scholarly journals An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity

1990 ◽  
Vol 266 (2) ◽  
pp. 497-504 ◽  
Author(s):  
R J Edwards ◽  
A M Singleton ◽  
B P Murray ◽  
D Sesardic ◽  
K J Rich ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid Sequences ◽  
Enzyme Inactivation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Variable Regions ◽  
Aryl Hydrocarbon ◽  
Hepatic Microsomes ◽  
Peptide Antibody ◽  
A Site ◽  
Cytochrome P 450

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.

Differential sensitivity of hepatic microsomal aryl hydrocarbon hydroxylase to the inhibitory effect of SKF 525-A in rats of varying age, hormonal- and drug-induced status

1982 ◽  
Vol 60 (8) ◽  
pp. 1083-1088 ◽  
Author(s):  
L. S. Gontovnick ◽  
G. D. Bellward
Keyword(s):  
Inhibitory Effect ◽  
Differential Sensitivity ◽  
Male Rats ◽  
Aryl Hydrocarbon Hydroxylase ◽  
Drug Induced ◽  
Adult Rats ◽  
Aryl Hydrocarbon ◽  
Hepatic Microsomes ◽  
Cytochrome P 450

The inhibition of hepatic microsomal aryl hydrocarbon hydroxylase (AHH) activity by SKF 525-A in vitro was studied in hepatic microsomes from rats of varying age, sex hormone status, and drug treatment. The concentration of SKF 525-A required to produce a 50% inhibition of AHH activity, the IC50, was determined for the various microsomal preparations. Microsomes from adult female and immature rats exhibited a similar sensitivity to SKF 525-A. However, microsomes from adult male rats were significantly more sensitive to the inhibitory effect of the drug. AHH activity from pseudohermaphroditic male rats was found to have the same sensitivity to SKF 525-A as in the female littermates. Microsomes obtained from adult rats that had been pretreated with various cytochrome P-450 inducing agents yielded significantly different IC50 values from untreated controls. These differences in IC50 values indicated that the predominant form(s) of cytochrome P-450 that hydroxylate benzo(a)pyrcne varied with the age, hormonal, and induced status of the animal.

scholarly journals Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes

1992 ◽  
Vol 287 (3) ◽  
pp. 863-870 ◽  
Author(s):  
P Puccini ◽  
S Menicagli ◽  
V Longo ◽  
A Santucci ◽  
P G Gervasi
Keyword(s):  
Polyclonal Antibodies ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Amino Acid Sequences ◽  
High Rate ◽  
Hepatic Microsomes ◽  
Syrian Golden Hamsters ◽  
Km Value ◽  
High Degree ◽  
Cytochrome P 450

A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.

Subtyping ofNeisseria meningitidisstrains isolated in Quebec, Canada: correlation between deduced amino acid sequences and serosubtyping techniques

1997 ◽  
Vol 43 (3) ◽  
pp. 234-238 ◽  
Author(s):  
Francis F. Arhin ◽  
France Moreau ◽  
Elaine L. Mills ◽  
James W. Coulton
Keyword(s):  
Amino Acid ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Outer Membrane Vesicles ◽  
Amino Acid Sequences ◽  
Complete Agreement ◽  
Enzyme Linked Immunosorbent Assay ◽  
Variable Regions ◽  
Whole Cells

Routine serosubtyping of Neisseria meningitidis relies upon reactivity of whole cells to monoclonal antibodies (mAbs). This procedure is limited in providing maximum serosubtype information because some epitopes in whole cells are masked and because mAbs are currently unavailable for some epitopes. To address masking of epitopes in whole cells, we isolated outer membrane vesicles (OMVs) from nine representative meningococcal strains that were isolated (1991–1993) in the province of Quebec, Canada; the OMVs were used in enzyme-linked immunosorbent assay for reactivity to mAbs, and improved serosubtyping information was obtained. A recent proposal assigns subtypes based on deduced amino acid sequences in the variable regions of the class 1 outer membrane protein. This scheme maintains the subtyping nomenclature that is based on reactivity to mAbs by defining the sequences in the epitopes recognized by the mAbs. We used this technique to assign subtypes to the meningococcal strains isolated in Quebec. For the strains tested, serosubtyping using mAbs and subtyping based on deduced amino acid sequences were in complete agreement. Subtyping using deduced amino acid sequences is superior because it does not depend on the availability of mAbs.Key words: Neisseria meningitidis, outer membrane proteins, serosubtyping, PorA.

Inhibitory Effects of Vitamin A on TCDD-induced Cytochrome P-450 1A1 Enzyme Activity and Expression

2005 ◽  
Vol 85 (1) ◽  
pp. 727-734 ◽  
Author(s):  
Yan-Mei Yang ◽  
Dong-Yang Huang ◽  
Ge-Fei Liu ◽  
Jiu-Chang Zhong ◽  
Kun Du ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Vitamin A ◽  
Inhibitory Effects ◽  
Cytochrome P 450
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