3-(Trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Journal Article · Tue Aug 26 04:00:00 EDT 1986 · Biochemistry; (United States)

OSTI ID:6846649

Frey, A B; Kreibich, G; Wadhera, A B; Clarke, L; Waxman, D J

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)TID). (/sup 125/I)TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of (/sup 125/I)TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by (/sup 125/I)TID in the correspondingly induced microsomes. Trypsin cleavage of (/sup 125/I)TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. (/sup 125/I)TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.

• Research Organization: New York Univ. Medical Center, NY
• OSTI ID: 6846649
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 17; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

3-METHYLCHOLANTHRENE
560300* -- Chemicals Metabolism & Toxicology
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANESTHETICS
ANIMALS
ANTICONVULSANTS
AROMATICS
AZINES
BARBITURATES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHEMICAL ACTIVATION
CHEMICAL REACTIONS
CYTOCHROMES
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
HETEROCYCLIC COMPOUNDS
HYDROCARBONS
HYPNOTICS AND SEDATIVES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
MICROSOMES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANOIDS
PHENOBARBITAL
PHOTOCHEMICAL REACTIONS
PIGMENTS
POLYCYCLIC AROMATIC HYDROCARBONS
PROTEINS
PYRIMIDINES
RADIOISOTOPES
RATS
REACTION KINETICS
RECEPTORS
RODENTS
SUBSTRATES
VERTEBRATES