b5 by hydroperoxides is readily demonstrable in microsomal fractions in presence of NADH. 3. Spectral evidence for a participation of the other microsomal hemoprotein, cytochrome P-450, in the hydroperoxide-linked effects was not obtained. Thus, in hepatocytes from phenobarbital-pretreated rats, no formation of cytochrome P-420, no displacement of a type I substrate, hexobarbital, and no major steady state redox change of cytochrome P-450 was detectable. However, when cytochrome P-450 was dithionite-reduced, an oxidation of this cytochrome occurred upon subsequent hydroperoxide addition. 4. Hydrogen peroxide addition to hepatocytes also leads to a lower steady-state degree of reduction of cytochrome b5. Evidence is provided with hepatocytes from rats pretreated with 3-amino-1,2,4-triazole that H2O2 generated intracellularly, e.g. from added glycolate, also causes a detectable oxidation of cytochrome b5. 5. The mechanism of these hydroperoxide effects remains to be established, and it is not clear whether cytochrome b5 reacts directly or indirectly. However, it is suggested that these effects may be of significance for the further study of cytochrome-b5-linked metabolic pathways.     

Interaction of cytochrome P-450 with a hydroperoxide derived from butylated hydroxytoluene. Mechanism of isomerization

Attack of O2 on the phenoxy radical derived from butylated hydroxytoluene resulted in the formation of 2,6-di-t-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BOOH). This hydroperoxide was rapidly consumed when incubated with rat liver microsomes in the absence of NADPH. The destruction of BOOH was accompanied by formation of the corresponding alcohol (BOH) and a derivative of the alcohol (B(OH)2) in which a t-butyl methyl group was hydroxylated. This diol was produced also when BOH was incubated with microsomes and NADPH, but at a slower rate. Mass spectral analyses of B(OH)2 formed from substrates labeled with either 2H or 18O, showed that oxygen was transferred from the peroxy group to a t-butyl group (via the heme iron of P-450) without migration of the intermediate alcohol from the enzyme active site. The results support a mechanism involving heterolytic O-O bond cleavage during isomerization of the hydroperoxide to B(OH)2. The chiral diol was produced from BOOH nonstereoselectively, but the NADPH/O2-supported hydroxylation of BOH resulted in the formation of a 20% excess of one enantiomer of B(OH)2. Analyses of products formed from the interaction of cumene hydroperoxide with cytochrome P-450 showed that this substrate undergoes rearrangement also; 2-phenyl-1,2-propanediol was produced, together with cumyl alcohol and acetophenone. These results indicate that isomerization competes with other pathways of hydroperoxide destruction by cytochrome P-450.     

The mechanism of cumene hydroperoxide-dependent lipid peroxidation: the function of cytochrome P-450

The addition of limiting amounts of cumene hydroperoxide to rat liver microsomes resulted in the rapid uptake of molecular oxygen, the formation of thiobarbituric acid reactive products, and the loss of hydroperoxide. The stoichiometry of lipid peroxidation and the yields of 2-phenyl-2-propanol (a major product of the reaction) and acetophenone (a minor product) observed with liver microsomes prepared from untreated rats is greater than that seen with liver microsomes from ciprofibrate-treated rats which, in turn, is greater than that observed with liver microsomes from phenobarbital-treated rats. The Km's and Vmax's of oxygen uptake varied with the type of rat liver microsomes used. Cytochrome P-450 substrates and inhibitors decreased the extents and initial rates of oxygen uptake and thiobarbituric acid reactive product formation. A mechanism is proposed involving the cytochrome P-450-catalyzed homolytic cleavage of the cumene hydroperoxide O-O bond to give the cumyloxyl radical. It is proposed that this oxygen-centered radical abstracts a hydrogen atom from an unsaturated fatty acid associated with a lipid (initiating lipid peroxidation) to give 2-phenyl-2-propanol or that the radical undergoes beta-scission to produce acetophenone and a methyl radical.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

Structure-activity relationships in the in vitro modulation of rat hepatic microsomal androst-4-ene-3,17-dione hydroxylase activities by derivatives of 5 alpha- and 5 beta-androstane

The relationships between structure and inhibitory potency toward microsomal cytochrome P-450 (P-450)-mediated androst-4-ene-3,17-dione hydroxylase activities were investigated in rat liver with a series of 5 alpha- and 5 beta-androstane derivatives. 5 beta-Reduced steroids (containing a cis-A/B ring junction) were more potent inhibitors than the 5 alpha-reduced epimers (containing a trans-A/B ring junction) except in the case of the 17 beta-hydroxy-substituted derivatives. The most effective inhibitor was 5 beta-androstane-3 beta-ol which exhibited I50 values of 7 and 27 microM against androstenedione 16 alpha- and 6 beta-hydroxylase activities, which are catalysed by P-450 IIC11 and IIIA2, respectively. In general, these two pathways of steroid hydroxylation were more susceptible to inhibition than the 7 alpha- and 16 beta-hydroxylase pathways. The 7 alpha-hydroxylase enzyme (P-450 IIA1) was only inhibited by 5 beta-reduced steroids that contained an oxygenated function at C17. All of the test compounds elicited type I spectral binding interactions with P-450 in oxidised microsomes. The most effective steroid inhibitors generally exhibited the greatest capacity to interact with P-450. Additional studies with one of the more potent compounds, 5 beta-androstane-3 beta-ol-17-one, revealed that the inhibition kinetics were competitive and that preincubation of the inhibitor with NADPH-supplemented microsomes prior to substrate (androstenedione) addition decreased the extent of inhibition observed. These findings are consistent with the assertion that the inhibition of hepatic steroid hydroxylases by 5 beta-androstanes involves an effective competitive interaction with the steroid substrate at the P-450 active site. Since the relative overproduction of 5 beta-reduced metabolites of certain androgens has been reported in clinical conditions, such as androgen insensitivity, it now appears important to investigate the hepatic drug oxidation capacity of patients with hormonal abnormalities.     

Oxidation of arylcyclopropanes by rabbit liver cytochrome P-450. Mechanistic implications of a multiple oxidation.

The arylcyclopropanes (cyclopropylarenes) cyclopropylbenzene and diphenylcyclopropane are oxidized by rabbit liver microsomal cytochrome P-450, both by the microsomal fraction and by the purified cytochrome in a reconstituted system. The products formed, principally benzoic acid, are due to an unusual triple oxidation of the substrate, which probably remains attached to the active site during the several steps of the oxidation. Both substrates were found to be inhibitors of the cytochrome P-450-dependent O-de-ethylation of 7-ethoxycoumarin. Model oxidation studies with cumene hydroperoxide as oxidizing agent and rabbit liver microsomal fraction as source of enzyme gave similar products to the microsomal and reconstituted systems. The significance of these results in the mechanism of oxidation catalysed by cytochrome P-450 is discussed.     

Hydroperoxide catalyzed liver microsomal aromatic hydroxylation reactions involving cytochrome P-450

Cumene hydroperoxide is capable of supporting the aromatic hydroxylation of a variety of compounds in the presence of hepatic microsomes. NADPH and molecular oxygen are not required. Cytochrome P-450 acts as the catalyst and could not be replaced by other hemoproteins. One mole of hydroperoxide is consumed for every mole of substrate hydroxylated. It is suggested that the oxenoid species of cytochrome P-450 involved in microsomal aromatic hydroxylation is present in a form equivalent to the ferryl from.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.     

Interactive binding at cytochrome P-450 of cell growth regulatory bioamines, steroid hormones, antihormones, and drugs

The virtually universal family of P-450 isozymes contribute to the regulation of cell growth by modulating the levels of steroids and other lipid messengers for cytoplasmic and nuclear processes, including gene expression. In microsomes from rat liver cells, the concentration ( approximately 1 nmole/mg protein) of cytochromes P-450 approximates that of intracellular binding sites (K(d) 1.0-50 microM) for histamine. The potencies of certain therapeutic drugs to inhibit catalytic activity of, and histamine binding to, cytochromes P-450 in vitro were previously shown by us to be predictive of relative propensities to modulate tumor growth in rodents. Also, we demonstrated that growth-regulating polyamines potently interact with histamine at P-450. We now show that several classes of steroid hormones, antiestrogens, and antiandrogens, as well as various arylalkylamine drugs, all potently inhibit (3)H-histamine binding to cytochrome P-450 (K(i) values: testosterone 0.28 microM, progesterone 0.56 microM, flutamide 1.7 microM, tamoxifen 9.0 microM). Furthermore, all the various hormone and drug ligands are mutually inhibitory in their binding to cytochrome P-450; e.g., K(i) values of androstenedione and progesterone, to inhibit imipramine binding to P-450 (determined by spectral analysis), are 11 nM and 26 nM, respectively. The K(i) value of imiprimine to inhibit binding of androstenedione to P-450 is 3.5 microM. We estimate the total P-450 content in microsomes to be greater in male than in female rats and correlated with the number of binding sites for histamine, but not for steroids and drugs that appear to be more selective for P-450 isozymes. Thus, for at least some isozymes, the homeostatic role of the monooxygenases may be governed by histamine, modulated by endogenous ligands, and perturbed by many foreign molecules.