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 language | English |
|---|---|
| Pages (from-to) | 39-48 |
| Number of pages | 10 |
| Journal | Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism |
| Volume | 1259 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 1995 Oct 26 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Biophysics
- Endocrinology
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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 journal › Article
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.
UR - http://www.scopus.com/inward/record.url?scp=0028789467&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0028789467&partnerID=8YFLogxK
U2 - 10.1016/0005-2760(95)00128-Y
DO - 10.1016/0005-2760(95)00128-Y
M3 - Article
C2 - 7492613
AN - SCOPUS:0028789467
VL - 1259
SP - 39
EP - 48
JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
SN - 1388-1981
IS - 1
ER -