Interaction between NADPH-cytochrome p-450 reductase and cytochrome p-450 in the membrane of phosphatidylcholine vesicles

Cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphatidylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80–90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the system in a similar way to the fast-phase reduction of cytochrome P-450, though the latter is not the rate-limiting step of the overall reaction.     

Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).     

Studies of the unique nature of the cytochrome P-450 active site. Electron spin resonance spectroscopy as a probe of the hemin iron of cytochrome P-450: the influence of buffer composition, alcohols, and nitrogenous ligands.

The electron spin resonance (esr) spectra of the low-spin form of hepatic microsomal cytochrome P-450 and of cytochrome P-450 isolated from Pseudomonas putida grown on d-camphor (P-450_cam) were studied in order to gain an understanding of the sensitivity of the hemin iron to changes in buffer. The shapes of the g_x and g_y esr signals of both the membrane-bound microsomal and soluble bacterial cytochromes P-450 were dependent upon buffer composition. With either system, the g_x and g_y signals were symmetric in some buffers and asymmetric in others. However, in potassium phosphate buffer, the esr spectra of low-spin cytochrome P-450 in microsomes isolated from phenobarbital (PB)- or 3-methylcholanthrene (3-MC-induced rats and cytochrome P-450_cam are similar with symmetric g_x and g_y signals. The esr spectrum of the low-spin form of cytochrome P-450 in isolated hepatocytes is similar to that of the microsomal and bacterial enzyme, again with a symmetric g_x signal. The effects of alcohols and nitrogenous ligands on the esr spectrum of the low-spin form were also investigated. The data indicate that extreme care must be exercised when interpreting esr spectra with respect to possible cytochrome P-450 heterogeneity in the microsomal membrane. The conditions for studying substrate interactions with microsomal cytochrome P-450 must also take into account these changes in symmetry of the esr spectrum.     

Binding of R and S warfarin to hepatic microsomal cytochrome P-450

The R and S enantiomers of the anticoagulant, warfarin, are metabolized to a series of monohydroxylated products by rat hepatic cytochrome P-450. The patterns of metabolites are a function of the warfarin enantiomer used and of the induction of the microsomal enzymes by phenobarbital (PB) and 3-methylcholanthrene (MC). We have studied the binding of R and S warfarin to cytochrome P-450 by difference spectrometry to probe the heterogeneity of cytochrome P-450 and to determine the role of this heterogeneity in the production of the patterns of warfarin metabolites. Uninduced cytochrome P-450 yielded modified type II spectra with R and S warfarin with equivalent binding constants, K_s = 1.50 mM. PB-induced cytochrome P-450 yielded modified type II spectra which varied biphasically with warfarin concentration with K_s(S) = 0.24 and 0.07 mm; K_s(R) = 0.79 and 0.12 mM. MC induction and 2-allyl-2-isopropylacetamide treatment yielded microsomes markedly enriched for cytochrome P-448 which, with R warfarin, yielded a type I spectrum, K_s = 0.24 mM, and with S warfarin a modified type II spectrum with K_s = 0.11 mM. The effects of the type I compound, hexobarbital, the type II compound, imidazole, or the opposite enantiomer to that being studied on the binding spectra of R and S warfarin to the variously induced cytochromes P-450 were investigated as an aid to elucidating the mode of interaction of cytochrome P-450 with warfarin. In all cases, prior binding of R or S warfarin influenced the binding of the opposite enantiomer. We conclude from these results that R and S warfarin bind to two separate forms of PB-induced cytochrome P-450 and two separate forms of MC-induced cytochrome P-448, all of which differ from uninduced cytochrome P-450. The variety of monohydroxylated metabolites of R and S warfarin is probably a consequence of the interactions with these different forms of cytochrome P-450.     

Rotational diffusion of cytochrome P-450 in the microsomal membrane—Evidence for a clusterlike organization from saturation transfer electron paramagnetic resonance spectroscopy

Rotational diffusion of cytochrome P-450 in rabbit liver microsomes has been studied by saturation transfer EPR spectroscopy. Sulfhydryl groups of cytochrome P-450 were selectively modified using a maleimide spin label. The effective rotational correlation time for the rotation of cytochrome P-450 was calculated to be about 480 μs which corresponds to a very strong immobilization thus evidencing protein aggregation within the membrane. The anisotropic character of the spectra indicates a nonspherical shape and/or anisotropic rotational motion of the cluster. The temperature dependence of the rotational correlation time shows a relatively sharp break at about 4 °C but only small changes above this temperature. The break at about 4 °C is probably caused by the onset of the cluster rotation. Below 4 °C the enzyme is almost immobilized. A similar complete immobilization can be achieved by treating the microsomes with glutaraldehyde.     

Electron paramagnetic resonance studies of cytochrome P-450 and adrenal ferredoxin in single whole rat adrenal glands. Effect of corticotropin

Low and high spin ferric cytochrome P-450 and reduced adrenal ferredoxin (adrenodoxin) have been directly studied by EPR techniques in whole rat adrenal glands. The spectra obtained correspond closely to those obtained from sub-cellular fractions except in the case of low spin ferric cytochrome P-450, where there are differences in the shape of the g = 2.41 line. The relative magnitudes of these peaks in anaerobic and aerobic rapidly frozen adrenals from control and corticotropin stimulated hypophysectomised rats were used to investigate the control and rate limiting steps in adrenal steroid biosynthesis via cytochrome P-450. All adrenals showed a close to maximal level of reduced adrenodoxin and aerobic and anaerobic glands from control rats and aerobic glands from corticotropin stimulated rats showed similar quantities of low spin ferric cytochrome P-450. On anaerobiosis the quantity of low spin ferric cytochrome in adrenals from corticotropin stimulated rats dropped to 30–40% of the aerobic level. Treatment of the rats with cycloheximide prior to administration of corticotropin prevented these changes. Approximately 0.4% of the total cytochrome P-450 was high spin ferric in control adrenals and in aerobic stimulated adrenals this rose to approximately 0.6%. These results demonstrate that association of substrate with cytochrome P-450 is the rate limiting step in adrenal steroidogenesis via cytochrome P-450. It is suggested on the basis of these and mitochondrial optical and EPR experiments that the limiting step being observed is cholesterol binding to cholesterol side chain cleavage cytochrome P-450, and that the rate of this association is stimulated by corticotropin.     

Molecular organization (topography) of cytochrome P-45011β in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450_11β from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450_11β following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11β hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450_11β is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450_11β on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.     

Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins)

The metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (1-8C) was studied in hepatic microsomes of control, phenobarbitone-pretreated (PB) and 3-methylcholanthrene-pretreated (3MC) C57/BL10 mice. Phenoxazone and its ethers were hydroxylated and O-dealkylated respectively to a common metabolite, resorufin. The three categories of microsomes differed greatly in activity for the metabolism and binding of the various substrate homologues. The most rapidly metabolised substrates for control microsomes were phenoxazone and its shortest-chain ethers, for PB microsomes phenoxazone and the pentyl ether, and for 3MC microsomes the ethyl and propyl ethers. The variations in activity occurred in Vmax rather than in the apparent K_m-value. All the ethers gave Type I cytochrome P-450-binding spectra. The substrates giving the largest Type I spectra were the same for all microsomes—the ethyl, propyl and butyl ethers—but the magnitudes of the spectra differed in the order 3MC- > PB- > control microsomes. Phenoxazone and resorufin gave Modified Type II cytochrome P-450-binding spectra. PB-induction was most marked for the depentylation reaction (increased 101-fold), whereas 3MC-induction was most marked for depropylation and debutylation (88- and 96-fold).The intermicrosomal differences were interpreted as reflecting the different metabolic specificities of variant forms of cytochrome P-450. Substrate lipophilicity increased with increasing ether chain length and was not a major influence on specificity. The main substrate influence on specificity was steric, due to the presence and length of the ether side chain. The preeminent effect of ether chain length was considered to be on the rate of substrate transformation rather than on substrate interaction with cytochrome P-450.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

Electron paramagnetic resonance spectra of mitochondrial and microsomal cytochrome P-450 from the rat adrenal

The electron paramagnetic resonance (EPR) spectra of rat adrenal zona fasciculata mitochondria showed peaks corresponding to low spin ferric cytochrome P-450 with apparent g values of 2.424, 2.248 and 1.917, and weak signals due to high spin ferric cytochrome P-450 with g_x values of 8.08 and 7.80. The former is attributed to cholesterol side chain cleavage cytochrome P-450, the latter to 11β-hydroxylase cytochrome P-450. On addition of deoxycorticosterone the g = 7.80 signal was elevated and there was an associated drop in the low spin signal. As the pH was reduced from 7.4 to 6.1, the g = 8.08 signal increased with again a drop in intensity of the low spin signal. Mitochondria from the zona glomerulosa showed similar spectral properties to those described above. Addition of succinate, isocitrate or pregnenolone caused a loss of the g = 8.08 signal. Addition of calcium increased the magnitude of the g = 8.08 signal, and caused a slight reduction in the magnitude of the low spin signal. Also, addition of deoxycorticosterone, pregnenolone, succinate or isocitrate caused slight shifts of the outer lines of the low spin spectrum. Interaction of mitochondrial cytochrome P-450 with metyrapone and aminoglutethimide modified the low spin parameters. Adrenal microsomal cytochrome P-450 had low spin ferric g values of 2.417, 2.244 and 1.919 and high spin ferric g_xy values of 7.90 and 3.85, distinct from the values obtained with mitochondria.     

Purification and characterization of pentobarbital-induced cytochrome P-450BM-1 from Bacillus megaterium ATCC 14581

When Bacillus megaterium ATCC 14581 is grown in the presence of barbiturates, a cytochrome P-450-dependent fatty acid monooxygenase (M_r 120 000) is induced (Kim, B.-H. and Fulco, A.J. (1983) Biochem. Biophys. Res. Commun. 116, 843–850). Gel filtration chromatography of a crude monooxygenase preparation from pentobarbital-induced B. megaterium indicated that not all of the induced cytochrome P-450 present in the extract was accounted for by this high-molecular-weight component. Further purification revealed the presence of two additional but smaller cytochrome P-450 species. The minor component, designated cytochrome P-450_BM-2, had a molecular mass of about 46 kDa, but has not yet been completely purified or further characterized. The major component, designated cytochrome P-450_BM-1, was obtained in pure form, exhibited fatty acid monooxygenase activity in the presence of iodosylbenzenediacetate, and has been extensively characterized. Its M_r of 38 000 makes it the smallest cytochrome P-450 yet purified to homogeneity. Although it is a soluble protein, a complete amino acid analysis indicated that it contains 42% hydrophobic residues. By the dansyl chloride procedure the NH₂-terminal amino acid is proline; the penultimate NH₂-terminal residue is alanine. The absolute absorption spectra of cytochrome P-450_BM-1 show maxima in the same general regions as do P-450 cytochromes from mammalian or other bacterial sources, but they differ in detail. The oxidized form of P-450_BM-1 has absorption maxima at 414, 533 and 567 nm, while the reduced form has peaks at 410 and 540 nm. The absorption maxima for the CO-reduced form of P-450_BM-1 are found at 415, 448 and 550 nm. Antisera from rabbits immunized with pure P-450_BM-1 strongly reacted with and precipitated this P-450, but showed no detectable affinity for either the 46 kDa P-450 or the 120 kDa fatty acid monooxygenase.     

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains.Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains.Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.     

Simultaneous purification of a cytochrome P-450 and cytochrome b5 from the house fly,Musca domestica L.

Cytochrome P-450, cytochrome b₅ and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b₅ and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b₅ and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b₅ fraction, by C8 reverse phase HPLC, resulted in a cytochrome b₅ fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa.     

Studies on cytochrome P-450-dependent lipid hydroperoxide reduction

A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction.     

Properties of an adrenal cytochrome P-450 (P-450scc) for the side chain cleavage of cholesterol

A highly purified (12 nmol of P-450-heme per milligram of protein) bovine adrenal cortex mitochondrial cytochrome P-450, termed P-450_sce, which cleaves the side chain of cholesterol to yield pregnenolone, is obtained in the substrate-bound ferric form with observed absorption maxima at 393 nm and 645 nm and a shoulder around 540 nm. The absorption spectra of the P-450_scc, whether in the substrate-bound ferric form or in the CO-complexed ferrous form, are subject to environmental perturbation. The addition of adrenal ferredoxin readily restores full ferric high spin type spectrum of the substrate-bound P-450_scc or, together with cholesterol and Tween 20, restores the CO-spectrum of the P-450_scc, exhibiting stable and typical spectra of cytochrome P-450. Tween 20, at concentration of 0.3%, remarkably increases the P-450_scc-catalyzed cholesterol side chain cleavage activity. Based on these findings, a highly reactive and reliable assay has been developed for the conversion of cholesterol to pregnenolone. The specific activity of the P-450_scc, thus determined in the presence of NADPH, NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1), adrenal ferredoxin, cholesterol, and molecular oxygen, is 16 mol of pregnenolone formed per minute per mole of P-450-heme and V of enzyme catalyzed reaction was 30 mol/min/mol of P-450-heme. Apparent K_m values are 120 μm for cholesterol and 1.5 μm for adrenal ferredoxin. The P-450_scc has a pH optimum at pH 7.2 and is most active at ionic strength of 0.1.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid)

Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.     

Comparative study of two highly purified forms of liver microsomal cytochrome P-450: circular dichroism and other properties.

The two major forms of rabbit liver microsomal cytochrome P-450, P-450_LM2 and P-450_LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450_LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450_LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450_LM4 has a much higher tryptophan content than P-450_LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450_LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450_LM2 gives a much stronger Type I difference spectrum than does P-450_LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450_LM2 and P-450_LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in α-helical content, particularly with P-450_LM4, in the presence of the phospholipid.     

Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes.

Antibodies prepared against purified cytochrome P-450 and P-448 from phenobarbital- and 3-methylcholanthrene-pretreated rats have been shown to recognize several forms of hepatic cytochrome P-450 (P. E. Thomas, A. Y. H. Lu, D. Ryan, S. B. West, J. Kawalek, and W. Levin, 1976, Mol. Pharmacol.12, 746–758). These antibodies have been made monospecific for a single form of cytochrome P-450 by immunoadsorption with partially purified solid-phase cytochrome P-450 from rats treated with a different inducer than that used for isolation of the antigen. Each monospecific antibody did not react with different forms of cytochrome P-450 present in the heterologous antigen preparation. These monospecific antibodies, covalently bound to Sepharose, were used to purify the antigens (catalytically inactive) from microsomes in a single step. The high specificity of these antibodies for a single form of cytochrome P-450 was used to quantitate two forms of cytochrome P-450 in rat liver microsomes by radial immunodiffusion. The percentage of the total cytochrome P-450 in microsomes that is represented by each of these two forms of cytochrome P-450 varied from 3 to 89% depending on the xenobiotic pretreatment of the rats.