Omega-1 Hydroxylation Research Articles

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.

Clofibrate-inducible rat hepatic P450s IVA1 and IVA3 catalyze the omega- and (omega-1)-hydroxylation of fatty acids and the omega-hydroxylation of prostaglandins E1 and F2 alpha.

Cytochromes P450IVA1 and IVA3 display 72% amino acid sequence similarity and are expressed in livers of rats treated with the hypolipidemic drug clofibrate. The catalytic activities of IVA1 and IVA3 were examined by cDNA-directed expression using vaccinia virus. cDNA-expressed IVA1 and IVA3 had relative Mrs of 51,500 and 52,000, respectively, on SDS-polyacrylamide gels. Both enzymes displayed reduced, CO-bound absorption spectra with lambda max of 452.5 nm. IVA1 and IVA3 hydroxylated lauric acid at the omega and omega-1 positions with equivalent omega/omega-1 ratios of about 12.5. IVA1 had a substrate turnover of 21 min-1 which was about fourfold higher than that of IVA3. The omega and omega-1 hydroxylation of palmitic acid was also catalyzed by these P450s with combined turnover numbers for both metabolites of 45 min-1 or 18 min-1 for IVA1 and IVA3, respectively. The omega/omega-1 oxidation ratio of IVA1 for palmitate was 1.25 which was almost fourfold higher than that obtained for IVA3. These enzymes also catalyzed omega oxidation of the physiologically important eicosanoids prostaglandins E1 and F2 alpha with turnover numbers of about one-tenth those calculated for fatty acid oxidations. No omega-1 hydroxy metabolites were produced. These studies indicate that the P450 enzymes IVA1 and IVA3 are able to catalyze the oxidations of both fatty acids and prostaglandins.

Microbial models of mammalian metabolism: biotransformations of phenacetin and itsO-alkyl homologues withCunninghamellaspecies

1. The analgesic compound phenacetin and its O-alkyl homologues were metabolized by Cunninghamella elegans to yield the O-dealkylation product paracetamol (acetaminophen), and metabolites resulting from omega-1 hydroxylation and further oxidations. 2. Structural identification was based upon physical, spectral and chromatographic comparisons of isolated metabolites with synthetic standards generated by alkylation of paracetamol with the appropriate alkyl halide, epoxide, or alpha,beta-unsaturated ketone. 3. The rank order of O-dealkylation within the homologous series based upon either substrate disappearance or phenol formation was found to be ethyl greater than isopropyl greater than n-propyl greater than n-butyl greater than methyl.

Urinary Excretion of Valproate and Some Metabolites in Chronically Treated Patients

Urinary excretion of the antiepileptic agent valproic acid (VPA) and major metabolites from its glucuronidation, beta-oxidation, and omega- and omega 1-hydroxylation pathways were studied under steady state conditions in 24 epileptic patients. Some 55 +/- 18% (SD) of the daily dose was recovered in urine, 33 +/- 14% in the form of VPA-glucuronide, 15 +/- 8% as beta-oxidation products, and 4 +/- 2% and 2 +/- 1% as products of the omega- and omega 1-hydroxylation pathways, respectively. Only 1 +/- 2% of the dose was excreted unchanged. The proportion metabolized by direct glucuronidation tended to increase with dose at the expense of the oxidative pathways, particularly beta-oxidation. However, the wide variation in the patterns of urinary metabolite excretion precludes use of routinely collected urinary excretion data as a basis for detecting any but severe noncomplicance with VPA therapy or abnormalities of VPA metabolism.

Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.

A unique cytochrome P-450-dependent fatty acid monooxygenase from Bacillus megaterium ATCC 14581 is strongly induced by phenobarbital (Narhi, L. O., and Fulco, A. J. (1982) J. Biol. Chem. 257, 2147-2150) and many other barbiturates (Kim, B.-H., and Fulco, A. J. (1983) Biochem. Biophys. Res. Commun. 116, 843-850). This monooxygenase has now been purified to homogeneity from pentobarbital-induced bacteria as a single polypeptide with a molecular weight of 119,000 +/- 5,000 daltons. In the presence of NADPH and O2, it can catalyze the oxygenation of long chain fatty acids without the aid of any other protein. The enzyme has a catalytic center activity of 4,600 nmol of fatty acid oxygenated per nmol of P-450 (the highest activity yet reported for a P-450-dependent monooxygenase) and also functions as a highly active cytochrome c reductase in the presence of NADPH. The purified holoenzyme is a soluble protein containing 40 mol % hydrophobic amino acid residues and 1 mol each of FAD and FMN/mol of heme. It is isolated and purified in the low spin form but is converted to the high spin form in the presence of long chain fatty acids. The enzyme, which catalyzes the omega-2 hydroxylation of saturated fatty acids and the hydroxylation and epoxidation of unsaturated fatty acids has its highest affinity (Km = 2 +/- 1 microM) for the C15 and C16 chain lengths.

Metabolism of leukotriene B 4 in hepatic microsomes

Leukotriene B 4 was metabolized in rat hepatic microsomes to two products. Mass spectral analysis of these two metabolites indicated that the major metabolite was the 20-hydroxy metabolite while the minor metabolite was the 19-hydroxy metabolite. The formation of these metabolites required NADPH and was linear with time (20 min) and protein (1.6 mg/ml). The K m apparent and V max for omega hydroxylation of LTB 4 was 14 uM and 0.138 nmol/min/mg protein. In contrast, the k m and V max for omega minus one hydroxylation was 54 uM and 0.093 nmol/min/mg protein. These results suggest that omega and omega minus one hydroxylations of LTB 4 may be mediated by different isozymes of hepatic P-450.

Urinary metabolites of 2-ethyl-2-methylsuccinimide (ethosuximide) studied by combined gas chromatography mass spectrometry.

Metabolites of 2-ethyl-2-methylsuccinimide (ethosuximide) have been studied by combined gas chromatography mass spectrometry in a urine sample from a patient treated for petit mal epilepsy with ethosuximide. A new metabolite, 2-carboxymethyl-2-methylsuccinimide, was identified in the urine. It is presumably formed by omega 1-hydroxylation of the ethyl sidechain of the drug followed by a further oxidation of the primary alcohol to a carboxyl group. A previously identified metabolite, 2-ethyl-3-hydroxy-2-methylsuccinimide, was shown in the present study to yield two gas chromatographic peaks (as the N-methylated compound), indicating the existence of a diastereoisomeric pair. Thus, the enzyme responsible for the ring hydroxylation is probably not stereospecific, or alternatively two enzymes with different stereospecificity may exist.