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A more recent version of this article appeared on July 1, 2006 Originally published In Press as doi:10.1194/jlr.M600117-JLR200 on April 12, 2006

Papers In Press, published online ahead of print April 3, 2006
J. Lipid Res., doi:10.1194/jlr.M600117-JLR200
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Submitted on March 9, 2006
Revised on March 30, 2006
Accepted on April 2, 2006

Enzyme activity assay for sterol 27-hydroxylase (CYP27A1) in mitochondria

Xiaobo Li, Philip Hylemon, William M. Pandak, and Shunlin Ren

Medicine, McGuire VA Medical Center, Richmond, VA 23249

Corresponding Author: shunlin.ren{at}va.gov

Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays an important role in the maintenance of intracellular cholesterol homeostasis. Cholesterol delivery to the mitochondrial inner membrane is believed to be a rate limiting step for the “acidic” pathway of bile acid synthesis. The present work reports that proteinase K treatment of mitochondria markedly increases CYP27A1 specific activity. With endogenous mitochondrial cholesterol, treatment with proteinase K increased CYP27A1 specific activity by 5-fold. Moreover, the addition of the exogenous cholesterol in -cyclodextrin plus proteinase K treatment increased the specific activity by 7-fold. Kinetic studies showed that the increased activity was time, proteinase K, and substrate concentration dependent. Proteinase K treatment decreased the apparent Km of CYP27A1 for cholesterol from 400 M to 150 M. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity as compared with mitochondria freshly isolated from rat liver tissue.


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