Abstract
A persistent problem in measurement of cholesterol 7α-hydroxylase (7α-OHase) activity by isotope incorporation has been solubilization of cholesterol substrate. Solubilization with Tween 20, for example, resulted in a 75% reduction in 7α-OHase activity after a 60 min incubation of substrate with microsomes. Incorporation of cholesterol substrate into small, unilamellar phospholipid vesicles (liposomes) prevented this effect, resulting in a 50% increase in activity over the same 60 min incubation at optimal concentrations. Using cholesterol in liposomes as substrate, standard assay conditions were determined to be: preparation of liposomes with 180 μM cholesterol substrate and 0.5 mg phospholipid/assay; incubation of these liposomes with 0.5 mg microsomal protein at 37 C for 60 min; addition of a NADPH generating system to start the reaction, and incubation at 37 C for 30 min before stopping the reaction and determining the amount of 7α-hydroxycholesterol formed. In addition to preventing the detergent-related inhibition of the enzyme, liposome-solubilized substrate also reduced the variation among replicates from a coefficient of 45% with Tween 20 to 4.2% with phospholipid. This method provides a sensitive and reliable alternative to methods which require more sophisticated equipment and allows total control of substrate concentration in a form readily accessible to the enzyme.
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This work was reported in part at the American Society of Biological Chemists Meeting, St. Louis, Missouri, June 1984.
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Junker, L.H., Story, J.A. An improved assay for cholesterol 7α-hydroxylase activity using phospholipid liposome solubilized substrate. Lipids 20, 712–718 (1985). https://doi.org/10.1007/BF02534392
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DOI: https://doi.org/10.1007/BF02534392