3-(trifluoromethyl)-3-(m-(/sup 125/I)iodopheynyl)diazirine ((/sup 125/I)TID) labels a substrate-binding site of rat hepatic cytochrome P-450 (P-450) form PB-4

Conference · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5389646

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

Rat liver microsomes were labeled with the hydrophobic photoactivable reagent (/sup 125/I)TID. /sup 125/I-incorporation into 3-methylcholanthrene (3-MC)- and phenobarbital (PB)-induced microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation into uninduced liver microsomes. The major hepatic P-450 forms inducible by PB and 3-MC (P-450 PB-4 and P-450 BNF-B, respectively) were shown to be the principal polypeptides labeled by (/sup 125/I)TID in the correspondingly induced microsomes. Extensive trypsin cleavage of (/sup 125/I)TID-labeled microsomal P-450 PB-4 yielded a single labeled peptide of M/sub r/ approx. 4000. (/sup 125/I)TID incorporation into microsomal P-450 PB-4 was fully inhibited by the P-450 PB-4 substrate benzphetamine (1mM). Unactivated TID was shown to be an effective inhibitor of P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced liver microsomes (IC/sub 50/ = 10..mu..M at 120..mu..M substrate). TID inhibited purified, reconstituted P-450 PB-4 with kinetics consistent with either competitive or mixed inhibition and a Ki of 2..mu..M. These findings indicate that TID is an active site-directed inhibitor of this hemeprotein and suggest that (/sup 125/I)TID may serve as a useful probe for the substrate binding site of rat hepatic P-450 PB-4.

• Research Organization: Harvard Medical School, Boston, MA
• OSTI ID: 5389646
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANESTHETICS
ANIMALS
ANTICONVULSANTS
AROMATICS
AZINES
BARBITURATES
BETA DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM DEPRESSANTS
CHOLANTHRENE
CONDENSED AROMATICS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DRUGS
ELECTRON CAPTURE RADIOISOTOPES
GLANDS
HETEROCYCLIC COMPOUNDS
HYPNOTICS AND SEDATIVES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIVER
MAMMALS
MEMBRANE PROTEINS
MICROSOMES
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANOIDS
ORGANS
PEPTIDES
PHENOBARBITAL
PROTEINS
PYRIMIDINES
RADIOISOTOPES
RATS
REACTION KINETICS
REAGENTS
RECEPTORS
RODENTS
TRACER TECHNIQUES
VERTEBRATES