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R J Edwards

Publications and source records attributed to R J Edwards.

At least 91 records · Page 5Linked to original sources
PubMed1996

Uptake and intracellular trafficking of asialoglycoprotein-polylysine-DNA complexes in isolated rat hepatocytes.

Receptor-mediated gene delivery has been reported for a number of different receptor systems although the intracellular fate of such systems has not been systematically investigated. In this study, we have determined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL296) was ionically complexed with pSV-CAT DNA at a molar ratio of 10:1. The resulting complex inhibited 125I-ASGP binding to rat hepatocytes but ASGP only partially inhibited the binding of complex. The ASGP-independent binding was due to the interaction of the PLL component of the complex with plasma membranes and could be minimised by replacing PLL296 with PLL19. Following internalisation, ASGP was cleaved from the complex and translocated to the lysosomes where it was degraded. The DNA, however, remained in an intracellular compartment that cosedimented with plasma membranes in Percoll density gradients. This study shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself, and second that the differential sorting of the two cleaved molecules leads to a rapid intracellular compartmentalisation of the DNA. Controlled release from this compartment may be a means for prolonged gene expression in gene therapy protocols.

Animals↗
PubMed1995

Distribution and induction of CYP3A1 and CYP3A2 in rat liver and extrahepatic tissues.

Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.

Amino Acid Sequence↗
PubMed1995

Enoxacin is an inducer of CYP1A2 in rat liver.

The induction of cytochrome P450 by enoxacin, ciprofloxacin, and ofloxacin was investigated in female Wistar rats. Animals were treated orally with daily doses ranging from 10 to 400 mg enoxacin per kg body wt, 400 mg ciprofloxacin, or 400 mg ofloxacin per kg body wt for up to 7 days. Activities of methoxyresorufin O-demethylase (MROD) and ethoxyresorufin O-deethylase (EROD) were determined fluorimetrically in hepatic microsomes. MROD activity was increased 2.6-fold after treatment with 100 mg enoxacin per kg body wt for 7 days. Lower doses of enoxacin did not induce MROD activity significantly. Antipeptide antibodies directed specifically against different rat cytochrome P450 enzymes demonstrated that CYP1A2, but not CYP1A1, was induced in rats treated with enoxacin. After ciprofloxacin or ofloxacin treatment, no induction of MROD or EROD activity was observed. Neither ciprofloxacin nor ofloxacin caused any change in CYP1A1 or CYP1A2 apoprotein levels. Further investigations with antipeptide antibodies showed that there was no induction of CYP2B1, CYP2B2, CYP2E1, CYP3A1, CYP3A2, CYP4A1, or CYP4A2 following treatment with enoxacin, ciprofloxacin, or ofloxacin. It is concluded that enoxacin, but not ciprofloxacin or ofloxacin, is an inducer of CYP1A2 in rat liver.

Animals↗
PubMed1995

Extra-nuclear location of histones in activated human peripheral blood lymphocytes and cultured T-cells.

Dextrin-2-sulphate (D2S) is a sulphated polysaccharide which inhibits human immunodeficiency virus type 1 infection of T-cells by binding to the cell surface. During our investigations of the nature of this interaction, a cell membrane fraction was prepared by ultracentrifugation from the T-cell line, HPB-ALL. Separation of membrane proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and analysis for binding proteins using ligand blotting showed that 3H-D2S bound, in a saturable and displaceable manner, to two regions corresponding to molecular weights of 14,000-18,000 and 28,000-32,000. The N-terminal sequences of two of the major protein components in the 14,000-18,000 region were consistent with those of histones H2B and H3. The presence of histone H2B in the cell membrane preparation was confirmed by immunoblotting and enzyme-linked immunosorbent assay using a specific antibody. Histone standards were used to determine the level of each histone in the cell membrane fraction. In addition, the binding of 3H-D2S to purified histone standards was quantified. These results show that all of the binding of 3H-D2S to proteins in the 14,000-18,000 region of the cell membrane preparation can be attributed to the histones present. In contrast to HPB-ALL cells, a cell membrane fraction from freshly isolated human peripheral blood lymphocytes contained very low levels of histones. However, after culture with phytohaemagglutinin for 3 days the cell membrane fraction contained greatly increased levels of histones. To exclude the possibility of contamination of the cell membrane preparation with histones derived from the nucleus, cell membranes were also prepared using an affinity-based method using polyethyleneimine-cellulose. Immunoblotting of adsorbed plasma membranes showed the presence of histone H2B. SDS-polyacrylamide gels stained for protein also indicated that the preparation contained histones H1, H2A, H3 and H4. In further experiments whole cells were used to avoid contamination from nuclear proteins. Lactoperoxidase mediated 125I labelling, a method specific for radiolabelling cell surface proteins, confirmed the presence of histones H2B, H3 and H4 on the surface of HPB-ALL cells. Also, incubation of HPB-ALL cells or phytohaemagglutinin-activated peripheral blood lymphocytes with D2S caused displacement of histones from the cell surface into the supernatant without altering cell viability. In addition, immunocytochemistry of freshly isolated peripheral blood lymphocytes showed that histone H2B was located predominantly in the nucleus. However, in phytohaemagglutinin-activated peripheral blood lymphocytes immunoreactive material was also prominent in the endoplasmic reticulum and on the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression and distribution of cholesterol 7 alpha-hydroxylase in rat liver.

The hydroxylation of cholesterol by cholesterol 7 alpha-hydroxylase (CYP7) to 7 alpha-hydroxycholesterol is the rate-limiting step in the production of bile acids. An anti-peptide antibody targeted to the C-terminus of CYP7 was produced by immunising rabbits with the synthetic peptide Tyr-Lys-Leu-Lys-His. The antibody bound to a single band of 54 kDa from rat hepatic microsomal fractions. The intensity of the band was subject to a diurnal variation and showed a significant increase (P < 0.01) in apoprotein at night. Treatment of rats with cholestyramine increased CYP7 apoprotein in the morning (P < 0.005) and at night (P < 0.005), but diurnal variation was maintained. CYP7 catalytic activity, measured using a specific gas chromatography/mass spectrometry assay, showed similar changes in the pattern of diurnal variation and induction. The distribution of CYP7 in rat liver tissue sections was investigated by immunocytochemistry. In sections from rats treated with cholestyramine, there was an even distribution of immunoreactivity, except in the proximal perivenous hepatocytes where immunoreactivity was slightly more intense. A similar distribution was found in sections from untreated rat liver, except immunoreactivity was overall slightly less intense. This study shows that the C-terminus of CYP7 is a useful epitope for the targeting of anti-peptide antibodies.

Acetates↗
PubMed1995

Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes.

An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290-296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse. A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin. Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse. To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay. These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn. Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins. However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus. Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290-296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2. As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6. The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences. All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human. It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.

Amino Acid Sequence↗
PubMed1995

Oval cell differentiation into hepatocytes in the acetylaminofluorene-treated regenerating rat liver.

When hepatocyte regeneration after a two-thirds partial hepatectomy (PH) in rats is blocked by oral gavage of acetylaminofluorene, a proliferation of ductular cells ensues that results in a profusion of neoductules radiating from each portal tract. To examine the possibility that this population of newly emerging cells harbors cells capable of differentiating into hepatocytes, we have looked in these cells for expression of functional markers of hepatocyte commitment at both the RNA and protein levels. Expression of albumin and alpha-fetoprotein (alpha-FP) messenger RNA (mRNA) transcripts were sought in situ using antisense riboprobes, and the expression of a number of cytochrome P450 enzymes was examined immunohistochemically. Before any signs of differentiation the ductular cells strongly expressed cytokeratins 7, 8, 18, and 19 in the same manner as authentic bile ducts, but unlike the latter also expressed vimentin. In situ hybridization studies showed that small bile ducts close to the limiting plate, as well as the newly formed ducts, expressed albumin and alpha-fetoprotein messenger RNAs, and immunocytochemistry showed that the distribution of the respective proteins was similar. Beginning at 1 week after partial hepatectomy, areas of differentiation could be found in the new ducts, with cells resembling either columnar intestinal-type epithelia or hepatocytes. Intestinal-like cells expressed neither albumin, alpha-FP, nor cytochrome P450 enzymes, whereas ductular cells appearing like hepatocytes with the typical membranous distribution of cytokeratin 8 strongly expressed a variety of cytochrome P450 enzymes normally associated with functional hepatocytes. These observations further support the belief that reactive ductules, sprouted from small ducts, can represent an adaptive response of the liver to replenish lost hepatocytes, although some of the newborn cells appear to differentiate along intestinal lines.

2-Acetylaminofluorene↗
PubMed1995

The resistant hepatocyte model of carcinogenesis in the rat: the apparent independent development of oval cell proliferation and early nodules.

The early cellular changes in the Solt-Farber resistant hepatocyte model of carcinogenesis have been studied to clarify the relationship of oval cell proliferation to the development of early hepatocyte nodules. Cellular proliferation, intermediate filament profiles and the expression of specific cytochrome P450 enzymes were examined. At 24 h after partial hepatectomy (PH) many of the bile ductular cells were in S phase, but over the next few days DNA synthesis progressively decreased in the portal bile ducts and was more common in arborizing ductules (oval cells) radiating from the portal areas. These cells strongly expressed cytokeratins 8 and 19 and vimentin, and from 1 week after PH they frequently underwent differentiation either into hepatocytes, expressing cytochrome P450 enzymes, or into intestinal-type cells. Five days after PH, numerous basophilic foci were discernible, and these expanded rapidly. The ductular cells swirled around the foci, but their antigenic profile clearly indicated that these cells were not involved in the development of these early nodules. In normal hepatocytes, cytokeratin 8 immunoreactivity was distinctly membranous in location, and could only be readily detected in periportal hepatocytes. In the basophilic hepatocyte foci, overexpression of cytokeratin 8 was consistently associated with cells organizing into acini, with expression reminiscent of authentic bile ducts, possibly indicating a structure-function relationship. In conclusion, early foci and nodules in this model are derived from resistant hepatocytes and not ductular oval cells, the latter being a facultative multipotential stem cell compartment.

Acetates↗
PubMed1995

Determination of human hepatic cytochrome P4501A2 activity in vitro use of tacrine as an isoenzyme-specific probe.

Oxidative metabolism of the cognition activator tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is thought to be catalyzed by cytochrome P4501A2 (CYP1A2). In this study, the use of tacrine as a specific substrate to measure CYP1A2 activity in vitro was investigated. Tacrine metabolism was assessed in 16 human liver microsomal samples. Initially, the percentage conversion of tacrine to stable metabolites (i.e. 1-, 2-, 4-, and 7-hydroxytacrine) at a single time point was correlated with levels of CYP1A2 apoprotein. Apoprotein was detected by immunoquantification using a monospecific CYP1A2 antipeptide antibody. Significant correlations were seen between CYP1A2 content and the degree of 1-hydroxylation (r = 0.81, p < 0.001), 7-hydroxylation (r = 0.70, p < 0.001), and metabolism to all stable products (r = 0.82, p < 0.001). The major metabolite formed in all livers was 1-hydroxytacrine. The conversion of tacrine to this metabolite was examined in more detail. The rate of formation varied from 19.2 pmol min-1 mg-1 to 101.0 pmol min-1 mg-1. There was a significant correlation (r = 0.84, p < 0.001) between the rate of formation and CYP1A2 levels. Tacrine metabolism was also compared with the rate of formation of 3-methylxanthine, from theophylline, a reaction previously shown to be catalyzed by CYP1A2. Significant correlations were found between 3-methylxanthine formation and all quantified tacrine metabolites. The rate of 3-methylxanthine generation also correlated with CYP1A2 apoprotein levels. It is concluded, therefore, that tacrine is a valuable probe for the determination of human hepatic CYP1A2 activity in vitro.

Amino Acid Sequence↗
PubMed1995

Enzymatic studies of the activation of heterocyclic food mutagens in man.

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are amongst the most abundant of the heterocyclic aromatic amines formed during the cooking of beef. Both compounds are genotoxic and carcinogenic in rodents. These effects are manifested only after activation of the amines by P450. Human liver is very active at converting these amines to mutagenic products. Studies in vitro have established that, for both amines, mutagenicity with human liver microsomes is entirely via the N-hydroxylamine, essentially the only oxidation product of either amine. Both N-hydroxylation and mutagenicity of the amines can be almost completely inhibited by furafylline, a potent and highly selective inhibitor of CYP1A2 in man. These data, together with the work of others, show that the N-hydroxylation and hence the mutagenicity of both MeIQx and PhIP in man is catalyzed almost exclusively by CYP1A2. Liver from cynomolgus monkeys, unlike that from humans and marmosets, is very poor at activating MeIQx or PhIP to mutagenic products. Studies with anti-peptide antibodies of defined specificity revealed that this is due to the absence of CYP1A2, suggesting that this species should not be used to assess the possible risk posed to man by these amines. The marmoset would be a better species for this purpose.

Animals↗
PubMed1994

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine is a potent mutagen in the mouse small intestine.

Mutations in long lived stem cells are critical events in carcinogenesis. The Dlb-1 assay detects intestinal stem cell mutation at the Dlb-1 locus in Dlb-1a/b heterozygous mice by visualizing mutated clones of epithelial cells in situ which do not bind the lectin Dolichos biflorus agglutinin. We have used this assay to show that the food-derived heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a potent intestinal mutagen when administered either i.p. or p.o. This contrasts with the inactivity of the structurally related mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the assay which we have described previously. Immunocytochemical localization of the P-450 enzyme CYP1A2, which is responsible for the primary activation of these mutagens, shows that in untreated mice it is present in liver hepatocytes and in occasional villus epithelial cells but is absent from the target intestinal stem cell population. In addition, liver microsomes, unlike intestinal microsomes, were able to convert PhIP to the proximate mutagen N-hydroxy-PhIP. CYP1A2 immunoreactivity in beta-napthoflavone-induced animals was elevated in liver hepatocytes and increased to a lesser extent in duodenal villus epithelial cells. Treatment with beta-napthoflavone produced an unexpected 46% decrease in the number of Dlb-1 mutations in response to PhIP. Following treatment with PhIP, there was no difference in the number of Dlb-1 locus mutations between the proximal and distal ends of the small intestine in uninduced animals, indicating that the bile duct is unlikely to be responsible for transport of mutation inducing metabolites of PhIP to the small intestine. Our results demonstrate that metabolic activation of an indirect acting genotoxic agent can occur at a site other than the target tissue, and absence of the enzymes required for activation of a mutagen does not necessarily protect that tissue from its genotoxic effects.

Animals↗
PubMed1994

Contribution of CYP1A1 and CYP1A2 to the activation of heterocyclic amines in monkeys and human.

The activation of heterocyclic amines to mutagenic products by hepatic microsomal fractions from cynomolgus monkey, marmoset monkey and man was compared with the respective levels of cytochrome P450 enzymes CYP1A1 and CYP1A2. The rate of activation of 2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to mutagens by hepatic microsomal fraction from cynomolgus monkey was very low. This was associated with a lack of constitutive expression of CYP1A1 and CYP1A2. In contrast, human hepatic microsomal fraction readily activates these heterocyclic amines and this is associated with constitutive expression of CYP1A2. Treatment of cynomolgus monkey with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a very modest induction of CYP1A2, and a small increase in the activation of MeIQx and IQ. However, there was marked induction of CYP1A1 which was accompanied by > 10-fold increases in PhIP activation and 7-ethoxyresorufin O-deethylase (EROD), 7-methoxyresorufin O-demethylase (MROD) and aryl hydrocarbon hydroxylase activities. Following treatment of cynomolgus monkey with 3-methylcholanthrene, induction of CYP1A1, but not CYP1A2, was evident. In untreated marmoset monkey the activations of MeIQx and PhIP, as well as phenacetin O-deethylase, EROD, MROD and aryl hydrocarbon hydroxylase activities, are similar to those in man, although the activations of IQ and coumarin 7-hydroxylase activity are lower than in man. The presence of constitutive CYP1A2, and the absence of CYP1A1, in the liver of this species correspond to the situation in man. Treatment of marmoset monkey with TCDD results in increased CYP1A2 levels (4-fold), accompanied by proportional increases in the activation of MeIQx and IQ and phenacetin O-deethylase, EROD and MROD activities. The activation of PhIP is increased disproportionately, by 8-fold, most likely due to the activity of CYP1A1 which is also induced by TCDD in this species. Overall, the hepatic metabolism of heterocyclic amines by CYP1A enzymes in the untreated marmoset monkey resembles that in human more closely than that in the cynomolgus monkey. Therefore, marmoset monkey may be a more suitable model than the cynomolgus monkey for carcinogenicity studies involving MeIQx and PhIP, but not IQ.

Animals↗
PubMed1994

Infection by HIV-1 blocked by binding of dextrin 2-sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T-cells.

1. Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1. Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested. 2. In saturation binding studies, [3H]-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells. It bound to other human T-cell lines in a similar manner. 3. There was very little binding of [3H]-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of [3H]-D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of [3H]-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells. The Kd of binding did not change significantly during the course of these experiments.4. [3H]-D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h.5. These results suggest that there is a relationship between the expression of the [3H]-D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV- 1.

Anticoagulants↗
PubMed1993

Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man.

An anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283-294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286-290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.

Amino Acid Sequence↗