Mechanistic studies of lanosterol C-32 demethylation. Conditions which promote oxysterol intermediate accumulation during the demethylation process

Abstract

Conditions have been established which promote the accumulation of the dihydrolanosterol C-32 demethylation intermediates lanost-8-en-3 beta,32-diol and 3 beta-hydroxylanost-8-en-32-aldehyde with intact hepatic microsomes. Accumulation of dihydrolanosterol-derived oxysterols occurs with a variety of assay manipulations which include short incubation times, limiting enzyme amounts, high pH, and increasing substrate concentration. In addition, competitive inhibition of dihydrolanosterol demethylation by lanosterol, or the reciprocal inhibition of lanosterol demethylation by dihydrolanosterol, leads to oxysterol accumulation at the expense of demethylated end product. Similarly, the nonsteroidal demethylase inhibitors miconazole and ketoconazole promote oxysterol accumulation in a concentration-dependent manner. Finally, cholesterol loading of isolated microsomes results in changes in the measured kinetic constants, Km and Vmax, and results in enhanced oxysterol accumulation above that seen in control microsomal preparations. The major oxysterol intermediate accumulated under all the conditions described above is the C-32 aldehyde in an approximate 3:1 ratio to the C-32 alcohol. These data support the conclusion that a single enzyme species is responsible for all three oxidations of the C-32 demethylation sequence. In addition, intermediates which do not routinely accumulate during demethylation are freely diffusible from the enzyme when appropriate conditions are established to prevent their further metabolism.