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Journal of Lipid Research, Vol. 44, 2400-2405, December 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Methods

Assay method for mitochondrial sterol 27-hydroxylase with 7-hydroxy-4-cholesten-3-one as a substrate in the rat liver

Yoshikazu Ota^*, Tada-Aki Eto^*, Shun-Ichi Tanaka^*, Hideto Sueta^*, Hironori Shiotsuki^*, Yorio Maeda^*, Mizuho Une and Kazuo Chijiiwa^1,*

^* Department of Surgery I, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Hiroshima 734-8553, Japan

¹ To whom correspondence should be addressed. e-mail: kazuochi{at}med.myazaki-u.ac.jp

Mitochondrial sterol 27-hydroxylase (EC 1.14.13.15) is an important enzyme, not only in the formation of bile acids from cholesterol intermediates in the liver but also in the removal of cholesterol by side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium-labeled tracers. These methods may be inaccurate for measuring enzyme activity, because the amount of electron-transferring proteins may be insufficient for maximal velocity. To solve this problem, after solubilization of the enzyme from rat liver mitochondria with n-octyl-ß-D-glucopyranoside (OGP), we measured the enzyme activity by incubating the solubilized enzyme with saturated amounts of electron-transferring proteins. In our assay system, using 7-hydroxy-4-cholesten-3-one (HCO) as a substrate, we could easily measure the product, 7,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportionate to the time up to 5 min and the protein concentration up to 0.5 mg of protein/ml. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than previously reported values.

A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.

Abbreviations: C-triol, 5ß-cholestane-3,7,12-triol; HCO, 7-hydroxy-4-cholesten-3-one; GC-SIM, gas chromatography-mass spectrometry with selected ion monitoring; OGP, n-octyl-ß-D-glucopyranoside; TMS, trimethylsilyl

Supplementary key words adrenodoxin • NADPH-adrenodoxin reductase • 7,27-dihydroxy-4-cholesten-3-one • high-performance liquid chromatography • solubilization • CYP27A1 • n-octyl-ß-D-glucopyranoside • cholesterol • 5ß-cholestane-3,7,12-triol

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