Kinetics of two cytochrome P450-dependent aromatic amine hydroxylases from lung and liver microsomes of rabbit

ABSTRACT

The kinetics of 2-aminofluorene [AF] and 4-Aminobiphenyl [4-AB] N-hydroxylase reactions were determined using eight highly purified forms of rabbit liver cytochrome-P450 [Cyt-P450], with control and 2,3,7,8-tetrchloro-dibenzo-p-dioxin [TCDD] induced rabbit lung and liver microsomes.Both AF and 4-AB N-hydroxylases were best defined by two enzymatic systems, showing a high affinity and low capacity and a low affinity; high capacity system in control and TCDD pretreated microsomes. TCDD pretreatment differently modifies the apparent Km and Vmax. of the arylmine N-hydroxylases in the two tissues microsomal enzymes and in a substrate-dependent manner. Cyt-P450 [form 4] showed the following AF N-hydroxylase activity 3417 +/- 326 pmol N-OH-AF/nmol Cyt-450/min. In contrast to the microsomal situation, the kinetics studies of AF and 4-AB N-hydroxylases with the form 4, were best described by one enzymatic system. liver microsomal AF [0.25 micro M] N-Hydroxylase activity, where as lung microsomal N-hydroxylase activity was only decreased 35%. Co-incubation of 2-diethyl-aminoethyl-2,2-diphenylvalerate [SKF 525 A] with AF stimulated liver microsomal AF N-/hydroxylase activity by 25% whereas a 50% decrease in the N-hydroxylase activity was observed when lung microsomes were assayed