Purification and properties of a cytochrome P450 arachidonic acid epoxygenase from rabbit renal cortex.

A rabbit cytochrome P450 which catalyzes the epoxidation of arachidonic acid to two of the four possible regioisomeric epoxyeicosatrienoic acid metabolites was purified from renal cortex. A small amount of the unresolved omega/omega-1 hydroxylated eicosatetraenoic acid products were also produced. The enzyme had a specific content of 8.4 nmol of P450/mg of protein and exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after silver staining. Sequencing revealed a single NH2-terminal amino acid sequence with the first 20 residues identical to rabbit cytochrome P450 2C2. We suggest this enzyme be termed P450 2CAA (for arachidonic acid) until the complete sequence and substrate selectivity are established. Purified P450 2CAA was in the low spin state as evidenced by an absorption maximum at 415 nm; the reduced-carbonyl complex exhibited a maximum at 451 nm. The specific activity for metabolism of 7 microM arachidonic acid was 1.1 nmol of product formed/min/nmol of P450. About 75% of the metabolites were two of the four possible epoxyeicosatrienoic acids identified as the 11,12- and 14,15-epoxyeicosatrienoic acids by coelution with synthetic and commercial standards on reversed and normal-phase high pressure liquid chromatographic separations. The ratio of the 11,12- to 14,15-epoxyeicosatrienoic acids was 1.5:1. The purified enzyme exhibited no significant activity toward 7-ethoxyresorufin or progesterone, but demethylated aminopyrine and benzphetamine. Other fatty acids were also substrates for the enzyme. Oleic, linoleic, and lauric acids, all at about 10 microM, were metabolized at rates of 0.32, 0.72, and 0.73 nmol/min/nmol of P450, respectively. Monoclonal antibody that cross-reacts with P450 2C2 inhibited 63% of the microsomal epoxidation activity from renal cortex microsomes from phenobarbital-treated rabbits. The production of the epoxide metabolites of arachidonic acid suggests that P450 2CAA may have a significant role in arachidonic acid-mediated intra- and intercellular signalling pathways.     

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

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.     

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.     

Cytochrome P-450 enzyme-specific control of the regio- and enantiofacial selectivity of the microsomal arachidonic acid epoxygenase

Chiral analysis of the rat liver microsomal arachidonic acid epoxygenase metabolites shows enantioselective formation of 8,9-, 11,12-, and 14,15-cis-epoxyeicosatrienoic acids in an approximately 2:1, 4:1, and 2:1 ratio of antipodes, respectively. Animal treatment with the cytochrome P-450 inducer phenobarbital increased the overall enantiofacial selectivity of the microsomal epoxygenase and caused a concomitant inversion in the absolute configurations of its metabolites. These effects of phenobarbital were time-dependent and temporally linked to increases in the concentration of microsomal cytochrome P-450 enzymes. Reconstitution of the epoxygenase reaction utilizing several purified cytochrome P-450 demonstrated that the asymmetry of epoxidation is under cytochrome P-450 enzyme control. These results established that the chirality of the hepatic arachidonic acid epoxygenase is under regulatory control and confirm cytochromes P-450 IIB1 and IIB2 as two of the endogenous epoxygenases induced in vivo by phenobarbital.     

NH2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15-20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65-87% hydrophobic amino acids, and only one charged amino acid (Asp) in the amino-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11-12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes (P-450a-e).     

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.     

Cytochrome P-450 arachidonic acid epoxygenase. Regulatory control of the renal epoxygenase by dietary salt loading.

The rat kidney microsomal epoxygenase catalyzed the asymmetric epoxidation of arachidonic acid to generate as major products: 8(R),9(S)-, 11(R),12(S)- and 14(S),15(R)-epoxyeicosatrienoic acids with optical purities of 97, 88, and 70%, respectively. Inhibition studies utilizing a panel of polyclonal antibodies to several rat liver cytochrome P-450 isoforms, indicated that the renal epoxygenase(s) belongs to the cytochrome P-450 2C gene family. Dietary salt, administered either as a 2-2.5% (w/v) solution in the drinking water or as a modified solid diet containing 8% NaCl (w/w), resulted in marked and selective increases in the renal microsomal epoxygenase activity (416 and 260% of controls, for the liquid and solid forms of NaCl, respectively) with no significant changes in the microsomal omega/omega-1 oxygenase or in the hepatic arachidonic acid monooxygenase reaction. Immunoblotting studies demonstrated that dietary salt induced marked increases in the concentration of a cytochrome P-450 isoform(s) recognized by polyclonal antibodies raised against human liver cytochrome P-450 2C10 or rat liver cytochrome P-450 2C11. Comparisons of the stereochemical selectivity of the induced and non-induced microsomal epoxygenase(s) with that of purified rat liver cytochrome P-450 2C11 suggest that the salt-induced protein(s) is catalytically and structurally different from liver cytochrome P-450 2C11. The in vivo significance of dietary salt in regulating the activities of the kidney endogenous arachidonic acid epoxygenase was established by the demonstration of a salt-induced 10-20-fold increase in the urinary output of epoxygenase metabolites. These results, in conjunction with published evidence demonstrating the potent biological activities of its metabolites, suggest a role for the epoxygenase in the renal response to dietary salt.     

A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs.     

Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.     

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with -naphthoflavone (-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH₂ terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.Abbreviations -NF -napthoflavone - SDS-PAGE Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis - 3-MC 3-Methyl Cholanthrene - PEG Poly Ethylene Glycol - DTT Dithiothreitol - PMSF Phenyl Methyl Sulfonylfluoride - EDTA disodium ethylenediaminetetraacetate - NADPH reduced nicotinamide adenine dinucleotide phosphate - P-450 cytochrome P450, PB-1, PB-4, PB-5 and P-450 isozymes purified from phenobarbital induced rat liver - HPLC High Pressure Liquid Chromatography - B[]P benzo[]pyrene - CM Carboxymethyl Sephadex - PTH-amino acid phenylthiohydantoin amino acid, Cytochrome P-450 EC 1.14.14.1, NADPH Cytochrome P-450 (c) reductase ED 1.6.2.4     

Cytochrome P-450C-M/F, a new constitutive form of microsomal cytochrome P-450 in male and female rat liver with estrogen 2- and 16 alpha-hydroxylase activity

A new cytochrome P-450 isozyme, P-450C-M/F, has been purified from untreated rat liver microsomes. The purified preparation was electrophoretically homogeneous and contained 12-15 nmol of P450/mg of protein and had a minimum molecular weight of 48,500. The NH2-terminal amino acid sequence of P-450C-M/F was different from that of other P-450's. Immunoblot analysis of microsomes demonstrated that P-450C-M/F was present in the liver of untreated male as well as female rats. Treatment of rats with phenobarbital, 3-methylcholanthrene, or beta-naphthoflavone did not induce P-450C-M/F. Cytochrome P-450C-M/F exhibited little activities of 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation or hydroxylation of arylhydrocarbon, testosterone, androstenedione, and progesterone. In contrast, it was highly active in N-demethylation of ethylmorphine and benzphetamine and in 2- and 16 alpha-hydroxylation of estrogens, particularly that of estradiol. These studies establish that cytochrome P-450C-M/F is constitutively present in both male and female rats and suggest that it may be involved in the oxidative metabolism of estradiol, particularly in the formation of estriol, the uterotropic metabolite of estradiol.     

Isolation, partial purification, and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris.

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein. 2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE. 3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris. 4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a Vmax of 2.12 pmol resorufin/min.mg protein and a Km of 770 nM benzyloxyresorufin at 25 degrees C, pH 8.0. 5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein. 6. The apparent molecular weight of the threefold-purified reductase was 63,000.     

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.     

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system

[3H]Corticosterone was incubated with cytochrome P-45011 beta purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [3H]18-hydroxycorticosterone was incubated with cytochrome P-45011 beta, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.     

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.     

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: isozyme specificity and role of cytochrome b5

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process.     

Evidence for functional and structural multiplicity of pregnenolone-16 alpha-carbonitrile-inducible cytochrome P-450 isozymes in rat liver microsomes

Administration of pregnenolone-16 alpha-carbonitrile (PCN) to adult female rats caused a 2-fold increase in total liver microsomal cytochrome P-450 along with 5-7-fold increases in four in vitro monooxygenase activities considered diagnostic for the major PCN-inducible cytochrome P-450 isozyme. However, upon administration of chloramphenicol to PCN-treated rats, these monooxygenase activities could be resolved into three groups. Thus, the ability of the microsomes to convert triacetyloleandomycin to a metabolite that forms a spectral complex with the reduced heme iron was decreased by 80% by chloramphenicol, whereas only a 50% decrease was observed in the rate of conversion of (R)-warfarin to its 9,10-dehydro metabolite and in the rate of 6 beta-hydroxylation of androstenedione. More strikingly, the 10-hydroxylation of (R)-warfarin was actually enhanced 2-fold by the chloramphenicol treatment. Fractionation studies were carried out on liver microsomes from PCN-treated adult male rats, and two highly purified cytochromes P-450, referred to as PCNa and PCNb, were recovered. PCNb was found to be identical in the sequence of the first 15 amino acid residues with a PCN-inducible isozyme, the complete amino acid sequence of which has recently been deduced in another laboratory [Gonzalez, F. J., Nebert, D. W., Hardwick, J. P., & Kasper, C. B. (1985) J. Biol. Chem. 260, 7435-7441]. The other isozyme, PCNa, differed in amino acid sequence in three of the first 15 positions from PCNb. Upon immunoblot analysis, polyclonal antibodies raised to PCNb also recognized PCNa. Thus, the PCN-inducible family of rat liver cytochrome P-450 comprises at least two separate proteins.     

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.     

Chiral resolution of the epoxyeicosatrienoic acids, arachidonic acid epoxygenase metabolites

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.