出版社：American Society for Biochemistry and Molecular Biology
摘要：Mitochondrial cholesterol 27-hydroxylase (CYP27A1) plays animportant role in the maintenance of intracellular cholesterolhomeostasis. Cholesterol delivery to the mitochondrial innermembrane is believed to be a rate-limiting step for the "acidic"pathway of bile acid synthesis. This work reports that proteinaseK treatment of mitochondria markedly increases CYP27A1 specificactivity. With endogenous mitochondrial cholesterol, treatmentwith proteinase K increased CYP27A1 specific activity by 5-fold.Moreover, the addition of the exogenous cholesterol in ß-cyclodextrinplus proteinase K treatment increased the specific activityby 7-fold. Kinetic studies showed that the increased activitywas time-, proteinase K-, and substrate concentration-dependent.Proteinase K treatment decreased the apparent Km of CYP27A1for 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 mitochondriafreshly isolated from rat liver tissue.Supplementary key words cholesterol metabolism • ß-cyclodextrin • proteinase K • proteolysis
