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

Y. K. Paik, J. T. Billheimer, R. L. Magolda, J. L. Gaylor

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Abstract

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.

Original languageEnglish
Pages (from-to)6470-6477
Number of pages8
JournalJournal of Biological Chemistry
Volume261
Issue number14
Publication statusPublished - 1986 Dec 1

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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