Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

David J. Shapiro; Jeffrey L. Nordstrom; Jonathan J. Mitschelen; Victor W. Rodwell; Robert T. Schimke

doi:10.1016/0005-2744(74)90098-9

Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

David J. Shapiro, Jeffrey L. Nordstrom, Jonathan J. Mitschelen, Victor W. Rodwell, Robert T. Schimke

Research output: Contribution to journal › Article › peer-review

Abstract

A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.

Original language	English (US)
Pages (from-to)	369-377
Number of pages	9
Journal	BBA - Enzymology
Volume	370
Issue number	2
DOIs	https://doi.org/10.1016/0005-2744(74)90098-9
State	Published - Dec 29 1974
Externally published	Yes

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title = "Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts",

abstract = "A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.",

author = "Shapiro, {David J.} and Nordstrom, {Jeffrey L.} and Mitschelen, {Jonathan J.} and Rodwell, {Victor W.} and Schimke, {Robert T.}",

note = "Funding Information: This work was supported by Research Grant 71-860 from the American Heart Association, by a grant from the Indiana Heart Association, by Research Grant GM 14931 from the National Institute of General Medical Sciences, and Research Grant NP148F from the American Cancer Sooety D J S is a Helen Hay Whitney Foundation Postdoctoral Fellow",

year = "1974",

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doi = "10.1016/0005-2744(74)90098-9",

language = "English (US)",

volume = "370",

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T1 - Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

AU - Shapiro, David J.

AU - Nordstrom, Jeffrey L.

AU - Mitschelen, Jonathan J.

AU - Rodwell, Victor W.

AU - Schimke, Robert T.

N1 - Funding Information: This work was supported by Research Grant 71-860 from the American Heart Association, by a grant from the Indiana Heart Association, by Research Grant GM 14931 from the National Institute of General Medical Sciences, and Research Grant NP148F from the American Cancer Sooety D J S is a Helen Hay Whitney Foundation Postdoctoral Fellow

PY - 1974/12/29

Y1 - 1974/12/29

N2 - A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.

AB - A rapid, highly reproducible, micro-incubation, direct thin-layer chromatographic assay for 3-hydroxy-3-methylglutaryl-CoA reductase is described. The isolation and quantitation of mevalonic acid is simplified by direct application of the deproteinized, acidified incubation mixture to thin-layer chromatography sheets. The tedious and variable extraction of mevalonic acid into ether is eliminated by this procedure. Background levels are lower than those obtained with comparable assays and permit quantitation of as little as 30 pmoles of mevalonic acid. The method is illustrated by analysis of 3-hydroxy-3-methylglutaryl CoA reductase from rat liver, cultured L cells, and solubilized microsomal reductase. The rapid inactivation of reductase by MgATP and the low levels of reductase activity in cholesterol-treated L cells are accurately determined by using the micro-incubation, direct thin-layer chromatography method.

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Micro assay for 3-hdyroxy-3-methylglutaryl-CoA reductase in rat liver and in L-cell fibroblasts

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