A comparison of some properties of microsomal cytochrome P-450 from normal, methylcholanthrene-, and phenobarbital-treated rats.

Abstract The differences in spectral properties of hepatic microsomal cytochrome P-450 between uninduced and methylcholanthrene (MC)-induced rats were examined in detail. In order to explain the difference in the ethyl isocyanide (EtNC) difference spectrum of reduced P-450 between uninduced and induced rats by a possible difference in the environment of the heme moiety between the two cases, the properties of P-450 in microsomes from both types of animals were compared. P-450 in microsomes from MC-injected animals was more resistant to treatment with n-propanol or KSCN, a reagent which attacks hydrophobic bonds and causes the conversion of P-450 to P-420, than P-450 from PB-treated or untreated animals. On the other hand, the P-450's in all these animals showed almost the same sensitivity to HgCl2. Addition of n-heptanol to microsomes caused a change in the equilibrium between the two states of the EtNC compound; however, microsomes from MC-treated animals were less affected by the addition of the alcohol than those from PB-treated or untreated animals. These findings suggest that the environment of P-450 heme is more hydrophobic in microsomes from MC-treated rats than those from PB-treated or untreated animals. In order to learn whether the difference in the environment of P-450 heme between uninduced and MC-induced microsomes is ascribed to two molecular species of P-450 or two physical forms of a single molecular species, the response of P-450 to in vivo treatment with MC and other chemicals was examined. The time courses of changes in the P-450 contents and in the ratios of the two ethyl isocyanide compounds of reduced P-450 (the “455” and the “430” states) were determined after MC- or phenobarbital (PB) administration and after cessation of the injections. After MC administration, the 455 to 430 ratio began to increase prior to the increase in P-450 content. The administration of aminotriazole (AT), a competitive inhibitor of heme biosynthesis, together with MC, caused a complete inhibition of the increase in the P-450 content but no change in the increased 455 to 430 ratio. On the other hand, when cycloheximide (CH) was injected jointly with MC, the increase in the P-450 content was completely inhibited, but the increase in the 455 to 430 ratio was only partly inhibited. These findings indicate that synthesis of P-450 heme is not necessarily required for the change in the 455 to 430 ratio which is induced by MC, and the synthesis of certain protein(s) of microsomal membranes other than the P-450 apoprotein is necessary for the increase in the 455 to 430 ratio. They further suggest that there exist two physical forms of a single molecular species of cytochrome P-450. Careful examination of similar inductive changes in the properties of P-450 showed that PB-administration does cause similar changes to those induced by MC-administration, although to a smaller extent. Based on all these findings, the differences in the two forms of cytochrome P-450 are discussed with relevance to the microsomal membrane localization of the protein.