Oxidation and protein binding of aromatic amines by rat liver microsomes

Abstract

The binding of DMA and AF to protein by rat liver microsomes was studied under various conditions and compared with the utilization of DMA for oxidative demethylation and N-oxide formation. According to current evidence only the former reaction involves cytochrome P-450 as oxygen activator. 1. (1)|When the binding was tested with detoxication inhibitors (CO, SKF 525-A, mercurials) known to differentiate between demethylation and N-oxidation, it gave an intermediate or biphasic response. A similar result was obtained after graded, structural disorganization of the membranes by sonication or cholate treatment. While a major part of the binding approximated the demethylation in its susceptibility to inhibition, a minor, less well-defined part was more resistant. On the basis of the difference in CO susceptibility, the former type of binding was considered cytochrome-dependent, while the latter may include cytochrome-independent components. 2. (2)|The binding of DMA was not in all respects a reflection of the demethylation or N-oxide formation. For instance, the binding of DMA was not inhibited (but in fact stimulated) at high substrate concentrations or after pretreatment of the microsomes with moderate concentrations of trypsin, while the demethylation was markedly reduced under these conditions. Similarly, the binding was not inhibited in microsomes after preincubation in plain medium, although the N-oxide formation greatly diminished after this treatment. These and other data suggest that the binding was related to intermediates in the oxygenation reactions, rather than to end products. The possibility is discussed that cytochrome-dependent and cytochrome-independent binding are related to the enzyme-substrate complexes involved in C- and N-oxygenation, respectively. Anomalous decomposition of these complexes may lead to a disconnection of bound reaction intermediates in the form of free radicals. 3. (3)|Under the conditions studied, the binding of AF was closely correlated with that of DMA.