Expression and induction of cytochrome P450s in rabbit parotid glands.

Earlier, we isolated and purified five different P450 isoforms from rabbit kidney cortex microsomes, three of which are members of the CYP4A subfamily (CYP4A5, CYP4A6, and CYP4A7), with the others being CYP2B4 and CYP1A1. In contrast, P450s in parotid glands were unknown. The fact that the parotid glands bear a marked morphological and functional resemblance to kidney tissue prompted us to investigate P450s in these glands. The present study was undertaken to determine which P450 isoforms are expressed in this tissue. Microsomes from parotid glands of untreated rabbits were found to contain 42.3 pmol of P450/mg protein and to catalyze the omega-hydroxylation of laurate. Administration of di(2-ethylhexyl) phthalate (DEHP) resulted in a 7-fold increase of laurate omega-hydroxylation. This enzyme activity was greatly inhibited by pretreatment with antibodies against CYP4A5. Furthermore, parotid gland CYP4A5, CYP4A6, and CYP4A7 mRNAs were identified by RT-PCR. Moreover, the CYP4A enzymes were demonstrated immunohistochemically to be localized exclusively in the ducts of these glands. In addition to the CYP4A enzymes, immunoblot analysis revealed that CYP2B4 is constitutively present, and that CYP1A1 is induced in these glands by treatment with 3-methylcholanthrene. Taken together, we can conclude that the P450 isoforms expressed in rabbit kidney cortex and parotid glands are identical in composition.

Unique heme environment at the putative distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis (peroxygenase P450(SPalpha).

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C >/= 5) induced changes in the spectrum of ferric P450(SPalpha) to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450(SPalpha) in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450(SPalpha) to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450(SPalpha) combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450(SPalpha), in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450(SPalpha). The unique properties of the hydroxylase reaction catalyzed by P450(SPalpha), where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its alpha position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450(SPalpha) is proposed.

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.

Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: substrate structure required for alpha-hydroxylation.

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The Km value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10-C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, Km values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.

Human fatty acid omega-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein.

The gene of the human fatty acid omega-hydroxylase, CYP4A11, has been isolated from a human BAC library, and its complete genomic sequence has been determined. The CYP4A11 gene spanned 12,568 bp and contained 12 exons. The known PPAR recognition elements (PPRE), which were reported to be involved in the induction of CYP4A6 by clofibric acid, were not observed within the 5'-flanking region of the CYP4A11 gene. The recombinant CYP4A11 protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 6.4 nmol of P450/mg of protein. This P450 exhibited omega-hydroxylation activity toward laurate, with a turnover number of 14.7 nmol/min/nmol of P450. The apparent K(m) and V(max) values were 56.7 microM and 15.2 nmol/min/nmol of P450, respectively. It also showed omega-hydroxylation activity toward palmitate, with a turnover number of 0.78 nmol/min/nmol of P450. Although several reports from other groups described that CYP4A11 preparations catalyzed omega-hydroxylation of arachidonic acid, our purified recombinant protein exhibited no activity toward arachidonic acid nor prostaglandin A(1).

Expression and catalytic activity of mouse leukotriene B4 omega-hydroxylase, CYP4F14.

We have isolated a cDNA for a mouse leukotriene B4 omega-hydroxylase, CYP4F14. The cDNA encoded a protein with 524 amino acids, whose sequence similarity is 95% that of rat CYP4F1. The microsomes from yeast cells transfected with CYP4F14 expression vector showed 0.1 nmol P450/mg protein and catalyzed omega-hydroxylations of leukotriene B4, 6-trans-leukotriene B4, lipoxin A4, prostaglandin A1, and several hydroxyeicosatetraeonic acids (HETEs), with 8-HETE being the most active substrate. In contrast, no activity was detected toward lipoxin B4, laurate, and arachidonate. The mRNA for CYP4F14 had three different 5' untranslated sequences. Analysis of the CYP4F14 gene showed that two exon I sequences with different transcription start sites are located in the gene, and two splicing signals on the 3' end of intron I are alternatively used. The mRNA for this P450 was detected only in the liver by Northern blot analysis, whereas a small amount of the mRNA was detected in the brain using RT-PCR. Administration of clofibrate had no effect on microsomal 6-trans-leukotriene B4 omega-hydroxylase activity, but resulted in a marked reduction in the content of mRNA for this P450 in the liver. These findings indicate that CYP4F14 is very similar to CYP4F1 except for its expression in the brain and 5' untranslated sequences.

Expression and molecular cloning of human liver leukotriene B4 omega-hydroxylase (CYP4F2) gene.

Human liver leukotriene B4 (LTB4) omega-hydroxylase (CYP4F2) plays an important role in the metabolic inactivation and degradation of LTB4, a potent mediator of inflammation. The regulatory mechanism for the transcription of CYP4F2 has not yet been clarified. Here, we report that CYP4F2 is constitutively expressed in a human hepatoma cell line, HepG2, and is not induced by clofibrate. We isolated the gene encoding CYP4F2 and determined its genomic organization and the functional activity of its promoters. The CYP4F2 gene contains at least 13 exons with its open reading frame being encoded from exon II to exon XIII. Exon I includes 49 bp of a 5' untranslated sequence. The structure of this gene is very similar to that of the CYP4F3 gene earlier reported by Kikuta et al. (DNA Cell Biol 1998;17:221-230). The 5' flanking sequence downstream from -165 of the CYP4F2 gene has 75% similarity to the corresponding region of the CYP4F3 gene. However, common putative regulating elements in the two human CYP4F genes were not detected except for the TATA box. The elements recognized by nuclear receptors were not observed within its 5' flanking region. Deletion of the 5' flanking regions containing putative regulating elements recognized by HNF-3beta, CDP CR, and p300 caused alterations in the transcriptional activity. The region from -83 to -67 was necessary for transcription, but the TATA sequence was not. Our results indicate that the human two CYP4F genes evolved by duplication and alterations of the transcription regulation region and the site of exon III.

Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.

Purification and characterization of recombinant human neutrophil leukotriene B4 omega-hydroxylase (cytochrome P450 4F3).

Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity.

Further characterization of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed alpha-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 microM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2 mM, but CO did not. Imidazole, an inhibitor of plant alpha-oxidation, showed no inhibitory effect at 1 mM.

Phytanic acid alpha-hydroxylation by bacterial cytochrome P450.

Fatty acid alpha-hydroxylase, a cytochrome P450 enzyme, from Sphingomonas paucimobilis, utilizes various straight-chain fatty acids as substrates. We investigated whether a recombinant fatty acid alpha-hydroxylase is able to metabolize phytanic acid, a methyl-branched fatty acid. When phytanic acid was incubated with the recombinant enzyme in the presence of H2O2, a reaction product was detected by gas chromatography, whereas a reaction product was not detected in the absence of H2O2. When a heat-inactivated enzyme was used, a reaction product was not detected with any concentration of H2O2. Analysis of the methylated product by gas chromatography-mass spectrometry revealed a fragmentation pattern of 2-hydroxyphytanic acid methyl ester. By single-ion monitoring, the mass ion and the characteristic fragmentation ions of 2-hydroxyphytanic acid methyl ester were detected at the retention time corresponding to the time of the product observed on the gas chromatogram. The Km value for phytanic acid was approximately 50 microM, which was similar to that for myristic acid, although the calculated Vmax for phytanic acid was about 15-fold lower than that for myristic acid. These results indicate that a bacterial cytochrome P450 is able to oxidize phytanic acid to form 2-hydroxyphytanic acid.

Protein expression, characterization, and regulation of CYP4F4 and CYP4F5 cloned from rat brain.

We previously reported the cDNA cloning of three new forms of P450, CYP4F4, CYP4F5, and CYP4F6, from a rat brain cDNA library. In the present study, we expressed CYP4F4 and CYP4F5 in Escherichia coli using the pCWOri expression vector with a modification of their N-terminal amino acid sequences and the incorporation of a C-terminal [His]4 tag to aid in purification. CYP4F5 recombinant protein was purified to a specific content of 7.7 nmol/mg protein from the membrane fraction of E. coli and showed omega-hydroxylation activity toward leukotriene B4 (LTB4), a chemical mediator of inflammation. On the other hand, the solubilized membrane fraction of CYP4F4-expressed recombinant protein catalyzed the omega-hydroxylation of prostaglandin A1, prostaglandin E1, and 6-trans-LTB4 as well as LTB4. The effects of the peroxisome proliferator, clofibrate, on mRNA expression of CYP4F4, 4F5, and 4F6 were studied by Northern blot analysis. The expression levels of the mRNA of these CYP4Fs were shown to be reduced by clofibrate in liver.

Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid. FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes. Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium. The isolated gene encoded 415 amino acids. A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH. Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion. Overall, FAAH has no significant identity to the known P450s. CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm. These results suggest bacterial FAAH is a novel member of the P450 superfamily.

Purification and characterization of two new cytochrome P-450 related to CYP2C subfamily from rabbit small intestine microsomes.

Two forms of cytochrome P-450, designated P-450id and P-450ie, were purified to specific contents of 14.3 and 15.0 nmol of P-450/mg of protein, respectively, from small intestine mucosa microsomes of rabbits. P-450id and P-450ie showed apparent molecular weights of 50 and 49 kDa, respectively, on SDS-PAGE. Both P-450s catalyzed N-demethylation of nitrosodimethylamine. The NH2-terminal amino acid sequence (first 19 residues) of P-450id exhibited 74-90% identity with those of six members of the rabbit P-450 2C subfamily, except for P-450 2C3. Similarly, the NH2-terminal sequence (first 22 residues) of P-450ie showed 73-86% identity with those of the same members of the rabbit P-450 2C subfamily. The peptide mapping patterns of the two P-450s were quite different from each other. In addition, P-450id did not cross-react with the guinea-pig antibodies against P-450ie. The results indicate that rabbit small intestine mucosa contain two new distinct forms of P-450s, both of which may be classified into the 2C subfamily.

Purification and characterization of rabbit small intestinal cytochromes P450 belonging to CYP2J and CYP4A subfamilies.

A new form of P450 designated P450ib2 was purified from rabbit small intestine microsomes. This P450 had properties very similar, to P450ib (CYP2J1), and showed 88% identity with CYP2J1 in its first 20 NH2-terminal amino acid sequence, excluding 3 undetermined residues. Both P450ib and P450ib2 were immunohistochemically detected in the mucosal epitherial cells of the duodenum, jejunum, and ileum in the small intestine, whereas no immunoreactivity was observed in other tissues including liver, kidney, lung, colon, and stomach. The results support that the two closely related P450s are specifically localized in the rabbit small intestine. Another small intestinal P450, P450ia, was found to hydroxylate a wide variety of fatty acids including straight-chain, branched-chain, unsaturated, or hydroxy fatty acids, and prostaglandin A at the omega and (omega-1) positions. P450ia was identical with a rabbit kidney fatty acid omega-hydroxylase, CYP4A7, in its 25 NH2-terminal amino acid sequence, excluding 2 undetermined residues. The results identify P450ia as CYP4A7.

Direct involvement of hydrogen peroxide in bacterial alpha-hydroxylation of fatty acid.

We have reported that fatty-acid alpha-hydroxylase partially purified from Sphingomonas paucimobilis required NADH and molecular oxygen. In this study, we found that the reaction was greatly inhibited by catalase. Glutathione and glutathione peroxidase also inhibited alpha-hydroxylation, but superoxide dismutase and mannitol did not. Replacement of NADH and molecular oxygen by hydrogen peroxide increased the alpha-hydroxylation activity. In the presence of hydrogen peroxide, molecular oxygen was not required for the activity. These findings suggest that hydrogen peroxide was essential for bacterial alpha-hydroxylase.

Cloning and expression of a novel form of leukotriene B4 omega-hydroxylase from human liver.

We have isolated and sequenced a cDNA for human liver LTB4 omega-hydroxylase. The cDNA encoded a protein of 520 amino acids with a molecular weight of 59,853 Da. The cDNA-deduced amino acid sequence showed 87.3% homology to that of human polymorphonuclear leukocytes (PMN) LTB4 omega-hydroxylase (CYP4F3). Northern blot analysis revealed that the mRNA hybridized to the specific cDNA fragment is expressed in human liver, but not in human PMN. The microsomes from yeast cells transfected with the cDNA catalyzed the omega-hydroxylation of LTB4 with a Km of 44.8 microM. These results clearly show that a new form of the CYP4F LTB4 omega-hydroxylase exists in human liver.

Purification and cDNA cloning of human liver CYP4A fatty acid omega-hydroxylase.

Laurate omega-hydroxylase activity of human liver microsomes was strongly inhibited by an antibody against rabbit fatty acid omega-hydroxylase P450 4A5, and Western blot analysis with this antibody showed the presence of two immunochemically related proteins with apparent molecular weights of approximately 50 and 52 kDa in all of 14 human liver specimens examined. A fatty acid omega-hydroxylase (designated P450HL omega) was purified to a specific content of 15 nmol of P450/mg of protein from microsomes of a single human liver on the basis of its laurate omega-hydroxylase activity and its reactivity with the P450 4A5 antibody. This P450HL omega showed an apparent molecular weight of 52 kDa on SDS-PAGE. Furthermore, a cDNA clone (designated HL24) has been isolated from a human liver cDNA library by using the cDNA for P450 4A5 as a probe. The sequence of residues 5 through 25 deduced from cDNA HL24 was identical to the NH2-terminal amino acid sequence of P450HL omega except for one undetermined residue. This cDNA encoded a protein of 519 amino acids with a molecular weight of 59,347. The amino acid sequence predicted from the cDNA showed 82% identity with that of P450 4A5. Northern blot analysis showed that the mRNA hybridized to the cDNA is expressed in the human liver and kidney. (ABSTRACT TRUNCATED AT 250 WORDS)

Separation and partial characterization of soluble fatty acid alpha-hydroxylase from Sphingomonas paucimobilus.

The crude extracts from Sphingomonas paucimobilus containing 2-hydroxy fatty acid-rich sphingolipids were found to oxidize [1-14C]-myristate to 2-hydroxymyristate in the presence of NADH. The myristate-oxidation activity was partially purified about 290-fold from the cell-free extracts by ammonium sulfate fractionation and hydroxylapatite chromatography. When laurate, myristate, and palmitate were used as the substrates, the reaction products were identified as the corresponding 2-hydroxy fatty acids by gas-chromatography (GC), HPLC, and GC-mass spectrometry. The enzyme preparation required NADH and molecular oxygen for its alpha-hydroxylation activity, suggesting that the bacterial alpha-hydroxylation is typical of monooxygenase reactions. Of fatty acids tested, myristate was the most efficient substrate.

Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega-1)-hydroxylating P450s, P450 2C2 and P450 2E1.

P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C9-C18 fatty acids at the omega-1 position and to a much lesser extent at the omega and omega-2 positions, P450 2C2 hydroxylated C9-C13 fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of omega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C9-C12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as omega-1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)