Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe–2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe–2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe–S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)^?1min^?1 for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)^?1 min^?1 for Pux. This difference mainly arises from weak CYP199A2–PuxB binding (K _m 34.3 vs. 0.45 μM for Pux) rather than slow electron transfer (k _cat 19.1 vs. 37.9 s^?1 for Pux). Comparison of the 2.0-Å-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin–P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe–2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.     

Crystal structure of CYP199A2, a para-substituted benzoic acid oxidizing cytochrome P450 from Rhodopseudomonas palustris

CYP199A2, a cytochrome P450 enzyme from Rhodopseudomonas palustris, oxidatively demethylates 4-methoxybenzoic acid to 4-hydroxybenzoic acid. 4-Ethylbenzoic acid is converted to a mixture of predominantly 4-(1-hydroxyethyl)-benzoic acid and 4-vinylbenzoic acid, the latter being a rare example of CC bond dehydrogenation of an unbranched alkyl group. The crystal structure of CYP199A2 has been determined at 2.0-Å resolution. The enzyme has the common P450 fold, but the B′ helix is missing and the G helix is broken into two (G and G′) by a kink at Pro204. Helices G and G′ are bent back from the extended BC loop and the I helix to open up a clearly defined substrate access channel. Channel openings in this region of the P450 fold are rare in bacterial P450 enzymes but more common in eukaryotic P450 enzymes. The channel is hydrophobic except for the basic residue Arg246 at the entrance, which probably plays a role in the specificity of this enzyme for charged benzoates over neutral phenols and benzenes. The substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues. Computer docking of 4-ethylbenzoic acid into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the β-methyl group is located over the heme iron by Phe185, the side chain of which is only 6.35 Å above the iron in the native structure. This binding orientation is consistent with the observed product profile of exclusive attack at the para substituent. Putidaredoxin of the CYP101A1 system from Pseudomonas putida supports substrate oxidation by CYP199A2 at ∼6% of the activity of the physiological ferredoxin. Comparison of the heme proximal faces of CYP199A2 and CYP101A1 suggests that charge reversal surrounding the surface residue Leu369 in CYP199A2 may be a significant factor in this low cross-activity.     

Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)⁻¹ min⁻¹. Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Drosophila melanogaster CYP6A8, an insect P450 that catalyzes lauric acid (omega-1)-hydroxylation

Only a handful of P450 genes have been functionally characterized from the approximately 90 recently identified in the genome of Drosophila melanogaster. Cyp6a8 encodes a 506-amino acid protein with 53.6% amino acid identity with CYP6A2. CYP6A2 has been shown to catalyze the metabolism of several insecticides including aldrin and heptachlor. CYP6A8 is expressed at many developmental stages as well as in adult life. CYP6A8 was produced in Saccharomyces cerevisiae and enzymatically characterized after catalytic activity was reconstituted with D. melanogaster P450 reductase and NADPH. Although several saturated or non-saturated fatty acids were not metabolized by CYP6A8, lauric acid (C12:0), a short-chain unsaturated fatty acid, was oxidized by CYP6A8 to produce 11-hydroxylauric acid with an apparent V(max) of 25 nmol/min/nmol P450. This is the first report showing that a member of the CYP6 family catalyzes the hydroxylation of lauric acid. Our data open new prospects for the CYP6 P450 enzymes, which could be involved in important physiological functions through fatty acid metabolism.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.     

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.     

Comparison of P450s from human and fugu: 420 million years of vertebrate P450 evolution

The fugu (pufferfish) genome has been sequenced, and a second genome assembly was released 17 May 2002. Exhaustive searches were made to identify all P450 genes and pseudogenes from the earlier release of 26 October 2001. P450 genes assembled as completely as possible from these data were used to do additional searches of the newer assembly and all P450 genes and pseudogenes in the available fugu sequence data have been identified, compared to human P450s, and assigned names. There are 54 P450 genes in fugu and 1 nearly intact pseudogene (CYP3A50P). CYP1A is missing much of its N-terminal half; however, 45 P450 genes are completely assembled. Eight others are lacking only one or two exons or less. CYP2X4 is known only from an EST. This may be a 55th P450 gene if it represents an accurate sequence. In addition to 2X4, there are 16 other pseudogene fragments or small pieces of P450 genes. At the P450 family level, 17 of 18 mammalian families are found in fugu. CYP39 is the only CYP family missing and it is not seen in any other fish sequence data either. The CYP2 family shows the largest degree of divergence. In the CYP2 family, only CYP2R1 and CYP2U1 are conserved as recognizable subfamilies across species. Intron-exon boundaries are largely preserved across 420 million years of evolution.     

Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.     

Crystal structure of a ferredoxin reductase for the CYP199A2 system from Rhodopseudomonas palustris

Cytochrome P450‐199A2 from Rhodopseudomonas palustris oxidizes para‐substituted benzoic acids and may play a role in lignin and aromatic acid degradation pathways in the bacterium. CYP199A2 has an associated [2Fe‐2S] ferredoxin, palustrisredoxin (Pux) but not a ferredoxin reductase. A genome search identified the palustrisredoxin reductase (PuR) gene. PuR was produced in Escherichia coli and shown to be a flavin‐dependent protein that supports efficient electron transfer from NADH to Pux, thus reconstituting CYP199A2 monooxygenase activity (k_cat = 37.9 s^–1 with 4‐methoxybenzoic acid). The reduction of Pux by PuR shows K_m = 4.2 μM and k_cat = 262 s^–1 in 50 mM Tris, pH 7.4. K_m is increased to 154 μM in the presence of 200 mM KCl, indicating the importance of ionic interactions in PuR/Pux binding. The crystal structure of PuR has been determined at 2.2 Å resolution and found to be closely related to that of other oxygenase‐coupled NADH‐dependent ferredoxin reductases. Residues on the surface that had been proposed to be involved in ferredoxin reductase‐ferredoxin binding are conserved in PuR. However, Lys328 in PuR lies over the FAD isoalloxazine ring and, together with His11 and Gln41, render the electrostatic potential of the surface more positive and may account for the greater involvement of electrostatic interactions in ferredoxin binding by PuR. Consistent with these observations the K328G mutation weakened Pux binding and virtually eliminated the dependence of PuR/Pux binding on salt concentration, thus confirming that the FAD si side surface in the vicinity of Lys328 is the ferredoxin binding site. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.     

Reduction of toxic metabolite formation of acetaminophen

Acetaminophen is a widely used over-the-counter drug that causes severe hepatic damage upon overdose. Cytochrome P450-dependent oxidation of acetaminophen results in the formation of the toxic N-acetyl-p-benzoquinone-imine (NAPQI). Inhibition of cytochrome P450 enzymes responsible for NAPQI formation might be useful--besides N-acetylcysteine treatment--in managing acetaminophen overdose. Investigations were carried out using human liver microsomes to test whether selective inhibition of cytochrome P450s reduces NAPQI formation. Selective inhibition of CYP3A4 and CYP1A2 did not reduce, whereas the inhibition of CYP2A6 and CYP2E1 significantly decreased NAPQI formation. Furthermore, selective CYP2E1 inhibitors that are used in human therapy were tested for their inhibitory effect on NAPQI formation. 4-Methylpyrazole, disulfiram, and diethyl-dithiocarbamate were the most potent inhibitors with IC(50) values of 50 microM, 8 microM, and 33 microM, respectively. Although cimetidin is used in the therapy of acetaminophen overdose as an inhibitor of cytochrome P450, it is not able to reduce NAPQI formation.     

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 (Ki = 14+/- 4 microg/mL), and to a lesser extent, CYP1A2 (Ki = 106 +/- 24 microg/mL), CYP2E1 (Ki = 127 +/- 42 microg/mL), and CYP3A4 (Ki = 155 +/- 43 microg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 (Ki = 15 +/-6 microg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 (Ki's between 4.9 and 55 microg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC50 < 40 microg/mL).     

Effect of homomeric P450-P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.     

Finding homes for orphan cytochrome P450s: CYP4V2 and CYP4F22 in disease states

The cytochrome P450 (CYP) 4 family of enzymes contains several recently identified membersthat are referred to as “orphan P450s” because their endogenous substrates are unknown.Human CYP4V2 and CYP4F22 are two such orphan P450s that are strongly linked to ocular andskin disease, respectively. Genetic analyses have identified a wide spectrum of mutations in the CYP4V2gene from patients suffering from Bietti’s crystalline corneoretinal dystrophy, and mutations in theCYP4F22 gene have been linked to lamellar ichthyosis. The strong gene–disease associations provideunique opportunities for elucidating the substrate specificity of these orphan P450s and unraveling thebiochemical pathways that may be impacted in patients with CYP4V2 and CYP4F22 functional deficits.     

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

Several P450 enzymes localized in the endoplasmic reticulum and thought to be involved primarily in xenobiotic metabolism, including mouse and rat CYP1A1 and mouse CYP1A2, have also been found to translocate to mitochondria. We report here that the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces enzymatically active CYP1A4/1A5, the avian orthologs of mammalian CYP1A1/1A2, in chick embryo liver mitochondria as well as in microsomes. P450 proteins and activity levels (CYP1A4-dependent 7-ethoxyresorufin-O-deethylase and CYP1A5-dependent arachidonic acid epoxygenation) in mitochondria were 23-40% of those in microsomes. DHET formation by mitochondria was twice that of microsomes and was attributable to a mitochondrial soluble epoxide hydrolase as confirmed by Western blotting with antiEPHX2, conversion by mitochondria of pure 11,12 and 14,15-EET to the corresponding DHETs and inhibition of DHET formation by the soluble epoxide hydrolase inhibitor, 12(-3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). TCDD also suppressed formation of mitochondrial and microsomal 20-HETE. The findings newly identify mitochondria as a site of P450-dependent arachidonic acid metabolism and as a potential target for TCDD effects. They also demonstrate that mitochondria contain soluble epoxide hydrolase and underscore a role for CYP1A in endobiotic metabolism.     

Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

Cytochrome P450-catalyzed brassinosteroid pathway activation through synthesis of castasterone and brassinolide in Phaseolus vulgaris

The last reaction in the biosynthesis of brassinolide has been examined enzymatically. A microsomal enzyme preparation from cultured cells of Phaseolus vulgaris catalyzed a conversion from castasterone to brassinolide, indicating that castasterone 6-oxidase (brassinolide synthase) is membrane associated. This enzyme preparation also catalyzed the conversions of 6-deoxocastasterone and typhasterol to castasterone which have been reported to be catalyzed by cytochrome P450s, CYP85A1 of tomato and CYP92A6 of pea, respectively. The activities of these enzymes require molecular oxygen as well as NADPH as a cofactor. The enzyme activities were strongly inhibited by carbon monoxide, an inhibitor of cytochrome P450, and this inhibition was recovered by blue light irradiation in the presence of oxygen. Commercial cytochrome P450 inhibitors including cytochrome c, SKF 525A, 1-aminobenzotriazole and ketoconazole also inhibited the enzyme activities. The present work presents unanimous enzymological evidence that cytochrome P450s are responsible for the synthesis of brassinolide from castasterone as well as of castasterone from typhasterol and 6-deoxocastasterone, which have been deemed activation steps of BRs.     

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.