J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M600117-JLR200 on April 12, 2006 Originally published In Press as doi:10.1194/jlr.M600117-JLR200 on April 3, 2006

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Journal of Lipid Research, Vol. 47, 1507-1512, July 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Enzyme activity assay for cholesterol 27-hydroxylase in mitochondria

Xiaobo Li*,{dagger},§, Philip Hylemon*,{dagger}, William M. Pandak* and Shunlin Ren1,*

* Division of Gastroenterology, Department of Medicine, Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA
{dagger} Division of Gastroenterology, Department of Microbiology, Veterans Affairs Medical Center and Virginia Commonwealth University, Richmond, VA
§ Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai, China

Published, JLR Papers in Press, April 12, 2006.

1 To whom correspondence should be addressed. e-mail: 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. This 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 ß-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 to 150 µM. Using this new assay, we found that during rat hepatocyte preparation and cell culture, mitochondria gradually lose CYP27A1 activity compared with mitochondria freshly isolated from rat liver tissue.

Supplementary key words cholesterol metabolism • ß-cyclodextrin • proteinase K • proteolysis


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