Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Interaction of aliphatid alcohols with cytochrome P-450 from rat liver microsomes]

The interaction of alyphatic alcohols and cyclohexanol with cytochrome P-450 in microsomes has been investigated. All alchohols induced the modified 11 type spectral changes by mixing with microsomes. These changes are characterized by lambdamax = 412 and lambdamin = 380-382 nm in difference spectra. The dissociation constants of the alcohol cytochrome P-450 complexes are determined. On this dissociation constants influence pH and Triton X-100 presence. The interaction of the alcohols with cytochrome P-450 in phosphate buffer pH = 6,0 in the detergents absence is characterized by one dissociation constant for MeOH, EtOH, n-BuOH and cyclohexanol and by two dissociation constants for i-PrOH, i-BuOH and tert.-BuOH. The interaction of the alcohols with cytochrome P-450 in Tris-HCL-buffer (pH 7.5) in the Triton X-100 presence is characterized for all above alcohols by the dissociations constants, which are described by Taft equation with coefficient rho =-1.55. This fact confirms the interaction of alcohols HO-groups with heme iron of cytochrome P-450. The scheme of interaction of alcohols with cytochrome P-450 is discussed.     

Oxidation of uroporphyrinogen by methylcholanthrene-induced cytochrome P-450. Essential role of cytochrome P-450d.

We have previously shown that uroporphyrinogen is oxidized to uroporphyrin by microsomes (microsomal fractions) from 3-methylcholanthrene-pretreated chick embryo liver [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We report here that a specific antibody to chick liver methylcholanthrene-induced cytochrome P-450 (P-450) inhibited both uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in chick-embryo liver microsomes. 3-Methylcholanthrene-pretreatment of rats and mice markedly increased uroporphyrinogen oxidation in hepatic microsomes as well as P-450-mediated ethoxyresorufin de-ethylation. In rodent microsomes, uroporphyrinogen oxidation required the addition of NADPH, whereas chick liver microsomes required both NADPH and 3,3',4,4'-tetrachlorobiphenyl. Treatment of rats with methylcholanthrene, hexachlorobenzene and o-aminoazotoluene increased uroporphyrinogen oxidation and P-450d, whereas phenobarbital did not increase either. The contribution of hepatic P-450c and P-450d to uroporphyrinogen oxidation and ethoxyresorufin O-de-ethylation in methylcholanthrene-induced microsomes was assessed by using specific antibodies to P-450c and P-450d. Uroporphyrinogen oxidation by methylcholanthrene-induced rat liver microsomes was inhibited up to 75% by specific antibodies to P-450d, but not by specific antibodies to P-450c. In contrast, ethoxyresorufin de-ethylation was inhibited only 20% by anti-P450d but 70% by anti-P450c. Methylcholanthrene-induced kidney microsomes which contain P-450c but non P-450d did not oxidize uroporphyrinogen. These data indicate that hepatic P-450d catalyses uroporphyrinogen oxidation. We suggest that the P-450d-catalysed oxidation of uroporphyrinogen has a role in the uroporphyria caused by hexachlorobenzene and other compounds.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Induction of cytochrome P-450 in rat liver microsomes by perfluorodecalin

Perfluorodecalin was incorporated into phospholipid liposomes and injected intraperitoneally in various dozes. The maximal cytochrome P-450 induction is reached 48 hours after perfluorodecalin injection. Cytochrome P-450 content increases 4 times after perfluorodecalin injection in dose of 0.6 ml/kg in homogenate, and 6 times after perfluorodecalin injection in a dose of 0.4 ml/kg in microsomes. Phenobarbital and perfluorodecalin induce several cytochrome P-450 isozymes and cause the appearance of a new isozyme with mass 56 kD absent in microsomes of intact CBA mice. Perfluorodecalin induction strongly increased the rate of NADPH-dependent aminopyrine nN-demethylation (6-7 times per mg of microsomal protein and 1.5 times per nmol cytochrome P-450). The rate of NADPH-dependent hydroxylation of aniline was not affected by perfluorodecalin induction.     

Inhibition of uroporphyrinogen decarboxylase activity. The role of cytochrome P-450-mediated uroporphyrinogen oxidation.

It was previously shown that uroporphyrinogen oxidation is catalysed by a form of cytochrome P-450 induced by 3-methylcholanthrene [Sinclair, Lambrecht & Sinclair (1987) Biochem. Biophys. Res. Commun. 146, 1324-1329]. We have now measured uroporphyrinogen oxidation and uroporphyrinogen decarboxylation simultaneously in 10,000 g supernatants from the livers of methylcholanthrene-treated mice and chick embryos incubated with an NADPH-generating system. We found that uroporphyrinogen oxidation is associated with inhibition of uroporphyrinogen decarboxylase activity. The decreased uroporphyrinogen decarboxylase activity was not due to depletion of substrate, since decarboxylase activity was not increased by a 2.6-fold increase in uroporphyrinogen. Uroporphyrinogen oxidation and the associated inhibition of decarboxylase activity were also observed with liver supernatant from methylcholanthrene-treated chick embryo; both actions required the addition of 3,3',4,4'-tetrachlorobiphenyl. Uroporphyrinogen oxidation catalysed by microsomes from a methylcholanthrene-treated mouse inhibited the uroporphyrinogen decarboxylase activity in the 100,000 g supernatant. Ketoconazole, an inhibitor of cytochrome P-450, prevented both uroporphyrinogen oxidation and the inhibition of uroporphyrinogen decarboxylation. The addition of ketoconazole to mouse supernatant actively oxidizing uroporphyrinogen inhibited the oxidation and restored decarboxylation. The latter finding suggested that a labile inhibitor was formed during the oxidation. These results suggest uroporphyrinogen oxidation may be important in the mechanism of chemically induced uroporphyria.     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.     

Electron paramagnetic resonance studies of cytochrome P-450 in plant microsomes.

The technique of electron paramagnetic resonnance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study. At 77 K in the oxidised state, signals were observed at g values of 2.40, 2.25 and 1.93, characteristic of cytochrome P-450 in the low-spin state, and also at g = 4.27, attributable to ferric iron in a rhombic environment. The signals at g = 2.40, 2.25 and 1.93 disappeared upon reduction with sodium dithionite. At 10 K in the oxidised state, signals at g = 8.3 and 3.3 appeared, and these were attributed to high-spin cytochrome P-450. At this temperature a further signal at g = 6, due to cytochrome P-420, was seen in aged tulip-bulb microsomes. Redox titration of both high-spin and low-spin cytochrome P-450 gave the same apparent midpoint potential of -315 +/- mV at pH 6.8 and 25 degrees C. The significance of this value is discussed. Addition of "type I" or "type II" ligands to oxidized cytochrome P-450 caused an increase and a decrease, respectively, in the ratio of the high-spin to the low-spin form. A second effect of aniline, a type II ligand of cytochrome P-450, was to remove the g = 6 signal, suggesting that it also interacts with cytochrome P-420. No iron-sulphur proteins similar to those found in some other cytochrome P-450 electron-transport chains could be detected in any of the microsomes analysed.     

Identification of cross-linked cytochrome P-450 in rat liver microsomes by enzyme-immunoassay

The topography of microsomal proteins was studied by 2-dimensional gelelectrophoresis. The second dimension was run in the presence of 2-mercaptoethanol, thus allowing detection of proteins previously cross-linked by disulfide bonds as off-diagonal spots. With hepatic microsomes from phenobarbital pretreated rats, several off-diagonal spots were seen. The most intense spot, with a molecular weight of 52,000, was derived from a dimer of this protein. It was identified as cytochrome P-450 (P-450) by a double antibody enzyme-immunoassay. The dimer is probably formed by oxidation of sulfhydryl groups of P-450 molecules during the preparation of microsomes. P-450 can also be cross-linked to form 105,000, 167,000, and 240,000 dal oligomers by treating microsomes with dithiobis(succinimidyl propionate) at 0°C. Cross-linking of P-450 to other proteins was also observed with one-dimensional gel-electrophoresis. The results suggest that the cross-linked proteins are close neighbors of P-450 in the membrane.     

Participation of cytochrome P-450 in nicotine oxidation

Addition of nicotine to phenobarbital-inducible cytochrome P-450 caused a shift of maximum of Soret peak toward the red approximately 3 nm. The difference spectrum produced by nicotine showed a type 2 spectral change with a peak at 427 nm and a trough at 393 nm. A spectral dissociation constant of phenobarbital-inducible cytochrome P-450 was found to be 0.16 mM for nicotine. Nicotine oxidation in the reconstituted system depended on cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH. These results indicate that phenobarbital-inducible cytochrome P-450 participates in nicotine oxidation.     

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.     

Spectroscopic quantitation of cytochrome P-450 in human lung microsomes

The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.     

Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.