Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

Steroid-8-ene isomerase that catalyzes isomerization of Δ⁸- to Δ⁷-sterols has been solubilized from rat liver microsomes with a mixture of two detergents, octylglucoside and sodium taurodeoxycholic acid. During the a 40-fold enrichment of the solubilized enzyme, other enzymes of cholesterol biosynthesis, endogenous lipids, and electron carriers are removed. A comparison of properties of the solubilized and partially purified isomerase with the membrane-bound enzyme shows they are essentially identical with respect to pH profile, effect of inhibitors and cofactors, substrate specificity, and K(m) values. Addition of phospholipid to the partially purified enzyme stimulates activity as much as 1.8-fold over control rates. Although the relative rate of isomerization of cholesta-8,24-dien-3β-ol is six times that observed with cholest-8-en-3β-ol, the Δ⁸ to Δ⁷ ratio at equilibrium is approximately equal. The reversibility of the reaction has been demonstrated by the direct conversion of cholest-7-en-3β-ol to cholest-8-en-3β-ol; at equilibrium the Δ⁷-isomer is predominant (19/1). The purified enzyme does not catalyze isomerization of cholesta-8,14-dien-3β-ol and cholest-8(14)-en-3β-ol under conditions that result in equilibrium mixtures of isomers from cholest-8(9)-en-3β-ol. These results are consistent with the earlier suggestion that Δ⁸(14)-sterols are neither formed nor metabolized by the same microsomal enzymes that catalyze transformation of lanosterol to cholesterol.