Catalytic activity of isolated cytochromes P-450d and P-450c

Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined.     

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.     

A prostaglandin omega-hydroxylase cytochrome P-450 (P-450PG-omega) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin omega-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes (Powell, W.S. (1978) J. Biol. Chem. 253, 6711-6716). Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450I (LM2) and P-450II (LM5), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450PG-omega, regioselectively hydroxylates prostaglandins at the omega-position in reconstituted systems with a turnover of 1-5 min-1. Titration with purified pig liver cytochrome b5, demonstrated a 4-fold maximum stimulation at a cytochrome b5 to a P-450 molar ratio of 1-2. Rabbit lung P-450PG-omega formed a typical type I binding spectrum upon the addition of prostaglandin E1 with a calculated K8 of 1 microM, which agreed reasonably well with the kinetically calculated Km of 3 microM. Cytochrome P-450PG-omega was isolated as a low-spin isozyme with a lambda max (450 nm) in the CO-difference spectrum distinguishable from P-450I (451 nm) and P-450II (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450PG-omega had a relatively low specific content (12.1 nmol mg-1), it appeared homogeneous with a calculated minimum Mr of 56,000, intermediate between rabbit LM4 and LM6. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a Mr identical to P-450PG-omega, was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450I and P-450II. P-450PG-omega appears to be a novel rabbit P-450, possessing high activity towards omega-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Selective loss of pulmonary cytochrome P-450I in rabbits pretreated with polychlorinated biphenyls

The polychlorinated biphenyls mixture, Aroclor 1254, generally considered a powerful inducer of rat hepatic and pulmonary microsomal monooxygenases, caused a 70% decrease in ethylmorphine N-demethylase activity, a 31% decrease in benzo(a)pyrene hydroxylase activity, and a 42% decrease in cytochrome P-450 content in rabbit lung microsomes. When pulmonary cytochrome P-450 was solubilized and subjected to column chromatography, the elution profiles of the two forms of the hemeprotein showed a marked decrease in cytochrome P-450I in treated rabbits, with no significant alteration in cytochrome P-450II content. These data were confirmed by subjecting the two cytochromes to gel electrophoresis and staining the electrophoretic bands for protein and heme-associated peroxidase activity. Cytochromes P-450I and P-450II isolated from Aroclor 1254-treated rabbits showed differences in spectral properties as well as in their stabilities. The CO difference spectral determinations showed absorbance maxima at 452 and 450 nm for cytochromes P-450I and P-450II, respectively. At room temperature, cytochrome P-450II was much more stable than P-450I. The present studies provide evidence not only for species differences in the biological actions of the polychlorinated biphenyls but also demonstrate differential effects of the environmental pollutant on the two major forms of cytochrome P-450 and associated enzymic activities in rabbit lungs.     

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.     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

NADPH-cytochrome P-450 reductase. Circular dichroism and physical studies.

NADPH-cytochrome P-450 reductase was purified from hepatic microsomes of phenobarbital and hydrocortisone-treated rats by detergent solubilization and column chromatography. This membrane protein contains 31 mol per cent hydrophobic amino acid residues, 6 half-cystine residues, and a single tryptophan residue as determined by amino acid analysis after mineral or organic acid hydrolysis. The free mobility of cytochrome P-450 reductase in sodium dodecyl sulfate was identical to that of several soluble proteins used as standards (i.e. ovalbumin, bovin serum albumin, erythrocuprein, beta-galactosidase). Molecular weight estimates from sedimentation equilibrium studies in the presence of guanidine hydrochloride (76,500) are consistent with those determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at various per cent gel concentrations (79,000 to 80,000). Computer analysis of circular dichroism spectra of cytochrome P-450 reductase in the far ultraviolet region indicated the presence of 34 per cent alpha helical and 16 per cent beta structure. The amount of random structure was calculated to be 50 per cent.     

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

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

Purification of cytochrome P-450 from pig testis by aniline-sepharose 4B and isoelectric focusing

Testicular cytochrome P-450 was purified by a procedure including preparative isoelectrofocusing. The cytochrome P-450 was determined to have an isoelectric point of 6.47 on analytical isoelectric focusing. The purified cytochrome P-450 was found to be homogeneous and its molecular weight was estimated to be 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectrum with a peak at 448 nm exhibited absorption spectrum of a typical cytochrome P-450. 284-fold purification was achieved with an yield of 10.6%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing Aniline-Sepharose 4B column chromatography and preparative isoelectric focusing.     

The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450

The complete covalent structure of the constitutive cytochrome P-450, form 3b, from rabbit liver microsomes was determined. The apocytochrome contains 490 amino acid residues in a single polypeptide chain, Mr = 55,860. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. The constitutive form is only 46% homologous to the phenobarbital-induced cytochrome P-450 (Heinemann, F. S., and Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201) and contains a deletion at position 22. Strongly conserved regions include Cys435 and a previously identified tryptic peptide, residues 345-357. The distribution of hydrophobic segments is used to predict the membrane topology of the protein, and four possible orientations of this protein in the membrane are presented.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. We have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

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.     

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.