Enzymic formation of esters of methyl sterol precursors of cholesterol

For investigation of the reactions of cholesterol biosynthesis, a number of workers use the 10,OOOg supernatant fraction (or similar preparations) obtained from cell-free homogenates of rat liver. We have found that esters of methyl sterol biosynthetic intermediates are formed by this crude source of enzymes. Eskers of (230-, Czn-, CZS-, and C?,-sterol intermediates have been isolated by silicic acid chromatography of an acetone extract of incubation mixtures. Competition be- tween ester formation and demethylation of the Czs-sterol intermediate has been demonstrated. With 4a-methyl-5a- cholest-7-en-3/3-01 as substrate, maximal velocities of ester formation (0.36 nmole/30 min per mg of protein) were almost equivalent to maximal velocities of demethylation (0.45 nmole/30 min per mg of protein). Ester formation may be eliminated by carrying out incubations with microsomal preparations; ester formation may be restored completely upon addition (to the microsomes) of either coenzyme A and ATP or the supernatant fraction resulting from centrifugation at 105,000 g. Ester formation has been examined similarly with broken- cell preparations of rat skin. With 4a-methyl-5a-choles:-7-en- 3/3-01 as substrate, the rate of ester formation was more than six times the rate of methyl sterol demethylation. The very significant competition between esterification and demethyla- tion of methyl sterol intermediates of skin suggests that sterol intermediates accumulate in rat skin because of the rapid formation of esters that may not be further metabolized.