Rat-liver cholesterol 7α-hydroxylase. Pt. 1

A new assay is described to measure the activity of cholesterol 7α-hydroxylase and compared to the conventional 14C 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-3H]cholesterol is incubated at 370C and in the presence of molecular O2, in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9,000 x g supernatant), NADPH, MgCl2 and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 μM) in the incubation mixture to saturate the ezyme (K(m) = 100 μM). 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 [4-14C]cholesterol technique (r = 0.96; P < 0.001). The main advantage of the [7α-3H]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. (orig.)