Inactivation of purified rat liver cytochrome P-450 2B1 and rabbit liver cytochrome P-450 2B4 by N-methylcarbazole.
Metabolism of N-methylcarbazole by purified rat liver cytochrome P-450 2B1 or rabbit liver P-450 2B4 resulted in the inactivation of these enzymes in a time-dependent, pseudo-first order manner as assayed spectrally by the decrease in the reduced CO spectrum at 450 nm. The inactivation was saturable with respect to the concentration of N-methylcarbazole, and a Ki = 5.2 microM and kINACT = 0.14 min-1 were determined for the inactivation of P-450 2B1. For P-450 2B4 inactivation, the Ki was 23 microM and the kINACT = 0.21 min-1. There was no increase in the reduced CO spectrum at 420 nm accompanying the inactivation, and the slight loss of the P-450 heme prosthetic group, as determined by the spectrum at 418 nm, was not sufficient to account for the loss of the reduced CO spectrum at 450 nm. The metabolism of N-methylcarbazole by P-450 did not result in the formation of a metabolic intermediate complex, which could also be responsible for the loss of cytochrome P-450 activity. Loss of catalytic activity for further substrate metabolism was also observed after preincubation of enzyme with N-methylcarbazole and the loss of catalytic activity correlated with the loss of the reduced CO spectrum. Accompanying the loss of spectrally detectable P-450 2B1 and P-450 2B4 catalytic activity, there was an increase in the NADPH oxidation rate. This increased rate persisted on subsequent addition of NADPH.
Kuemmerle
S C
SC
Department of Pathology, Northwestern University Medical School.
Shen
T
T
Hollenberg
P F
PF
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002227:Carbazoles / Q000494:pharmacology*; D002248:Carbon Monoxide / Q000032:analysis / Q000378:metabolism; D002273:Carcinogens / Q000494:pharmacology*; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism*
D002227:Carbazoles; D002273:Carcinogens; D009249:NADP; C038545:N-methylcarbazole; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C050356:steroid 15-alpha-hydroxylase