A monoclonal antibody raised to rat liver cytochrome P-448 (form c) which recognises an epitope common to many other forms of cytochrome P-450

Boobis, Alan R.; McQuade, Jamie; Sesardic, Dorothea; Robson, Richard T.; Hayward, Chris; Lock, Edward A.; Elcombe, Cliff; Rose, Michael S.; Davies, Donald S.

doi:10.1016/0006-2952(85)90633-1

Cited by 23 publications

(9 citation statements)

References 43 publications

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“…The hepatic expression of PAH-inducible forms of cytochrome P450 (products of the P450IA gene sub-family) has been investigated in man, using a monoclonal antibody (MAb 3/4/2) which, as previously reported (Boobis et al, 1985b), was raised to rat cytochrome P450c. This antibody recognises analogous form(s) of cytochrome P450 in many other species, including man .…”

Section: Discussionmentioning

confidence: 96%

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A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O‐deethylation of phenacetin and is inducible by cigarette smoking.

Sesardic

Boobis

Edwards

et al. 1988

Brit J Clinical Pharma

214

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1. In previous studies (Boobis et al., 1985b) it was shown that a monoclonal antibody (MAb 3/4/2), raised against rat cytochrome P450 form c, reacts with an isoenzyme(s) of cytochrome P450 in human liver. It was predicted that the epitope with which this antibody reacts should be present on both isoenzymes of the P450IA gene sub‐family (the orthologues of forms c and d) in man (Edwards et al., 1987). 2. This antibody was used to probe 45 different samples of human liver, by the technique of Western blotting. With one exception, all of the samples contained immunoreactive protein, a single band at Mr 54,000 (orthologous to rat form d), which ranged in content from less than 0.5 to 33.5 pmol mg‐1 microsomal protein. The content of the human orthologue of form c was below 0.5 pmol mg‐1, the limit of detection of the assay. 3. Thirteen of the samples were from patients of known smoking status. Immunoreactive P450 content was 3.5‐fold higher, and phenacetin O‐deethylase activity was four‐fold higher, in the smokers than in the non‐smokers. 4. There was a highly significant correlation between the amount of immunoreactive cytochrome P450 and the high affinity component of phenacetin O‐deethylase activity in both smokers and non‐smokers. 5. It is concluded that the high affinity component of phenacetin O‐deethylase activity in man is catalysed by the orthologue of rat cytochrome P450d, and that this isoenzyme is inducible by cigarette smoking. 6. In a number of previous publications it has been suggested that there is an association between the poor metaboliser (PM) phenotype for debrisoquine and impaired phenacetin O‐deethylation. In the present study it was shown that not all subjects PM for debrisoquine are poor metabolisers of phenacetin.

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Section: Discussionmentioning

confidence: 96%

“…Monoclonal antibody (MAb) 3/4/2, the production and characterisation of which have previously been described (Boobis et al, 1985b; (1970). Western blotting of the separated proteins was performed as previously described , using a modification of the method of Towbin et al (1979).…”

Section: Preparation Of Monoclonal Antibodymentioning

confidence: 99%

A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O‐deethylation of phenacetin and is inducible by cigarette smoking.

Sesardic

Boobis

Edwards

et al. 1988

Brit J Clinical Pharma

214

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“…The production, purification, and characterization of the specificity of the antibodies used in this study (MAb 107 and 3/4/2) have been described previously (30,31). These antibodies were derived from the fusion of the mouse myeloma cell line P3.NSl.l-Ag4-1 (NS1 cells) with splenic lymphocytes obtained from Balblc mice immunized with preparations of either CYPlAl or 1A2, partially purified from the livers of MC-treated rats or rabbits, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Microsomal proteins were separated by SDS-PAGE with 9% polyacrylamide and electrophoretically transferred to 0.45-pm nitrocellulose membranes as described by Boobis et al (31). Dot-blotting was performed with a Bio-Dot microfiltration apparatus from Bio-Rad Laboratories (Hemel Hempstead, UK).…”

Section: Methodsmentioning

confidence: 99%

Ontogenetic development of the distribution of constitutive and 3-methylcholanthrene-induced CYP1A1 and CYP1A2 in rabbit liver.

Rich

Foster²,

Edwards³

et al. 1993

J Histochem Cytochem.

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We investigated the expression, distribution, and inducibility of 3-methylcholanthrene (MC)-inducible P450 enzymes, CYPlAl and 1A2, in livers of rabbits at different stages of development, ranging from 4 days before birth (-4 days of age) to adulthood. These enzymes were identified by immunoblotting and immunocytochemistry and quantified by dot-blotting, utilizing previously characterized monoclonal antibodies, 107 and 31412, specific for CYPIA2 and both CYPlAl and 1A2, respectively, and a polydonal antibody that recognizes both enzymes. Expression of CYPIA2 is always greater than that of CYPlAl in livers of untreated rabbits, regardless of age. Moreover, immunocytochemistry showed that CYPlAl is evenly distributed throughout the liver at all ages, whereas CYPIA2 is highly localized to only a few scattered cells at 1 day before birth. More hepatocytes express this enzyme perinatally. By 6 days of age, expression of CYPIA2 is confined to a narrow band of centrilobular ceb, but with increasing age the enzyme is expressed in more hepatocytes until weaning, when all hepatocytes are posi-

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“…production of a monoclonal antibody (designated 3/4/2) which was clearly specific for the target P450 in the rat, The role that P450 plays in the metabolic activation of carcinogenic chemicals is well established and has although it was not inhibitory [36]. Fortunately, this antibody did recognise a single band in Western blotting been reviewed by a number of authors [24][25][26].…”

Section: Launched a Dramatic New Line Of Research Intomentioning

confidence: 99%

Dissecting the function of cytochrome P450

Boobis

Edwards

Adams

et al. 1996

Brit J Clinical Pharma

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1The role that P450 plays in the metabolism of drugs and other chemicals is often pivotal in determining the duration and magnitude of their biological effects, so that it is important to identify the specific forms of the enzyme involved. 2The similarity between different forms of P450 is such that polyclonal antibodies raised by conventional means, even against a homogeneous preparation of the enzyme will often react with several different P450s. 3Whilst monoclonal antibodies are often more specific, their selectivity cannot be determined prior to their use. 4The use of short sequences of amino acids (4–7 residues) predicted to occur in surface loop regions of the protein as immunogens, affords a means of targeting antibodies to specific forms of P450. 5The terminal amino acid of a peptide, here the C‐terminal residue, is very immunodominant in determining the specificity of the resultant antibody. Hence, antibodies against the C‐terminal residues of P450 have a high affinity and, in most cases, will be specific. 6Application of anti‐peptide antibodies to the major forms of P450 in human liver revealed the anticipated interindividual variation. However, evidence was found that the polymorphism in CYP3A5 expression is quantitative rather than qualitative. 7A putative pro‐inhibitory region on the surface of mammalian P450, possibly involved in intramolecular electron transport, has been identified, using a combination of directed anti‐peptide antibodies and sequence alignment based on secondary structure. 8Antibodies directed against defined regions of P450 enzymes are proving invaluable in exploring the regulation, specificity and function of these key enzymes.

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A monoclonal antibody raised to rat liver cytochrome P-448 (form c) which recognises an epitope common to many other forms of cytochrome P-450

Cited by 23 publications

References 43 publications

A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O‐deethylation of phenacetin and is inducible by cigarette smoking.

A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O‐deethylation of phenacetin and is inducible by cigarette smoking.

Ontogenetic development of the distribution of constitutive and 3-methylcholanthrene-induced CYP1A1 and CYP1A2 in rabbit liver.

Dissecting the function of cytochrome P450

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