Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b₅ and NADPH-cytochrome $\text{c}$ reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome $\text{c}$ reductase and phosphatidylcholine.     

Heterogeneity of liver microsomal cytochrome P-450: the spectral characterization of reactants with reduced cytochrome P-450.

The aerobic metabolism of benzphetamine by liver microsomes, during a cytochrome P-450-catalyzed mixed-function oxidation reaction, results in the formation of an easily detected spectral complex with an absorption band maximum at 456 nm. Electron paramagnetic resonance studies, as well as studies with the chemical reductant, sodium dithionite, or the oxidant, potassium ferricyanide, indicate that the spectral complex results from the formation of a product adduct with reduced cytochrome P-450. The spectral properties of this product complex of cytochrome P-450 have been compared to those observed with carbon monoxide, metyrapone, and ethylisocyanide. The reaction of these reagents to specific pools of microsomal cytochrome P-450 permits the identification of at least two major and two minor types of cytochrome P-450 in liver microsomes prepared from phenobarbital-treated rats.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

Novel epoxides formed during the liver cytochrome P-450 oxidation of arachidonic acid

Arachidonic acid is enzymatically oxidized by the rat liver microsomal mixed-function oxidase system, in the presence of NADPH and oxygen, to a wide variety of products. We report here, the identification of the major organic-soluble metabolites. They are the 5,6-,8,9-,11,12-, and 14,15-epoxy-eicosatrienoic acid derivatives of arachidonic acid.     

Mouse liver cytochrome P-450 (P-450IIIAM1): its cDNA cloning and inducibility by dexamethasone.

A full-length cDNA complementary to mouse liver mRNA coding for one of the cytochromes P-450 (P-450) in the P-450IIIA family, namely P-450IIIM1, was isolated and completely sequenced. The sequence of this cDNA clone, pMDex13, revealed that it encoded a polypeptide of 504 deduced amino acid residues (Mr = 57,853). The deduced amino acid sequence showed 87.3 and 84.9% identity with rat P-450IIIA1 and P-450IIIA2, respectively. The NH2-terminal 24 amino acid sequences of P-450IIIAM1 were completely identical with purified mouse P-450UT protein. RNA blot analysis showed that mRNA content of hepatic P-450IIIAM1 was remarkably increased by treatment of mice with dexamethasone.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Absolute configuration of the hydroxyeicosatetraenoic acids (HETEs) formed during catalytic oxygenation of arachidonic acid by microsomal cytochrome P-450

Rat liver microsomal cytochrome P-450 catalyzes the NADPH dependent metabolism of arachidonic acid to six regioisomeric cis,trans-dienols. The 12(R)-hydroxyeicosatetraenoic acid is generated with a high degree of enantioselectivity while the remaining regioisomers are produced as nearly racemic mixtures.     

Centrilobular expression of ethanol-inducible cytochrome P-450 (IIE1) in rat liver

Western blot analysis of digitonin eluates as well as immunohistochemical analysis revealed a 30-fold higher concentration of cytochrome P-450IIE1 in the centrilobular than in the periportal regions of the rat liver. Ethanol treatment caused a selective centrilobular induction of P-450IIE1, whereas phenobarbital induced P-450IIB1/2 in both liver lobule regions. The heterogeneous distribution pattern of P-450IIE1 was also observed in cells isolated from either region and correlated to the relative content of P-450IIE1 mRNA in the two cell types. The regiospecific expression and induction of P-450IIE1 may explain why several hepatotoxins, known to be metabolized by this isozyme, primarily damage the centrilobular region in the liver.     

Segmental homologies in the coding and 3' non-coding sequences of rat liver cytochrome P-450e and P-450b cDNAs and cytochrome P-450e-like genes

The nucleotide sequence of a cloned cDNA insert carried by pHDQ14 was determined and found to code for the 107 C-terminal amino acids of rat liver cytochrome P-450e. Comparison of the pHQ14 cDNA sequence with those of cloned cDNAs for cytochrome P-450b and of 2 P-450e-like genes revealed segmental homologies that may have resulted from gene conversion. These results suggest that gene conversion may generate sequence variants of genes for rat liver cytochrome P-450s.     

Purification and characterization of cytochrome P-450-dependent arachidonic acid epoxygenase from human liver

A novel human liver cytochrome P-450 isozyme (P-450-AA), which catalyzes arachidonic acid epoxidation, has been purified to electrophoretic homogeneity from human liver. As judged spectrally, the newly described isozyme is low spin in the oxidized state, with a soret band at 415 nm and an increased maximum at 451 nm in the CO-difference spectrum. Cytochrome P-450-AA appeared homogeneous as judged by the appearance of a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 53,100. Although cytochrome P-450-AA had a relatively low specific content of 10.8 nmol/mg, it possessed a high activity of arachidonic acid epoxidation. The P-450-AA oxidized arachidonic acid in a reconstituted system into the four regioisomeric epoxyeicosatrienoic acids (EETs) (5, 6-, 8, 9-, 11, 12-, 14, 15-EETs) at a rate of 2,010 pmol/nmol/min, a rate which is 37-fold higher than that observed with the crude microsomal preparation. Moreover, the purified cytochrome P-450-AA catalyzed the de-ethylation of 7-ethoxyresorufin at the rate of 2970 pmol/nmol/min, whereas other cytochrome P-450-dependent reactions were carried out at 23-2,000-fold lower rates and ranged between 0.3-130 pmol/nmol/min. The amino acid composition is different from that of other cytochrome P-450 isozymes. The NH2-terminal sequence of 20-amino acid residues was compared to that of LM2 and PB2-B2, the phenobarbital-induced forms in rabbit and rats, respectively. Comparison was also made with two forms of human cytochrome P-450, HLc and HLd. There were 7/20 identical residues for P-450-AA and LM2 and 4/20 for P-450-AA and PB2-B2. There were 2/20 identical residues for P-450-AA and HLd, and no identical residues were found for HLc. We conclude that the biologically active EETs, are formed by a distinct and unique P-450 isozyme from human liver and that arachidonic acid can serve as a screen for detection of the novel P-450 isozyme.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

Interaction of liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase in the presence and absence of lipid

Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid.     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

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.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.