Inhibitory effects of detergents on rat CYP1A1-dependent monooxygenase: Comparison of mixed and fused systems consisting of rat CYP 1A1 and yeast NADPH-P450 reductase

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes contai ning both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhi bition in both mixed and fused systems with K-i values of 24.6 and 21.5 muM , respectively. These results strongly suggest that Triton X-100 binds to t he substrate-binding pocket of CYP1A1, These K-i values are far below the c ritical micelle concentration of Triton X-100 (240 muM). Western blot analy sis revealed no disruption of the microsomal membrane by Triton X-100 in th e presence of 0-77 muM Triton X-100, On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K-i and K-i values of 1.2 and 5.4 m M of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solu bilized fused enzyme by Chaps preserved the activity whereas the solubilize d CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents. (C) 2001 Academic P ress.