scholarly journals Differences in adult and foetal human cytochrome P-450 forms recognized by monoclonal antibodies with specificity for the P450III family

1989 ◽  
Vol 260 (3) ◽  
pp. 635-640 ◽  
Author(s):  
T S Barnes ◽  
M D Burke ◽  
W T Melvin
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Microsomes ◽  
Covalent Modification ◽  
Adult Liver ◽  
Human Adult ◽  
Human Cytochrome ◽  
Foetal Liver ◽  
Cytochrome P 450 ◽  
Murine Monoclonal Antibodies

Six murine monoclonal antibodies raised against a major human adult liver cytochrome P-450 (P-450) of the PCN family (P450III) detected a protein in human foetal liver microsomes (microsomal fractions) which had an approx. 1 kDa higher molecular mass on SDS/polyacrylamide-gel electrophoresis than the protein recognized in human adult liver microsomes. Although each of the antibodies recognized both the adult and the foetal forms, antibody HL4 showed higher affinity for the foetal form. Recognition by the monoclonal antibodies of peptides generated by proteolytic cleavage of microsomal proteins showed different patterns for the adult and foetal forms. It is concluded that the foetal P-450 form recognized by antibodies to the major human adult liver form P450hA7, although structurally similar, is either a distinct P-450 isoenzyme or that the adult and foetal proteins have different covalent modification. Immunoquantification experiments showed comparable levels of the P-450 forms in adult and foetal liver, although there appeared to be less inter-individual variation in foetal livers.

scholarly journals Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16α-carbonitrile-inducible rat cytochromes P-450

1987 ◽  
Vol 248 (1) ◽  
pp. 301-304 ◽  
Author(s):  
T S Barnes ◽  
P M Shaw ◽  
M D Burke ◽  
W T Melvin
Keyword(s):  
Monoclonal Antibodies ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
The Other ◽  
Male Rat ◽  
Rat Liver Microsomes ◽  
Human Cytochrome ◽  
Male Specific ◽  
Cytochrome 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.

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

1987 ◽  
Vol 19 (10-11) ◽  
pp. 537-545 ◽  
Author(s):  
G. I. Murray ◽  
T. S. Barnes ◽  
H. F. Sewell ◽  
S. W. B. Ewen ◽  
W. T. Melvin ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Adult ◽  
Human Cytochrome ◽  
Foetal Liver ◽  
Normal Human ◽  
Cytochrome P 450

scholarly journals Porcine foetal and neonatal CYP3A liver expression

2011 ◽  
Vol 1 (1) ◽  
pp. 1 ◽  
Author(s):  
Marie Louise Hiort Hermann ◽  
Mette Tingleff Skaanild
Keyword(s):  
Cytochrome P450 ◽  
Liver Microsomes ◽  
Cytochrome P450 3A4 ◽  
Chain Reaction ◽  
Hepatic Expression ◽  
Human Cytochrome ◽  
Foetal Liver ◽  
Polymerase Chain ◽  
Cyp3a Enzyme ◽  
Exogenous Substances

Human cytochrome P450 3A7 (CYP3A7) and cytochrome P450 3A4 (CYP3A4) are hepatic metabolising enzymes which participates in the biotransformation of endo- and exogenous substances in foetuses and neonates respectively. These CYP3A enzymes display an inverse relationship: CYP3A7 is the dominant enzyme in the foetal liver, whereas the expression of CYP3A4 is low. After parturition there is a shift in the expression, thus CYP3A7 is down regulated, while the level of CYP3A4 gradually increases and becomes the dominant metabolising CYP3A enzyme in the adult. The minipig is increasingly being used as a model for humans in biomedical studies, because of its many similarities with the human physiology and anatomy. The aim of this study was to examine whether, as in humans, a shift is seen in the hepatic expression of a CYP3A7- like enzyme to cytochrome P450 3A29 (CYP3A29) (an orthologue to the human CYP3A4) in minipigs. This was elucidated by examining the hepatic mRNA expression of CYP3A7 and CYP3A29 in 39 foetuses and newborn Göttingen minipigs using quantitative real time polymerase chain reaction (qPCR). Furthermore the immunochemical level of CYP3A7-LE and CYP3A29 was measured in liver microsomes using western blotting. The expression of CYP3A29 was approximately 9- fold greater in neonates compared to foetuses, and a similar difference was reflected on the immunochemical level. It was not possible to detect a significant level of foetal CYP3A7 mRNA, but immunoblotting showed a visible difference depending on age. This study demonstrates an increase in the expression of CYP3A29, the CYP3A4 orthologue in perinatal minipigs as in humans, which suggests that the minipig could be a good model when testing for human foetal toxicity towards CYP3A4 substrates.

The localization of cytochrome P-450 in normal and pathological human liver by monoclonal antibodies to human cytochrome P-450

1987 ◽  
Vol 15 (4) ◽  
pp. 677-678 ◽  
Author(s):  
G. I. MURRAY ◽  
T. S. BARNES ◽  
S. W. B. EWEN ◽  
H. F. SEWELL ◽  
W. T. MELVIN ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Liver ◽  
Human Cytochrome ◽  
Cytochrome P 450

Metabolism of rifabutin and its 25-desacetyl metabolite, LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole.

1997 ◽  
Vol 41 (5) ◽  
pp. 924-926 ◽  
Author(s):  
C B Trapnell ◽  
C Jamis-Dow ◽  
R W Klecker ◽  
J M Collins
Keyword(s):  
Human Liver ◽  
High Performance ◽  
Opportunistic Infections ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Hepatic Enzymes ◽  
Microsomal Enzymes ◽  
Human Cytochrome ◽  
Immunodeficiency Virus ◽  
Cytochrome P 450

Rifabutin and fluconazole are often given concomitantly as therapy to prevent opportunistic infections in individuals infected with the human immunodeficiency virus. Recent reports have shown increased levels of rifabutin and its 25-desacetyl metabolite, LM565, in plasma when rifabutin is administered with fluconazole. Since fluconazole is known to inhibit microsomal enzymes, this study was undertaken to determine if this rifabutin-fluconazole interaction was due to an inhibition of human hepatic enzymes. The metabolism of both rifabutin and LM565 was evaluated in human liver microsomes and recombinant human cytochrome P-450 (CYP) 3A4 in the presence of fluconazole and other probe drugs known to inhibit CYP groups 1A2, 2C9, 2D6, 2E1, and 3A. The concentrations of rifabutin (1 microg/ml), LM565 (1 microg/ml), and fluconazole (10 and 100 microg/ml) used were equal to those observed in plasma after the administration of rifabutin and fluconazole at clinically relevant doses. High-performance liquid chromatography was used to assess the metabolism of rifabutin and LM565. Rifabutin was readily metabolized to LM565 by human microsomes, but the reaction was independent of NADPH and was not affected by the P-450 inhibitors. No rifabutin metabolism by recombinant CYP 3A4 was found to occur. LM565 was also metabolized by human microsomes to two products, but metabolism was dependent on NADPH and was affected by certain P-450 inhibitors. In addition, LM565 was readily metabolized by the recombinant CYP 3A4 to the same two products found with its metabolism by human microsomes. Therefore, rifabutin is metabolized by human microsomes but not via cytochrome P-450 enzymes, whereas LM565 is metabolized by CYP 3A4.

A human cytochrome P-450 characterized by inhibition studies as the sparteine–debrisoquine monooxygenase

1984 ◽  
Vol 62 (7) ◽  
pp. 860-862 ◽  
Author(s):  
T. Inaba ◽  
M. Nakano ◽  
S. V. Otton ◽  
W. A. Mahon ◽  
W. Kalow
Keyword(s):  
Correlation Coefficient ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Human Cytochrome ◽  
Competitive Inhibitors ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Cytochrome P 450

The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.

scholarly journals Three immunoidentical cytochromes P-450 from liver microsomes of phenobarbital-treated rats

1983 ◽  
Vol 215 (1) ◽  
pp. 83-89 ◽  
Author(s):  
H Sakai ◽  
Y Hino ◽  
S Minakami
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Liver Microsomes ◽  
Polyacrylamide Gel ◽  
Tryptic Digestion ◽  
Post Translational Modification ◽  
Cytochrome P 450

Three forms of cytochrome P-450 were purified to homogeneity from liver microsomes of Wistar-strain rats treated with phenobarbital. They had minimum mol.wts. of 52 000, 53 000 and 54 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and are designated as P-450(L), P-450(M) and P-450(H) respectively. They were shown to be immunoidentical by Ouchterlony double-diffusion analysis. Several criteria, such as isoelectric points, substrate specificities and sensitivities to tryptic digestion, however, indicated that these cytochromes are distinct isoenzymes of cytochrome P-450. Whereas P-450(L) was highly active on various substrates, P-450(H) had generally low catalytic activities, except on aminopyrine. The cytochromes purified by immunoaffinity chromatography using anti-P-450(L) showed a marked variation in their distribution depending on the strain and colony of rat. Limited tryptic digestion of P-450(H) gave one tryptic peptide showing the same mobility as P-450(L) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and their primary structures were very similar. The result suggests a possibility that such limited proteolysis is involved in the post-translational modification of the cytochrome or its destruction.

Monoclonal antibodies against human cytochrome P-450

1986 ◽  
Vol 14 (3) ◽  
pp. 621-621 ◽  
Author(s):  
TRISTAN S. BARNES ◽  
PETER M. SHAW ◽  
M. DANNY BURKE ◽  
WILLIAM T. MELVIN
Keyword(s):  
Monoclonal Antibodies ◽  
Human Cytochrome ◽  
Cytochrome P 450
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