Identification Of Cytochrome P450 Research Articles

Comparative transcriptome and metabolite profiling reveal diverse pattern of CYP-TS gene expression during corosolic acid biosynthesis in Lagerstroemia speciosa (L.) Pers.

Extensive leaf transcriptome profiling and differential gene expression analysis of field grown and elicited shoot cultures of L. speciosa suggest that differential synthesis of CRA is mediated primarily by CYP and TS genes, showing functional diversity. Lagerstroemia speciosa L. is a tree species with medicinal and horticultural attributes. The pentacyclic triterpene, Corosolic acid (CRA) obtained from this species is widely used for the management of diabetes mellitus in traditional medicine. The high mercantile value of the compound and limited availability of innate resources entail exploration of alternative sources for CRA production. Metabolic pathway engineering for enhanced bioproduction of plant secondary metabolites is an attractive proposition for which, candidate genes in the pathway need to be identified and characterized. Therefore, in the present investigation, we focused on the identification of cytochrome P450 (CYP450) and oxidosqualene cyclases (OSC) genes and their differential expression during biosynthesis of CRA. The pattern of differential expression of these genes in the shoot cultures of L. speciosa, elicited with different epigenetic modifiers (azacytidine (AzaC), sodium butyrate (NaBu) and anacardic acid (AA)), was studied in comparison with field grown plant. Further, in vitro cultures with varying (low to high) concentrations of CRA were systematically assessed for the expression of CYP-TS and associated genes involved in CRA biosynthesis by transcriptome sequencing. The sequenced samples were de novo assembled into 180,290 transcripts of which, 92,983 transcripts were further annotated by UniProt. The results are collectively given in co-occurrence heat maps to identify the differentially expressed genes. The combined transcript and metabolite profiles along with RT-qPCR analysis resulted in the identification of CYP-TS genes with high sequence variation. Further, instances of concordant/discordant relation between CRA biosynthesis and CYP-TS gene expression were observed, indicating functional diversity in genes.

Identification of cytochrome P450, odorant-binding protein, and chemosensory protein genes involved in Type II sex pheromone biosynthesis and transportation in the tea pest, Scopula subpunctaria

Sex pheromone-based pest management technology has been widely used to monitor and control insect pests in the agricultural, forestry, and public health sectors. Scopula subpunctaria is a widespread tea pest in China with Type II sex pheromone components. However, limited information is available on the biosynthesis and transportation of Type II sex pheromone components. In this study, we constructed an S. subpunctaria sex pheromone gland (PG) transcriptome and obtained 85,246 transcripts. Cytochrome P450 monooxygenases (CYPs) thought to epoxidize dienes and trienes to epoxides in the PG and odorant-binding proteins (OBPs) and chemosensory genes (CSPs) thought to be responsible for the binding and transportation of sex pheromone components. In present study, a total of 79 CYPs, 29 OBPs and 17 CSPs were identified. We found that SsubCYP341A and SsubCYP341B_ortholog1 belonged to the CYP341 family and were more highly expressed in the PG than in the female body. Of these, SsubCYP341A was the seventh-most PG-enriched CYP in the PG transcriptome. Two CYP4 members, CYP340BD_ortholog2 and CYP4G, were the top two most PG-enriched CYPs. Tissue expression and phylogenetic tree analysis showed that SsubOBP25, 27, and 28 belonged to the moth pheromone-binding protein family; they were distinctly expressed in the antennae and were more abundant in male antennae than in female antennae. SsubCSP16 was distributed into the same clade as CSPs from other moths that showed high binding affinities to sex pheromone components. It indicated that all the above-mentioned genes could be involved in sex pheromone biosynthesis or transportation. Our study provides large-scale PG sequence information that can be used to identify potential targets for the biological control of S. subpunctaria by disrupting its sex pheromone biosynthesis and transportation pathways.

CYPminer: an automated cytochrome P450 identification, classification, and data analysis tool for genome data sets across kingdoms

BackgroundCytochrome P450 monooxygenases (termed CYPs or P450s) are hemoproteins ubiquitously found across all kingdoms, playing a central role in intracellular metabolism, especially in metabolism of drugs and xenobiotics. The explosive growth of genome sequencing brings a new set of challenges and issues for researchers, such as a systematic investigation of CYPs across all kingdoms in terms of identification, classification, and pan-CYPome analyses. Such investigation requires an automated tool that can handle an enormous amount of sequencing data in a timely manner.ResultsCYPminer was developed in the Python language to facilitate rapid, comprehensive analysis of CYPs from genomes of all kingdoms. CYPminer consists of two procedures i) to generate the Genome-CYP Matrix (GCM) that lists all occurrences of CYPs across the genomes, and ii) to perform analyses and visualization of the GCM, including pan-CYPomes (pan- and core-CYPome), CYP co-occurrence networks, CYP clouds, and genome clustering data. The performance of CYPminer was evaluated with three datasets from fungal and bacterial genome sequences.ConclusionsCYPminer completes CYP analyses for large-scale genomes from all kingdoms, which allows systematic genome annotation and comparative insights for CYPs. CYPminer also can be extended and adapted easily for broader usage.

Expression of cytochrome P450 genes in human cord blood hematopoietic stem and progenitor cells (1139.7)

The human cytochrome P450 (CYP) superfamily comprises 57 genes, which encode enzymes important in the biotransformation of numerous endogenous and exogenous compounds. The aim of this study is to characterize the expression of all CYP genes in CD34(+) human cord blood hematopoietic stem and early progenitor cells (CBHSPCs), as a first step toward assessment of their biological functions in these cells. CD34(+) CBHSPCs were purified from umbilical cord blood via antibody affinity chromatography, using magnetically‐assisted chemical separation. Purity of CD34(+) cells was assessed using fluorescence‐activated cell sorting. RNA was isolated from purified CD34(+) cells for RNA‐PCR analysis, using primers specific for each of the 57 CYPs. Of the 57 CYPs, only 14 were detected by RNA‐PCR in CD34(+) CBHSPCs, including CYP2E1, CYP2U1, CYP2R1, CYP2S1, CYP2J2, CYP4F2, CYP20A1, and CYP27A1. CYP identification was confirmed by sequencing analysis of the detected, mRNA‐derived, PCR products. Preliminary quantitative analysis of CYP transcripts using real‐time RNA revealed preferential expression of multiple CYPs, such as CYP2R1, in CD34(+) CBHSPCs, compared to total lymphocytes or CD34(‐) cells from the cord blood. Additional studies on detection of CYP protein expression are underway.Grant Funding Source: Supported by State Scientific Key Projects for New Drug Research and Development(2011ZX09102‐010‐04)

Identification of Cytochrome P450 (CYP) genes in Zhikong scallop (Chlamys farreri)

Cytochrome P450 (CYP) superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop (Chlamys farreri) for CYP genes. In total, 88 non-redundant CYP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYP10 and CYP24 families. In comparison, protostomes (C. farreri, D. pluex, D. melanogaster) contained more CYP genes than deuterostomes (S. purpuratus and vertebrates) in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.

Evaluation of Time-Dependent Cytochrome P450 Inhibition in a High-Throughput, Automated Assay: Introducing a Novel Area Under the Curve Shift Approach

Early in the drug discovery process, the identification of cytochrome P450 (CYP) time-dependent inhibition (TDI) is an important step for compound optimization. Here we describe a high-throughput, automated method for the evaluation of TDI utilizing human liver microsomes and conventional CYP-specific mass spectrometer-based probes in a 384-well format. One of the key differences from other published TDI assays is the use of a shift in area the under curve of the percent activity remaining versus inhibitor concentration plot (AUC shift) rather than the traditional fold-shift in IC50, to determine the magnitude of TDI. An AUC shift of < 15% suggests negative TDI and > 15% suggests potential TDI. This AUC shift was used to achieve quantitative data reporting, even in the case of weak inhibitors for which IC50 values cannot be quantified. An Agilent Technologies BioCel 1200 System was programmed such that the TDI liability of up to 77 test compounds, incubated at four test concentrations, with and without NADPH in the pre-incubation, can be analyzed in a single run. The detailed automated methodology, assay validation, data reporting and the novel TDI AUC shift approach to describe magnitude of TDI are presented.

Identification of cytochrome P450 (CYP) isoforms involved in the metabolism of corynoline, and assessment of its herb–drug interactions

Corynoline, an isoquinoline alkaloid isolated from the genus Corydalis, has been demonstrated to show multiple pharmacological effects including inhibition of acetylcholinesterase, inhibition of cell adhesion, fungitoxic and cytotoxic activity. The present study focused on its metabolism and metabolism-based herb-drug interactions. After corynoline was incubated with human liver microsomes (HLMs) in the presence of NADPH, two metabolites (M-1 and M-2) were formed. Chemical inhibition experiments and assays with recombinant CYP isoforms showed that CYP2C9 was mainly involved in the formation of M-1 and CYP3A4 mainly catalysed the production of M-2. Among seven major CYP isoforms tested, corynoline showed strong inhibitory effects on the activities of CYP3A4 and CYP2C9, with an IC(50) of 3.3 ± 0.9 µm and 31.5 ± 0.5 µm, respectively. Kinetic analysis showed that inhibition of CYP3A4 by corynoline was best fit to a noncompetitive manner with K(i) of 3.2 µm, while inhibition of CYP2C9 by corynoline was best fit to a competitive manner with K(i) of 6.3 µm. Additionally, corynoline exhibited time-dependent inhibition (TDI) toward CYP3A4. The inactivation kinetic parameters (K(I) and k(inact) ) were calculated to be 6.8 µm and 0.07 min(-1) , respectively. These data are of significance for the application of corynoline and corynoline-containing herbs.

2,5-Dimethoxyamphetamine-derived designer drugs: Studies on the identification of cytochrome P450 (CYP) isoenzymes involved in formation of their main metabolites and on their capability to inhibit CYP2D6

The designer drugs 4-methyl-2,5-dimethoxy-amphetamine (DOM), 4-iodo-2,5-dimethoxy-amphetamine (DOI), 4-chloro-2,5-dimethoxy-amphetamine (DOC), 4-bromo-2,5-dimethoxy-amphetamine (DOB), 4-bromo-2,5-dimethoxy-methamphetamine (MDOB), and 2,4,5-trimethoxy-amphetamine (TMA-2) are potent serotonin 5HT 2 receptor agonists and have appeared on the illicit drug market. These drugs are mainly metabolized by O-demethylation or in case of DOM by hydroxylation of the methyl moiety. In an initial activity screening using microsomes of insect cells heterologously expressing human CYPs, CYP2D6 was found to be the only CYP isoenzyme involved in the above-mentioned main metabolic steps whereas the amounts of metabolites formed were very small. As inhibition of CYP2D6 by other amphetamines had been described, the inhibitory effects of the 2,5-dimethoxyamphetamine derivatives were studied using insect cell microsomes with heterologously expressed human CYP2D6 and pooled human liver microsomes (HLM) as enzyme sources and dextromethorphan O-demethylation as probe reaction. All studied drugs were observed to be non-mechanism-based competitive inhibitors of CYP2D6 with inhibition constants ( K i) from 7.1 to 296 μM using recombinant CYP2D6 and 2.7–19.9 μM using HLM. For comparison, the K i values for quinidine and fluoxetine were 0.0092 and 8.2 μM using recombinant CYP2D6 and 0.019 and 0.93 μM using HLM. As the K i values of the drugs were much higher than that of quinidine and, with the exception of DOI, higher than that of fluoxetine, interactions with other CYP2D6 substrates are possible but rather unlikely.

10.1007/s11828-008-1004-1

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.

Identification of cytochrome P450 and glutathione- S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L.

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione- S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CLint) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.3. CLint of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CLint of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CLint of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4.4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

Identification of CYP2D6 alleles by single nucleotide polymorphism analysis using pyrosequencing.

To develop a single nucleotide polymorphism (SNP) analysis for identification of cytochrome P450 (CYP)2D6 alleles by pyrosequencing. Swedish blood donors ( n=282) were typed for a partial CYP2D6 genotype comprising the alleles *1 (wild type), *2 (2850C>T), *3 (2549A>del), *4 (1846G>A) and *6 (1707T>del) using polymerase chain reaction (PCR) and pyrosequencing analysis. CYP2D6*5 (CYP2D6 deleted) was identified using an established long multiplex PCR method. Pyrosequencing is a sequencing-by-synthesis method in which a cascade of enzymatic reactions yields detectable light, which is proportional to the incorporated nucleotides. One feature of typing SNPs by pyrosequencing is that each allelic variant will give a unique sequence. These variants can be readily distinguished by pattern recognition software. Of 281 individuals analysed, 24 (8.5%) were found to be poor metabolisers with two non-functional alleles. This is in the range of 7-10%, previously reported for Caucasians. A total of 126 individuals (45%) had one functional and one non-functional allele and 131 individuals (47%) had two functional alleles. Pyrosequencing was found to be a fast and efficient tool for genotyping. The method is robust, reliable, rapid and has high throughput.

Identification of cytochromes P450 involved in human liver microsomal metabolism of ecabapide, a prokinetic agent

1. In vitro studies identified the hepatic cytochrome P450 (CYP) enzyme(s) involved in the major metabolism of ecabapide in human. 2. Ecabapide mainly underwent N-dealkylation to form M1 and 6-hydroxylation of the benzamide moiety to form M6. 3. The rates of formation of the major metabolites M1 and M6 were significantly correlated with CYP3A-selective testosterone 6beta-hydroxylase activities in 14 different human liver microsomes. The formation of both metabolites was markedly decreased by ketoconazole, miconazole or troleandomycin (TAO), CYP3A-selective inhibitors, and also was inhibited by anti-CYP3A antibodies. 4. These results strongly indicate that CYP3A is the predominant isozyme responsible for the major metabolism of ecabapide in human liver microsomes. 5. Marginal inhibition of the formation of M1 and M6 by nifedipine, a substrate of CYP3A with a Ki>100 muM, suggested that nifedipne has a limited potential to inhibit the major metabolic pathways of ecabapide.

Identification of Cytochrome P450 3A1/2 as the Major P450 Isoform Responsible for the Metabolism of Fentanyl by Rat Liver Microsomes

The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism. Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl.min-1.mg-1 microsomal protein, and the apparent Km values were 32 and 47 microM, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes.

Identification of Cytochrome P450 3A1/2 as the Major P450 Isoform Responsible for the Metabolism of Fentanyl by Rat Liver Microsomes

The metabolism of fentanyl was investigated using rat liver microsomes to determine whether fentanyl is metabolized by rat liver microsomal cytochrome P450 and, if so, which isoform is responsible for the metabolism.Microsomes isolated from rats pretreated with phenobarbital were more active in metabolizing fentanyl than were microsomes from saline controls. The major metabolic pathway of fentanyl was an oxidative N-dealkylation to norfentanyl, which was detected by a gas chromatograph-mass selective detector (GC-MSD) method. The apparent Vm values for microsomes isolated from saline- and phenobarbital-treated rats were 2 and 9 nmol norfentanyl centered dot min-1 centered dot mg-1 microsomal protein, and the apparent Km values were 32 and 47 micro Meter, respectively. Fentanyl metabolism was inhibited by antibodies specific for CYP3A1/2, as well as by chemical inhibitors specific for CYP3A. These results indicate that CYP3A1/2 plays a major role in the oxidation of fentanyl to norfentanyl by rat liver microsomes. (Anesth Analg 1996;82:936-41)

Characterization of six in vitro reactions mediated by human cytochrome P450: application to the testing of cytochrome P450-directed antibodies.

The identification of cytochrome P450 (CYP) isozymes mediating metabolic pathways of drugs has become increasingly important in anticipating pharmacokinetic drug interactions. The activity of individual CYPs can be monitored in vitro with human liver microsomes by means by relatively specific metabolic reactions: for CYP1A1/2, phenacetin O-deethylation; for CYP2C9, phenytoin 4-hydroxylation; for CYP2C19, S-mephenytoin 4'-hydroxylation; for CYP2D6, dextromethorphan O-demethylation; for CYP3A3/4, alprazolam 4-hydroxylation, and for CYP2E1, chlorzoxazone 6-hydroxylation. We determined the kinetic parameters (Vmax, Km) of these reactions and utilized them to test a monoclonal rat liver CYP1A1 antibody, a monoclonal rat liver CYP2E1 antibody, a polyclonal rabbit anti-rat liver CYP3A1 antibody, and a polyclonal goat anti-rat liver CYP2C11 antibody for their specificity and inhibitory capacity. The CYP1A1 monoclonal antibody (MAb), the CYP2E1 MAb, and the CYP3A1 polyclonal antibody (PAb) inhibited only their respective index reactions. The CYP2C11 PAb inhibited both phenytoin 4-hydroxylation and S-mephenytoin 4'-hydroxylation. At a microsomal versus antibody protein mass ratio of 1:15, 4-hydroxyalprazolam formation was reduced by 73.4% of control with the CYP3A1 PAb; 4'-hydroxymephenytoin formation decreased by 66.3% and 4-hydroxyphenytoin formation by 43.4% with the CYP2C11 PAb; phenacetin O-deethylation was reduced by 39.7% with the CYP1A1 MAb, and 6-hydroxychlorzoxazone formation decreased by 30.0% with the CYP2E1 MAb. Thus, all antibodies tested are at least CYP subfamily specific. The PAbs exhibited greater than 60% inhibition versus the CYP3A3/4- and the CYP2C19-mediated reactions, whereas the MAbs produced less than 50% inhibition for their respective index reactions. Because of their limited inhibitory capacity, MAbs may be correspondingly limited as tools for identification of human CYP enzymes via immunoinhibition studies.

Localization and identification of cytochrome P-450 in differentiating rat embryo cells in vitro and in foetal tissue in vivo by immunocytochemistry

The presence of the isoenzymes b, e and c of cytochrome P-450 in foetal rat limb-bud and mid-brain tissue has been investigated in vivo and in micromass cell cultures of limb-bud and mid-brain cells derived from rat embryos by a sensitive immunocytochemical technique. The cytochromes could not be detected by antibody staining at the start of the culture nor in 13-day-old embryos from which cultures were prepared. Two different antibodies directed against cytochrome P-450 revealed the ontogenic profile of the phenobarbitone-inducible b and e forms, which appeared at an earlier stage of development, day 1 of culture (equivalent to day 14 of gestation), than did the 3-methylcholanthrene-inducible c form, which appeared on day 3 of culture (equivalent to day 16 of gestation). These isoenzymes were not tissue specific. Comparison of the localization and intensity of staining of cells cultured in vitro for 5 days with tissue from the equivalent foetal developmental stage (day 18) in vivo revealed the presence of cytochrome P-450 in corresponding areas. In day 18 limb sections, cytochrome P-450 was localized in the perichondrial and myogenic tissue, which corresponded to the cells in the periphery of the chondrogenic foci in vitro. In mid-brain whole tissue, the enzyme was located in connective tissue and neurofibrils, corresponding to cells in the periphery of the foci of neurones in vitro. The correlation between in vitro and in vivo observations from time course, location and quantitative aspects, illustrated that the micromass culture technique is a valid model for metabolism studies with these specific isoenzymes.

Monoclonal antibody directed isolation and amino-terminal sequence analysis of phenobarbital induced rat liver cytochromes P-450

Cytochrome P-450 was isolated from liver microsomes of phenobarbital treated rats by an essentially single step immunopurification with a monoclonal antibody (MAb). The amino terminal sequence of the isolated cytochrome P-450 displayed a microheterogeneity of isozymes related to previously identified phenobarbital induced forms, indicating that each of these isozymes possess the MAb-specific epitope. This monoclonal antibody-based approach to isolation and subsequent identification of cytochrome P-450 may serve to classify different isozymes by their content of epitopes that bind to different MAbs.

Monoclonal antibody-directed immunopurification and identification of cytochromes P-450

Several rat liver cytochromes P-450 have been substantially purified in a one-step immunoadsorption procedure using Sepharose-bound monoclonal antibodies (MAbs) to the major forms of rat liver cytochrome P-450 induced by 3-methylcholanthrene and phenobarbital (MC-P-450 and PB-P-450, respectively). When mixed with solubilized rat liver microsomes the immunoadsorbent based on the MAb to MC-P-450 binds two polypeptides of MW 56,000 and 57,000 while the immunoadsorbent made with the MAb to PB-P-450 absorbs a species of MW 54,000. These polypeptides are readily desorbed by 0.1 M glycine (pH 3.0). Isolation of MAb-specific cytochrome P-450 isozymes by this method has applications in numerous phases of cytochrome P-450 research.

Identification of cytochrome P-450, and its distribution in the membrana granulosa of the preovulatory follicle, using quantitative cytochemistry.

The distribution of cytochrome P-450 (C-P450), an essential component of the oxidative enzyme system involved in the hydroxylation of steroids, was measured by quantitative cytochemistry in cryostat sections of preovulatory follicles obtained from rats in proestrous. The sections were reacted with medium saturated with carbon monoxide with or without the addition of sodium dithionite. The absorbance spectrum was measured from 400 to 500 nm and a difference spectrum calculated by subtracting the extinction obtained from incubations without sodium dithionite from that obtained in the presence of sodium dithionite. A distinct peak at 450nm was recorded in cells of the peripheral portion of the membrana granulosa (MG) but not in those of the cumulus, providing evidence for the presence and differential distribution of C-P450 in the MG of the preovulatory follicle.