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 containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM 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 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized 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.
Inouye
K
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Kondo
S
S
Yamamura
M
M
Nakanishi
D
D
Sakaki
T
T
D000818:Animals; D002793:Cholic Acids / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010078:Oxazines / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D002793:Cholic Acids; D003902:Detergents; D010078:Oxazines; D011993:Recombinant Fusion Proteins; C007649:ethoxyresorufin; D017830:Octoxynol; D019363:Cytochrome P-450 CYP1A1; D009251:NADPH-Ferrihemoprotein Reductase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate