Abstract

When the labeled synthetic progestin, [4-¹⁴C]- or [6,7-³H]norethynodrel, was incubated with rat liver microsomes, the steroid became tightly bound to proteins. The binding process required oxygen and NADPH, and was inhibited by carbon monoxide and SKF 525-A. Microsomes from animals treated with phenobarbital were more effective in causing binding. Precipitation of the proteins with trichloracetic acid, washing with organic solvents, dialysis against water, and filtration through Sephadex G-200 all failed to dissociate the radioactivity from the proteins. Unlabeled steroid added after the initiation of the incubation was unable to displace radioactivity from the proteins. Disc gel electrophoresis of the solubilized proteins indicated that the steriod was bound randomly to all proteins. Reduced glutathione and compounds that react with thiol groups inhibited radiolabeling of the proteins. Moreover, glutathione increased the water-soluble radioactivity at the expense of the protein bound radioactivity. Experiments using tritiated glutathione and [4-¹⁴C]norethynodrel demonstrated the formation of norethynodrel-glutathione conjugates. It was suggested that liver cytochrome P-450 oxygenase metabolized norethynodrel to a reactive intermediate(s) which could react with and bind covalently to proteins and glutathione. The implication of these findings is discussed.