Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 14-reductase

Chap Ki Kim, Kye Im Jeon, Dong Min Lim, Tae Neung Johng, James M. Trzaskos, James L. Gaylor, Young-Ki Paik

Research output: Contribution to journalArticle

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Abstract

We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of Δ8,14-diene or Δ7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis mammals (Paik et al. (1984) J. Biol. Chem. 259, ]3413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1 % lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01 % AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01 % AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 μM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a Mr = 70 000 protein that is composed of two equally-sized subunits having a Mr = 38 000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

Original languageEnglish
Pages (from-to)39-48
Number of pages10
JournalBiochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
Volume1259
Issue number1
DOIs
Publication statusPublished - 1995 Oct 26

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Lanosterol
Biosynthesis
Sterols
Purification
Rats
Oxidoreductases
Cholesterol
Liver
trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride
Nutrition
Diet
Cholestyramine Resin
Lovastatin
Mammals
Enzymes
Cycloheximide
Microsomes
NADP

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Endocrinology

Cite this

Kim, Chap Ki ; Jeon, Kye Im ; Lim, Dong Min ; Johng, Tae Neung ; Trzaskos, James M. ; Gaylor, James L. ; Paik, Young-Ki. / Cholesterol biosynthesis from lanosterol : regulation and purification of rat hepatic sterol 14-reductase. In: Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism. 1995 ; Vol. 1259, No. 1. pp. 39-48.
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abstract = "We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of Δ8,14-diene or Δ7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis mammals (Paik et al. (1984) J. Biol. Chem. 259, ]3413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5{\%} cholestyramine plus 0.1 {\%} lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5{\%} cholesterol or 0.01 {\%} AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01 {\%} AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 μM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a Mr = 70 000 protein that is composed of two equally-sized subunits having a Mr = 38 000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.",
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Cholesterol biosynthesis from lanosterol : regulation and purification of rat hepatic sterol 14-reductase. / Kim, Chap Ki; Jeon, Kye Im; Lim, Dong Min; Johng, Tae Neung; Trzaskos, James M.; Gaylor, James L.; Paik, Young-Ki.

In: Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism, Vol. 1259, No. 1, 26.10.1995, p. 39-48.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cholesterol biosynthesis from lanosterol

T2 - regulation and purification of rat hepatic sterol 14-reductase

AU - Kim, Chap Ki

AU - Jeon, Kye Im

AU - Lim, Dong Min

AU - Johng, Tae Neung

AU - Trzaskos, James M.

AU - Gaylor, James L.

AU - Paik, Young-Ki

PY - 1995/10/26

Y1 - 1995/10/26

N2 - We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of Δ8,14-diene or Δ7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis mammals (Paik et al. (1984) J. Biol. Chem. 259, ]3413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1 % lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01 % AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01 % AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 μM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a Mr = 70 000 protein that is composed of two equally-sized subunits having a Mr = 38 000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

AB - We have previously characterized the membrane-bound sterol 14-reductase (14-reductase) that catalyzes anaerobically NADPH-dependent reduction of the 14-double bond of Δ8,14-diene or Δ7,14-diene sterols that are sterol intermediates in cholesterol biosynthesis mammals (Paik et al. (1984) J. Biol. Chem. 259, ]3413-13423). To elucidate the regulatory mechanism as well as molecular characteristics of the 14-reductase, we extended our investigation on the consequences of alteration of the enzymic activity under various physiological conditions. The enzymic activity of rat hepatic sterol 14-reductase was induced more than 11-fold by feeding 5% cholestyramine plus 0.1 % lovastatin (the CL-diet) for 7 days but was severely suppressed by feeding 5% cholesterol or 0.01 % AY-9944 (an inhibitor of 14-reductase) for the same period. The increase or decrease in the 14-reductase activity also parallels the same change in the cholesterol synthetic rate in hepatocytes from rats that had been fed either the CL-diet or 0.01 % AY-9944. In vitro inhibition studies revealed that AY-9944 acts as a competitive inhibitor of the 14-reductase (Ki = 0.26 μM). A diurnal variation was observed for the 14-reductase with peak activity near the middle of the dark cycle (10 p.m.), which was abolished by administration of cycloheximide. With induced enzyme conditions 14-reductase has been further purified with chromatographic procedures to near homogeneity. Purified 14-reductase appears to be a Mr = 70 000 protein that is composed of two equally-sized subunits having a Mr = 38 000. All properties of the purified 14-reductase suggest that the solubilized enzyme is the principal 14-reductase of microsomes. Taken together, our results provide the first evidence in support of a previously unknown regulatory role for the 14-reductase in the overall cholesterol synthetic pathway.

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