Open AccessRat‐Liver Cholesterol 7α‐Hydroxylase
Author(s) -
CANTFORT Jacques,
GIELEN Jacques,
RENSON Jean
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb02134.x
Subject(s) - incubation , hydroxylation , chemistry , cholesterol oxidase , cholesterol , enzyme , tritium , microsome , tritiated water , substrate (aquarium) , biochemistry , potassium phosphate , chromatography , biology , ecology , physics , nuclear physics
A new assay is described to measure the activity of cholesterol 7α‐hydroxylase and compared to the conventional 14 C method used by other investigators. This method is based on the mechanism of the enzymic hydroxylation, i.e. a direct and stereospecific substitution of the 7α‐hydrogen by a hydroxyl group. [7α 3 ‐H]Cholesterol is incubated at 37 °C and in the presence of molecular O 2 , in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9000 × g supernatant), NADPH, MgCI 2 and cysteamine. Tween‐80 (1.5 mg/ml) is used to introduce enough substrate (300 μ) in the incubation mixture to saturate the enzyme ( K m = 100 μ). Under these conditions the tritiated water released into the incubation medium reflects accurately the enzymic activity. The results obtained with this method are similar to the one obtained with a [4‐ 14 C]cholesterol technique ( r = 0.96; P < 0.001). The main advantage of the [7α‐ 3 H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7α‐hydroxycholesterol, the tritiated water representing the entire cholesterol 7α‐hydroxylase activity.