Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450 k in rat kidney cortex : I. Characteristics of the hydroxylase system

Abstract

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450 K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450 K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450 K follow the same pattern of inactivation with increasing temperature. The apparent K m for the induced hydroxylase was 7.7 μ m and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450 K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10 −5 m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450 K-dependent aryl hydroxylase activity which differs from that present in the control rat.