Submitted on December 18, 2002
Revised on August 25, 2003
Accepted on August 25, 2003
Assay method of 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
Surgery I, Miyazaki Medical college, Miyazaki 889-1692
Corresponding Author: kazuochi{at}post.miyazaki-med.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 the side chain hydroxylation in extrahepatic tissues. The enzyme has been assayed by complicated methods using radiolabeled substrates or deuterium labeled tracers. In addition, these methods seem to be not accurate to measure the enzyme activity because amount of electron transferring proteins might be insufficient for maximal velocity. To solve these problems, after solubilization of the enzyme from the rat liver mitochondria with n-octyl-
-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 measured the product, 7,27-dihydroxy-4-cholesten-3-one, with HPLC monitoring absorbance at 240 nm. The product formation was proportional to time and protein concentration up to 5 min and up to 0.5 mg of protein/ml, respectively. The maximal velocity of the enzyme was 1.1 nmol/min/mg of protein, which was 4- to 16-fold higher than the reported values. A simple and accurate assay method for sterol 27-hydroxylase in rat liver mitochondria is herein described.
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