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.

Purification and Characterization of Hepatic P-450Iie1 from Acetone-Treated Mice

1993 ◽  
Vol 9 (3) ◽  
pp. 539-546 ◽  
Author(s):  
Vincenzo Longo ◽  
Silvia Menicagli ◽  
Michael Minks ◽  
Annalisa Santucci ◽  
Pier Giovanni Gervasi
Keyword(s):  
Polyclonal Antibodies ◽  
High Spin State ◽  
Purification And Characterization ◽  
Turnover Number ◽  
High Turnover ◽  
Hepatic Microsomes ◽  
Aminoacid Sequence ◽  
Reconstituted System ◽  
Cytochrome P 450

A new cytochrome P-450 isozyme (Mr = 52,000) was purified to apparent electrophoretic homogeneity from hepatic microsomes of mice treated with acetone and its biochemical, spectral, and immunological properties characterized. Several criteria indicated that the purified cytochrome was distinct from the known mouse P-450 isozymes. The absolute spectrum of its oxidized form indicated that it was in the high spin state. In a reconstituted system, it showed low catalytic activities towards 7-ethoxycoumarin, aminopyrine, and coumarin, whereas it catalyzed the oxidation of aniline, acetone, dimelhylnitrosoamine with high turnover number. The mouse enzyme was immunoreactive with polyclonal antibodies against rat P-45011E1 and exhibited an NH2-terminal aminoacid sequence with a high homology to that of rat-P-450IIEI. Based upon the above catalytic, spectral, immunological and structural properties, the purified mouse P-450 appears to be the ortholog of previously described P-450IIE1 (s) of other species.

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.

IN-VITRO METABOLISM OF 4-ANDROSTENE-3,17-DIONE BY HEPATIC MICROSOMES FROM THE RAINBOW TROUT (SALMO GAIRDNERII): EFFECTS OF HYPOPHYSECTOMY AND OESTRADIOL-17β

1981 ◽  
Vol 90 (1) ◽  
pp. 103-112 ◽  
Author(s):  
TIIU HANSSON ◽  
JAN-ÅKE GUSTAFSSON
Keyword(s):  
Rainbow Trout ◽  
Liver Microsomes ◽  
The Other ◽  
Sham Operation ◽  
Hydroxylase Activity ◽  
Oxidoreductase Activity ◽  
Hepatic Microsomes ◽  
Cytochrome P 450 ◽  
Reductive Metabolism

The metabolism of 4-androstene-3,17-dione by liver microsomes from juvenile rainbow trout, Salmo gairdnerii, was studied in vitro. Hypophysectomy of the fish significantly increased mean hepatic 17-hydroxysteroid oxidoreductase activity when compared with that from sham-operated fish but none of the other enzyme activities investigated were affected. Administration of oestradiol-17β resulted in a significant decrease in mean hepatic 6β-hydroxylase activity and total cytochrome P-450 content but had no effect on the 16-hydroxylation or on the reductive metabolism of androstenedione. The effect of oestradiol-17β on hepatic 6β-hydroxylase activity was as pronounced after hypophysectomy as after sham-operation indicating that these effects of oestradiol-17β are mainly direct and independent of the pituitary gland. The results indicate that hypophysial hormone(s) as well as oestradiol-17β play a role in the regulation of hepatic steroid metabolism in trout.

scholarly journals Modulation of CYP2C9 activity and hydrogen peroxide production by cytochrome b5

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Javier Gómez-Tabales ◽  
Elena García-Martín ◽  
José A. G. Agúndez ◽  
Carlos Gutierrez-Merino
Keyword(s):  
Salicylic Acid ◽  
Human Liver ◽  
Cytochromes P450 ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Catalytic Cycle ◽  
Km Value ◽  
Acid Hydroxylation ◽  
Metabolic Degradation ◽  
Cyp2c9 Activity

Abstract Cytochromes P450 (CYP) play a major role in drug detoxification, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono-oxygenation and detoxification reactions. Collateral reactions of this catalytic cycle can lead to a significant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti-inflammatory drugs. We studied modulation by microsomal membrane-bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti-cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH-dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates.

scholarly journals Rhesus Monkey (Macaca mulatta) Mucosal Antimicrobial Peptides Are Close Homologues of Human Molecules

2001 ◽  
Vol 8 (2) ◽  
pp. 370-375 ◽  
Author(s):  
Robert Bals ◽  
Christiane Lang ◽  
Daniel J. Weiner ◽  
Claus Vogelmeier ◽  
Ulrich Welsch ◽  
...  
Keyword(s):  
Rhesus Monkey ◽  
Antimicrobial Peptides ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Model Organisms ◽  
Immune Functions ◽  
Mucosal Surfaces ◽  
High Degree ◽  
Identification And Characterization

ABSTRACT One component of host defense at mucosal surfaces appears to be epithelium-derived antimicrobial peptides. Molecules of the defensin and cathelicidin families have been studied in several species, including human and mouse. We describe in this report the identification and characterization of rhesus monkey homologues of human mucosal antimicrobial peptides. Using reverse transcriptase PCR methodology, we cloned the cDNAs of rhesus monkey β-defensin 1 and 2 (rhBD-1 and rhBD-2) and rhesus monkey LL-37/CAP-18 (rhLL-37/rhCAP-18). The predicted amino acid sequences showed a high degree of homology to the human molecules. The expression of the monkey antimicrobial peptides was analyzed using immunohistochemistry with three polyclonal antibodies to the human molecules. As in humans, rhesus monkey antimicrobial peptides are expressed in epithelia of various organs. The present study demonstrates that β-defensins and cathelicidins of rhesus monkeys are close homologues to the human molecules and indicate that nonhuman primates represent valid model organisms to study innate immune functions.

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