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

Research output: Contribution to journalArticle

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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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Steroid Isomerases
Lanosterol
Isomerases
Biosynthesis
Purification
Steroids
Cholesterol
Isomerization
Enzymes
Sterols
Isomers
Taurodeoxycholic Acid
pH effects
Enzyme activity
Detergents
Liver
Liver Microsomes
Rats
Substrate Specificity
Phospholipids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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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.",
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Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase. / Paik, Y. K.; Billheimer, J. T.; Magolda, R. L.; Gaylor, J. L.

In: Journal of Biological Chemistry, Vol. 261, No. 14, 01.12.1986, p. 6470-6477.

Research output: Contribution to journalArticle

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T1 - Microsomal enzymes of cholesterol biosynthesis from lanosterol. Solubilization and purification of steroid 8-isomerase

AU - Paik, Y. K.

AU - Billheimer, J. T.

AU - Magolda, R. L.

AU - Gaylor, J. L.

PY - 1986/12/1

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