Oxygenation mechanism in the oxidation of xenobiotic aldehyde to carboxylic acid by mouse hepatic microsomes

11-Oxo-delta 8-tetrahydrocannabinol was oxidized to delta 8-tetrahydrocannabinol-11-oic acid by mouse hepatic microsomes. The oxygenation mechanism in the reaction was confirmed by the incorporation of oxygen-18 from molecular oxygen into delta 8-tetrahydrocannabinol-11-oic acid. The oxygenation of aldehyde to carboxylic acid represents a novel mechanism in biological oxidation of aldehyde to carboxylic acid.     

Hepatic microsomal oxygenation of aldehydes to carboxylic acids

Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under 18O2 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes.     

Isolation and characterization of cytochrome P-450meg.

The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.     

Characterization of a cytochrome P-450-dependent steroid hydroxylase system present in Bacillus megaterium.

Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.     

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.     

Microsomal oxygenase catalyzed oxidation of 11-hydroxy-delta 8-tetrahydrocannabinol to 11-oxo-delta 8-tetrahydrocannabinol.

Rabbit liver microsomes were found to catalyze oxidation of 11-hydroxy-Δ⁸-tetrahydrocannabinol to 11-oxo-Δ⁸-tetrahydrocannabinol. This enzyme reaction required NADPH and molecular oxygen, and it was partially inhibited by CO. Pyrazole, potassium cyanide and sodium azide showed no effect on this oxidation, but SKF-525 A caused a significant inhibition. Thus, it is concluded that this enzymatic reaction is mediated by a mixed function oxidase involving cytochrome P-450.     

Production of 19-hydroxy-11-deoxycorticosterone and 19-oxo-11-deoxycorticosterone from 11-deoxycorticosterone by cytochrome P-450(11)beta

Incubation of 11-deoxycorticosterone with a cytochrome P-450(11)beta-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450(11)beta was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450(11)beta. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450(11)beta and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M+ peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450(11)beta can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.     

Expression of cytochrome P-450d by Saccharomyces cerevisiae

Rat liver microsomal cytochrome P-450d was abundantly expressed in the yeast Saccharomyces cerevisiae by using a yeast-Escherichia coli shuttle vector consisting of rat liver P-450d cDNA and yeast acid phosphatase promoter. The expressed cytochrome P-450d was immunologically crossed with rat liver P-450d. The hydroxylase activity of estra-1,3,5(10)-triene-3, 17 beta-diol was 11 nmol/min per nmol P-450d, which is comparable to that reported previously for rat liver P-450d. The expressed P-450d content was nearlyt 1% of total yeast protein as estimated from immunoblotting, hydroxylase activity and optical absorpton of the reduced CO form.     

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.     

Isolation of four forms of acetone-induced cytochrome P-450 in chicken liver by h.p.l.c. and their enzymic characterization.

The purpose of this study was to purify and characterize the forms of cytochrome P-450 induced in chicken liver by acetone or ethanol. Using high performance liquid ion-exchange chromatography, we were able to isolate at least four different forms of cytochrome P-450 which were induced by acetone in chicken liver. All four forms of cytochrome P-450 proved to be distinct proteins, as indicated by their N-terminal amino acid sequences and their reconstituted catalytic activities. Two of these forms, also induced by glutethimide in chicken embryo liver, appeared to be cytochromes P450IIH1 and P450IIH2. Both of these cytochromes P-450 have identical catalytic activities towards benzphetamine demethylation. However, they differ in their abilities to hydroxylate p-nitrophenol and to convert acetaminophen into a metabolite that forms a covalent adduct with glutathione at the 3-position. Another form of cytochrome P-450 induced by acetone is highly active in the hydroxylation of p-nitrophenol and in the conversion of acetaminophen to a reactive metabolite, similar to reactions catalysed by mammalian cytochrome P450IIE. Yet the N-terminal amino acid sequence of this form has only 30-33% similarity with cytochrome P450IIE purified from rat, rabbit and human livers. A fourth form of cytochrome P-450 was identified whose N-terminal amino acid sequence and enzymic activities do not correspond to any mammalian cytochromes P-450 reported to be induced by acetone or ethanol.     

The catalytic site of rat hepatic lauric acid omega-hydroxylase. Protein versus prosthetic heme alkylation in the omega-hydroxylation of acetylenic fatty acids

Cytochrome P-450LA omega purified from clofibrate-induced rat liver oxidizes lauric acid to 11- and 12-hydroxydodecanoic acid in approximately a 1:17 ratio at a rate of 20 nmol/nmol P-450/min. In contrast, cytochrome P-450b oxidizes lauric acid much more slowly (0.5 nmol/nmol P-450/min) to an 8:1 mixture of the same metabolites. Western blot analysis indicates that P-450LA omega accounts for 1-2 and 16-30%, respectively, of the total cytochrome P-450 in uninduced and clofibrate-induced rat liver. Cytochrome b5 increases the efficiency of omega-hydroxylation but not the rate of catalytic turnover. Incubation of the enzyme with 10-undecynoic acid (10-UDYA) results in loss of approximately 45% of the enzymatic activity but none of the enzyme chromophore. Approximately 1 mol of 1,11-undecandioic acid is produced per mole of inactivated enzyme. This extraordinary inactivation efficiency is confirmed by NADPH consumption studies. Approximately 0.5 equivalents of label are covalently bound to the enzyme when it is incubated with 14C-labeled 10-UDYA. 11-Dodecenoic acid appears not to be a substrate for cytochrome P-450LA omega but is oxidized, presumably by a contaminating isozyme, to a 10:1 mixture of 11,12-epoxydodecanoic acid and 12-oxododecanoic acid. The results suggest the presence of two closely related P-450LA omega enzymes, only one of which is susceptible to inactivation by 10-UDYA. They also indicate that cytochrome P-450LA omega has a highly structured active site that sterically suppresses omega-1-hydroxylation in order to deliver the oxygen to the thermodynamically disfavored terminal carbon. Protein rather than heme alkylation follows from this reaction regiospecificity.     

Electrochemical investigations on the oxygen activation by cytochrome P-450.

The application of cytochrome P-450 in substrate conversion is complicated both due to the limited stability and the cofactor regeneration problems. To overcome the disadvantages of NADPH consumption the transfer of the reduction equivalents from an electrode into the cytochrome P-450-system was studied: 1. NADPH was cathodically reduced at a mercury pool electrode. By immobilization of NADP on dialdehyde Sephadex the reductive recycling was possible. 2. Different forms of reduced oxygen were produced by the cathode: a) The reaction of O2- with deoxycorticosterone yields a carboxylic acid derivative. In contrast the cytochrome P-450 catalyzed NADPH-dependent reaction with the same substrate gives corticosterone, O2- represents only an intermediate in the activation of oxygen and is not the "activated oxygen" species. b) Molecular oxygen was reduced to HO2- and H2O2, respectively. The interaction of adsorbed cytochrome P-450 on the electrode surface with the reduced oxygen species in the absence of NADPH was studied. The electrochemically generated peroxide seems to be more active than added H2O2. 3. In a model of electro-enzyme-reactor several substrates were hydroxylated by microsomal cytochrome P-450 with cathodically reduced oxygen which substitutes NADPH.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Cytochromes P-450 are extremely important in the oxidative metabolism of a variety of endogenous and exogenous compounds in pro- and eukaryotic organisms. Progress in understanding the structure and mechanism of action of this superfamily of enzymes has been hampered by the properties of the eukaryotic enzymes and the availability of only one well-characterized prokaryotic enzyme as a model. We report here the isolation of a Pseudomonas species which will utilize a monoterpene natural product, alpha-terpineol, as its sole source of carbon and energy. Approximately 1% of the soluble protein in the cell-free extract is a novel cytochrome P-450 (P-450terp). This enzyme and its associated iron sulfur protein electron carrier (terpredoxin) have been purified to homogeneity and their NH2-terminal amino acid sequences determined. The amino acid sequences of six tryptic peptide fragments of cytochrome P-450terp have also been determined. This sequence information was used to clone the gene encoding cytochrome P-450terp. Three clones representing approximately 8 kilobase pairs of unique sequences were selected and sequenced. Five non-overlapping open reading frames (ORFs) were found in the sequences, and the translated sequences were used to search the Protein Identification Resource for comparable proteins. The ORFs were identified as: 1) an alcohol dehydrogenase, 2) an aldehyde dehydrogenase, 3) cytochrome P-450terp, 4) terpredoxin reductase, and 5) terpredoxin. The identification of both the cytochrome P-450terp and terpredoxin DNA sequence was confirmed by the presence of each of the corresponding amino acid sequences found in the purified proteins. The five ORFs were bounded on both the 5' and 3' ends by consensus factor-independent terminator sequences. A consensus promoter sequence was found immediately 5' to the first ORF. These results indicate that we have sequenced the complete terp operon. Comparison of the amino acid sequence of cytochrome P-450terp to that of all other cytochromes P-450 has shown that it is the first member of the gene family CYP108. Preliminary characterization of the chemical and physical properties and the preparation of crystals of this new cytochrome P-450, suitable for x-ray diffraction analysis, indicate that it will be useful in comparison studies with other members of this class of proteins.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

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.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

The covalent-sorption reconstitution of steroid hydroxylases

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.