Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450

Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors.     

Preparation and characterization of polyclonal and monoclonal antibodies against human aromatase cytochrome P-450 (P-450AROM), and their use in its purification

Aromatase cytochrome P-450 (P-450AROM) was partially purified from human placental microsomes by hydrophobic affinity chromatography using Phenyl-Sepharose and ion-exchange chromatography on DEAE-cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome P-450 content and displayed a type I difference spectrum upon addition of the substrate androstenedione. When the cytochrome P-450-enriched fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained polyacrylamide gels and injected separately or together into three rabbits. An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the rabbit injected with the 55-kDa band or with both the 50- and 55-kDa bands inhibited aromatase activity of human placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of human fetal adrenal microsomes. In contrast, the serum of the rabbit injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the rabbit immunized with the 55-kDa protein bound specifically to a protein of 55 kDa in human placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of mice immunized against the 55-kDa protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of cytochrome P-450AROM. The finding that cytochrome P-450 and the 55-kDa protein were selectively retained by the affinity column and eluted with NaCl (2 M) and glycine (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified NADPH-cytochrome P-450 reductase and phospholipid, is indicative that the 55-kDa protein is indeed cytochrome P-450AROM. These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for human cytochrome P-450AROM.     

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Synthetic peptide antigens elicit monoclonal and polyclonal antibodies to cytochrome P450 IA2

Two peptide sequences from cytochrome P450 IA2 were synthesized, coupled to ovalbumin and used as antigens to generate anti-peptide monoclonal and polyclonal antibodies. Antisera to both peptides reacted with rat IA2 but not the structurally similar IA1 form as determined by enzyme-linked immunosorbent assay. However, antisera to both peptides detected both rat IA2 and IA1 on immunoblots. In addition immunoblots of human liver microsomes revealed that both antisera recognized human IA2, but not IA1. Monoclonal antibodies generated against one of the peptides recognized rat IA2 and IA1 but did not detect human IA2. These results demonstrate the utility of anti-peptide antisera as a practical approach for the generation of P450 specific antibodies.     

An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops

Hepatic microsomal cytochrome P-450 from the untreated coastal marine fish scup, Stenotomus chrysops, was solubilized and resolved into five fractions by ion-exchange chromatography. The major fraction, cytochrome P-450E (M_r = 54,300), was further purified to a specific content of 11.7 nmol heme/mg protein and contained a chromophore absorbing at 447 nm in the CO-ligated, reduced difference spectrum. NH₂-terminal sequence analysis of cytochrome P-450E by Edman degradation revealed no homology with any known cytochrome P-450 isozyme in the first nine residues. S. chrysops liver NADPH-cytochrome P-450 reductase, purified 225-fold (M_r = 82,600), had a specific activity of 45–60 U/mg with cytochrome c, contained both FAD and FMN, and was isolated as the one-electron reduced semiquinone.Purified cytochrome P-450E metabolized several substrates including 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene when reconstituted with lipid and hepatic NADPH-cytochrome P-450 reductase from either S. chrysops or rat. The purified, reconstituted monooxygenase system was sensitive to inhibition by 100 μM 7,8-benzoflavone, and analysis of products in reconstitutions with purified rat epoxide hydrolase indicated a preference for oxidation on the benzo-ring of benzo[a]pyrene consistent with the primary features of benzo[a]pyrene metabolism in microsomes. Cytochrome P-450E is identical to the major microsomal aromatic hydrocarbon-inducible cytochrome P-450 by the criteria of molecular weight, optical properties, and catalytic profile. It is suggested that substantial quantities of this aromatic hydrocarbon-inducible isozyme exist in the hepatic microsomes of some untreated S. chrysops. The characterization of this aryl hydrocarbon hydroxylase extends our understanding of the metabolism patterns observed in hepatic microsomes isolated from untreated fish.     

Inhibitory domain-specific antibodies to cytochrome P-450scc

Highly specific antibodies to cytochrome P-450scc and its F1 and F2 fragments, representing N- and C-terminal sequences of the hemeprotein respectively, were raised in rabbits. These antibodies were found to be inhibitory (up to 50-90%) for the cholesterol transformation into pregnenolone in the reconstituted system, indicating the involvement of both F1 and F2 domains formed by the respective fragments in monooxygenase catalysis. Cytochrome P-450scc in mitoplasts is not accessible for trypsin as revealed by immunological techniques. However, the treatment of submitochondrial particles with trypsin results in two main fragments identified by immunoblotting in the presence of the monospecific antibodies as F1 and F2 fragments. This indicates that the trypsin sensitive 250-257 region in cytochrome P-450scc molecule connecting both domains is exposed to the matrix side of the inner mitochondrial membrane.     

Preparation and characterization of monoclonal antibodies against a form of hamster liver cytochrome P-450 highly specific to aflatoxin B1

Monoclonal antibodies were prepared against a form of cytochrome P-450 (designated as cytochrome P-450-I) purified from 3-methylcholanthrene-treated hamster livers which is highly specific to aflatoxin B1. The cytochrome P-450-I was detected in ELISA and Western blots in liver microsomes from 3-methylcholanthrene-treated hamsters and also from non-treated and phenobarbital-treated hamsters in smaller amounts. However, none of the liver microsomes from 3-methylcholanthrene-treated rat, rabbit, guinea pig and Suncus murinus contained the cytochrome P-450-I. These results suggest that cytochrome P-450-I is specific to hamster and is induced mainly by 3-methylcholanthrene.     

Radiometric assay for direct quantitation of rat liver cytochrome P-450b using monoclonal antibodies

A simple and sensitive assay has been developed that is capable of detecting as little as 0.2 ng of the major isozyme of cytochrome P-450 (P-450b) isolated from the livers of phenobarbital-induced rats. This assay employs monoclonal antibodies generated against cytochrome P-450b to directly quantify the levels of this enzyme in various tissues. Separation of bound from free labeled antibody is achieved by using 6,9-diaminoacridine lactate (Rivanol). The useful range of the assay is between 1 and 100 ng of P-450b.     

Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450

Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.     

Immunochemical examinations of cytochrome P-450 in various tissues of human fetuses using antibodies to human fetal cytochrome P-450, P-450 HFLa

P-450 HFLa is a form of cytochrome P-450 purified from human fetal livers. The amounts of P-450 HFLa in several fetal tissues were determined immunochemically. Detectable amounts presented in livers, kidneys, adrenals, lungs and some other tissues of human fetuses. The amounts were the highest in livers. Activities of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase in livers but not in adrenals were inhibited by the anti-P-450 HFLa antibodies, probably suggesting that distinct forms of cytochrome P-450 are responsible for the oxidations in livers and adrenals.     

Electron paramagnetic resonance studies on hepatic microsomal cytochrome P-450 from a marine teleost fish

Hepatic microsomal cytochrome P-450 from fish (Stenotomus versicolor), untreated or treated with 3-methylcholanthrene, 5, 6-benzoflavone, or tricaine methanesulfonate, exhibited an absorption maximum at 450 nm when reduced and ligated to CO. Microsomes from all groups exhibited EPR spectra with g values near 2.4, 2.24 and 1.9, yielding crystal field parameters similar to those for cytochrome P-450 from a variety of other sources. Treatment with 3-methylcholanthrene or 5, 6-benzoflavone resulted in elevated levels of aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity yet produced no apparent change in the levels or optical properties of CO-ligated cytochrome P-450. Tricaine methanesulfonate, a common fish anaesthetic, caused a decrease in the levels of fish cytochrome P-450.     

Studies on species cross-reactivity of hemopexin by use of monoclonal and polyclonal antibodies

The extent of immunological cross-reactivity between hemopexins of four species (rat, human, rabbit and chicken) was assessed with four affinity purified polyclonal antibodies and three monoclonal antibodies using RIA, Western blotting and rocket immunoelectrophoresis. Neither the two monoclonal antibodies to rabbit hemopexin (Rb3D11 and Rb3H9), the monoclonal antibody (R4B3) to rat hemopexin nor any of the polyclonal antibodies showed shared antigenic determinants between avian and mammalian hemopexins as judged by RIA or rocket immunoelectrophoresis. Western blotting with polyclonal antibodies revealed some reactivity raising the possibility of a few shared, though distantly related, epitopes. Polyclonal antibodies, raised to the mammalian hemopexins cross-reacted to variable extents with the respective antigens by RIA, results paralleled by data obtained by Western blotting. Anti-rat monoclonal antibodies reacted only with rat hemopexin in Western blots and minimally with rabbit hemopexin in RIA. The anti-rabbit monoclonal antibodies recognized two distinct epitopes one of which is shared with human hemopexin and presumably highly conserved.     

Specificity of antisera directed against rat liver cytochrome P-448

A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b₅, NADH-cytochrome b₅ reductase, NADPH-cytochrome c reductase or epoxide hydrase.     

Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes

Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of azoreductase activity by antibodies against cytochromes P-450 and P-448

Hepatic microsomal azoreductase activity in mice was induced with phenobarbital (PB) and 3-methylcholanthrene (3-MC). Antibodies against cytochrome P-450 inhibited azoreductase activity of PB-treated animals while antibodies against cytochrome P-448 inhibited liver azoreductase activity of 3-MC-treated animals, each by about 90%. These antibodies also inhibited microsomal 7-ethoxycoumarin-O-deethylase activity to the same extent. It is concluded that hepatic microsomal azoreductase activity is almost totally dependent on cytochromes P-450 and P-448 and the contribution, if any, of other microsomal components is negligible.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.