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

Publisher Website
Google Scholar
Abstract
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-[125I]iodophenyl)diazirine [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]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 [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr .apprx. 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]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 .mu.M; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or .beta.-naphthoflavone-induced livers. P-450 PB-4 dependent microsomal androst-4-ene-3,17-dione 16.beta.-hydroxylase activity was also sensitive to inhibition by TID (IC50 .apprx. 5 .mu.M at 25 .mu.M steroid substrate), with lower sensitivities observed in the case of microsomal androst-4-ene-3,17-dione 6.beta.-hydroxylase (IC50 .times. 20 .mu.M), 16.alpha.-hydroxylase (IC50 > 100 .mu.M), or 7.alpha.-hydroxylase (IC50 > 100 .mu.M) activities, respectively catalyzed by cytochromes P-450 2a, 2c, and 3. TID inhibited 7-ethoxycoumarin O-deethylation catalyzed by purified and reconstituted P-450 PB-4 with kinetics consistent with mixed inhibition and a Ki of 2 .mu.M. These findings indicate that TID is an active site directed inhibitor of this heme protein and suggest that [125I]TID may serve as a useful probe for the substrate binding site of P-450 PB-4 and perhaps other cytochrome P-450 enzymes found in rat hepatic tissue.