Substrate-inducible Microsomal Aryl Hydroxylase in Mammalian Cell Culture

Abstract The aryl hydroxylase enzyme system is inducible in hamster fetus cell cultures. The enzyme system is localized in the 105,000 x g pellet (microsomal fraction), has an absolute requirement for NADPH and molecular O2, a pH optimum of pH 7.5, and a partial requirement for divalent cations. Exposure to carbon monoxide reduces the enzyme activity. Treatment with ethylenediaminetetraacetate or trypsin completely prevents enzymatic activity. The Km for the hydroxylase is approximately 0.6 µm with benzo[a]pyrene as substrate. Benz[a]anthracene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, dibenz[a,h]anthracene, and dibenz[a,c]anthracene are also substrates for the enzyme system. The spectrophotofluorometric determination of hydroxylated benzo[a]pyrene products is sufficiently sensitive to detect 10-12 mole per ml and, hence, has great utility in measuring the hydroxylase activity of cells grown in culture. This mammalian cell culture system is advantageous for the study of the mechanism of microsomal enzyme induction and the related areas of carcinogenesis and drug and steroid metabolism.