Rat-liver cholesterol 7$alpha$-hydroxylase. I. Development of a new assay based on the enzymic exchange of the tritium located on the 7$alpha$ position of the substrate
A new assay is described to measure the activity of cholesterol 7$alpha$- hydroxylase and compared to the conventional $sup 14$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 7$alpha$- hydrogen by a hydroxyl group. [7$alpha$$-$$sup 3$H]cholesterol is incubated at 37$sup 0$C and in the presence of molecular O$sub 2$, in a medium buffered by potassium phosphate at pH 7.4 and containing liver microsomes (or 9,000 x g supernatant), NADPH, MgCl$sub 2$ and cysteamine. Tween-80 (1.5 mg/ml) is used to introduce enough substrate (300 $mu$M) in the incubation mixture to saturate the ezyme (K(m) = 100 $mu$M). 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-$sup 14$C]cholesterol technique (r = 0.96; P < 0.001). The main advantage of the [7$alpha$$-$$sup 3$H]cholesterol method is a complete independence from further metabolism of the first enzymic product, the 7$alpha$-hydroxycholesterol, the tritiated water representing the entire cholesterol 7$alpha$-hydroxylase activity. (orig.)
- Research Organization:
- Liege Univ. (Belgium). Service de Chimie Medicale, Toxicologie et Hygiene
- NSA Number:
- NSA-33-009682
- OSTI ID:
- 4128684
- Journal Information:
- Eur. J. Biochem., v. 55, no. 1, pp. 23-31, Other Information: 8 figs.; 2 tabs.; 49 refs. Orig. Receipt Date: 30-JUN-76
- Country of Publication:
- Germany
- Language:
- English
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