Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes

doi:10.1016/0003-9861(79)90122-X

Archives of Biochemistry and Biophysics

Volume 192, Issue 2, February 1979, Pages 524-532

https://doi.org/10.1016/0003-9861(79)90122-X Get rights and content

Abstract

Antibodies prepared against purified cytochrome P-450 and P-448 from phenobarbital- and 3-methylcholanthrene-pretreated rats have been shown to recognize several forms of hepatic cytochrome P-450 (P. E. Thomas, A. Y. H. Lu, D. Ryan, S. B. West, J. Kawalek, and W. Levin, 1976, Mol. Pharmacol.12, 746–758). These antibodies have been made monospecific for a single form of cytochrome P-450 by immunoadsorption with partially purified solid-phase cytochrome P-450 from rats treated with a different inducer than that used for isolation of the antigen. Each monospecific antibody did not react with different forms of cytochrome P-450 present in the heterologous antigen preparation. These monospecific antibodies, covalently bound to Sepharose, were used to purify the antigens (catalytically inactive) from microsomes in a single step. The high specificity of these antibodies for a single form of cytochrome P-450 was used to quantitate two forms of cytochrome P-450 in rat liver microsomes by radial immunodiffusion. The percentage of the total cytochrome P-450 in microsomes that is represented by each of these two forms of cytochrome P-450 varied from 3 to 89% depending on the xenobiotic pretreatment of the rats.

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The sera were compared, and the best serum was used based on specificity and titer. Rabbit anti-27C1 antibody was purified by affinity chromatography with purified recombinant protein (84). Purified 27C1 was coupled to CNBr-activated Sepharose (GE Healthcare) according to the manufacturer's instructions.
Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all-trans-retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5–2.3 (on k_cat/K_m) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.
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Cytochrome P450 (P450) enzymes play dominant roles in the metabolism of toxic chemicals, as well as drugs, steroids, and several other important groups of compounds. The human genome contains 57 P450 (CYP) genes, about one-fourth of which can play potential roles in xenobiotic biotransformation. The P450s in experimental animal models are similar but not identical to the human P450s, in the context of gene organization, primary sequences, regulation, and catalytic activities. The major mechanism of P450 regulation is transcriptional control by heterodimeric receptors. The crystal structures of several important human P450s have now been determined, revealing similar folds, variability of certain regions giving rise to a 5-fold range in size of the active site, and plasticity that allows at least some ligands to bind with elements of an induced-fit process. The chemistry of P450 reactions is understood in the context of high-valent iron–oxygen chemistry and involves abstraction of hydrogen atoms or non-bonded electrons. Rate-limiting steps in catalysis vary with individual P450s and substrates. P450s play important roles in modulating the toxicity of chemicals, either detoxicating dangerous materials or activating non-toxic compounds to reactive forms. For instance, elimination of P450 2e1 (and 1a2) in mice greatly attenuates the in vivo toxicity of acetaminophen. P450s have also been implicated in several toxic drug–drug interactions in humans. Another area of toxicity in which P450s are important is chemical carcinogenesis, with good evidence for P450 contribution in animal models.
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Citation Excerpt :
Protein concentrations were estimated using a bicinchroninic acid (BCA) method according to manufacturer’s recommendations (Pierce, Rockford, IL). SDS–polyacrylamide gel electrophoresis/immunoblotting (“Western blotting”) was done as described elsewhere [36,37] using an immunoglobulin G fraction (0.1 mg protein/ml) of rabbit antiserum raised against purified rat P450 1A1 [38] and cross-absorbed against rat liver microsomes prepared from untreated rats coupled to Sepharose to remove antibodies reacting with other P450s [39]. Blots were developed with 4-chloro-1-naphthol and imaging was done using a flatbed scanner and the program ImageQuant (Molecular Dynamics, Sunnyvale, CA; now available from Amersham Biosciences, Piscataway, NJ), with the same size of rectangle used for every band.
Indigo and indirubin have been reported to be present at low levels in human urine. The possibility that indigoids are physiological ligands of the aryl hydrocarbon receptor (AhR) has been suggested by initial studies in yeast, where indirubin was found to be 50 times more potent than 2,3,7,8-tetrachlorodibenzo[p]dioxin (TCDD), and indigo was found to be equipotent. To demonstrate that these indigoids are bona fide agonists in mammalian systems, we employed a number of in vitro and in vivo measures of AhR agonist potency. In a hepatoma cell reporter system, indigo yielded an EC₅₀ of ∼5 × 10⁻⁶ M (indirubin 3^′-oxime EC₅₀ ∼5 × 10⁻⁷ M, indirubin EC₅₀ ∼1 × 10⁻⁷ M). A comparison of these EC₅₀ values with that of 2,3,7,8-tetrachlorodibenzofuran (TCDBF) (∼3 × 10⁻⁹ M) indicated that these compounds are less potent than classic halogenated-dibenzofurans or -dibenzo-p-dioxins. Competitive binding assays for AhR occupancy showed similar IC₅₀ values for indirubin and TCDBF (∼2 × 10⁻⁹ and 5 × 10⁻⁹ M), with the IC₅₀ values of indigo and indirubin 3^′-oxime being ∼10-fold higher. When rats were treated with these indigoids in the range of 1.5–50 mg/kg, induction of hepatic cytochrome P450 1A1 was detected. Differences in the rank-order of potency observed in vivo and in vitro could, in part, be explained by metabolism. Although their biological potencies are not as high as has been previously suggested, collectively the results show that these indole-derived pigments are agonists of AhR in vivo. The in vivo results suggest that solubility, distribution, and metabolism influence the response to the compounds.
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Expression and inhibition of cytochrome P450 (CYP) isozymes capable of forming an orphenadrine metabolite complex were studied in microsomes of untreated and inducer-treated male and female rats. High levels of complex-forming isozymes were found in microsomes of untreated male as compared to female rats. Treatment of male rats with several P450 inducers did not considerably increase the extent of in vitro complex formation. In female rats, however, phenobarbital or dexamethasone treatments led to pronounced induction. The isozyme specifity of complex formation was investigated by several approaches including: 1. inhibition by orphenadrine of isozyme-specific P450 activities, such as hydroxylation of testosterone, O-dealkylation of pentoxy- and ethoxyresorufin and complex formation with triacetyloleandomycin (TAO), 2. inhibition of orphenadrine complex formation by metyrapone, TAO, and cimetidine, and 3. correlation of complex levels with immunochemically, enzymatically, or spectroscopically determined amounts of P450 isozymes. Our data suggest that CYP2C11, a CYP3A isozyme and an unidentified P450 species are involved in complex formation with orphenadrine, but exclude the involvement of CYP1A1/2 and CYP2B1/2. The capability of CYP2C11 to form a metabolite complex with orphenadrine is strongly suggested for the following reasons: 1. Efficient inhibition of testosterone 2α- and 16α-hydroxylation by complex formation with orphenadrine in microsomes of untreated male rats, 2. high expression of orphenadrine-complexing isozymes in untreated male compared to female rats, 3. specific inhibition of in vitro complex formation by cimetidine, 4. suppression of complex-forming isozymes by 3-methylcholanthrene and β-naphthoflavone, and 5. concomitant induction of complex-forming isozymes, immunodetectable CYP2C11, and testosterone 2α-hydroxylase by stanozolol. That at least one, but not all, CYP3A isozymes is involved in complex formation is concluded from inhibition experiments with TAO that show that orphenadrine complexation can be significantly inhibited in microsomes of dexamethasone-treated, but not in microsomes of untreated rats. Furthermore, complex formation with TAO is not inhibited by orphenadrine in microsomes of phenobarbital (PB)-treated rats. In PB-treated female rats, a further unidentified complex-forming isozyme can be detected that is not inhibited by complex formation with TAO.
Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: Studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in escherichia coli
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Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues. In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides. FMO1 and FMO2 exhibited high affinities for these substrates (K_m<10 μM), in contrast to the low-affinity FMO3 form for which K_m values ranged between 100 and 280 μM. FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used. The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms. To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes. We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms. Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E. coli microsomes proceeded with apparent K_ms of 18 and 270 μM, respectively. FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity. Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent K_m values of 11 and 340 μM. The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 μM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM. The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively. Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

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^☆: A preliminary report of this work has been published in abstract form, Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1978) Fed. Proc.37, 766.

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Preparation of monospecific antibodies against two forms of rat liver cytochrome P-450 and quantitation of these antigens in microsomes☆

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