Reconstituted O-dealkylase systems containing various forms of liver microsomal cytochrome P-450

Abstract

Among the seven forms of purified liver microsomal cytochrome P-450 tested, P-4501, P-4502 (both from phenobarbital-treated rabbits), P-4504 (from phenobarbital-treated rats), and P-4482 (from methylcholanthrene-treated rats) could reconstitute significant activities catalyzing the O-deethylation of 7-ethoxycourmarin, but remarkable differences were seen among the catalytic properties of the four reconstituted systems. The systems containing P-4501, and P-4504 required the presence of cytochrome b5 for maximal activity, but no such requirement was observed with the other two systems. The Km value of the P-4502 system for ethoxycoumarin was of the order of 10(-7) M, whereas those of the other systems were about 1,000 times higher. The Vmax value determined for the P-4482 system was higher than those for the other systems by a factor of 10. 7-Methoxycoumarin was metabolized in a way similar to ethoxycoumarin by the systems containing P-4501, P-4502, and P-4504, but acted as a strong uncoupler in the P-4482-containing system. In the P-4482 system, however, ethoxycoumarin O-deethylation was almost completely coupled to NADPH oxidation. In the other systems, on the other hand, NADPH oxidation was partially uncoupled to similar extents with respect to the product formation with both ethoxy- and methoxycoumarins as substrates. The four systems could also be distinguished from one another with respect to the effects of several inhibitors. The P-4502-containing system, but not the other three systems, was progressively inactivated during methoxycoumarin O-demethylation and benzphetamine N-demethylation. Such inactivation was not observable during the ethoxycoumarin O-deethylation reaction. It is suggested that the active site of P-4502 reacted with formaldehyde produced by the demethylation reactions and was thus irreversibly inactivated. The results reported in this paper provide a clear example of different catalytic properties of multiple forms of hepatic microsomal cytochrome P-450.