Assay of cholesterol 7α-hydroxylase activity in rat hepatocytes in primary monolayer culture

article



Princen, H.M.G.

Meijer, P.

Kwekkeboom, J.

Kempen, H.J.M.
A sensitive and precise method is described to assay cholesterol 7α-hydroxylase activity in homogenates of rat hepatocytes cultured in monolayers for up to 76 h. The assay is based on measurement of the amount of radioactive cholesterol converted into 7α-[14C]-hydroxycholesterol. Sine no subcellular fractionation was applied to measure enzyme activity, this method is rapid and can be performed with cell protein, corresponding to as little as 1 to 2 million hepatocytes. Optimal assay conditions were determined and the reproducibility of this cholesterol 7α-hydroxylase determination was established. Exogenous cholesterol (105 μM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent K(m) of 56 μM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7α-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7α-hydroxylase activity in cultured hepatocytes revealed that after an initial loss of approximately 60% of the activity as compared with 287 pmol/h/mg for freshly isolated cells, the enzyme activity was increased to the initial level in hepatocytes cultured for 52 h. This result and the finding that the cholesterol 7α-hydroxylase activity was diminished by 94% after a 24-h incubation 5 μM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis. Since the cholesterol 7α-hydroxylase activity was a good reflection of the bile acid synthesis rate in hepatocytes during 76 h in culture, we conclude that this method may be a valuable tool in the study of the regulation of bile acid synthesis. Copyright © 1988 Published by Elsevier Inc.
Chemicals/CAS: cholesterol 7alpha monooxygenase, 9037-53-0; cholesterol, 57-88-5; Bile Acids and Salts; Carbon Monoxide, 630-08-0; Cholesterol 7-alpha-Hydroxylase, EC 1.14.13.17; Cholesterol, 57-88-5; Hydroxycholesterols; potassium fluoride, 7789-23-3; Potassium, 7440-09-7; Proteins; Steroid Hydroxylases, EC 1.14.-
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cholesterolcholesterol 7alpha monooxygenaseanimal cellenzyme activityliver cellnonhumanthin layer chromatographyAnimalBile Acids and SaltsCarbon MonoxideCells, CulturedCholesterolCholesterol 7-alpha-HydroxylaseChromatography, Thin LayerHydroxycholesterolsLiverPotassiumProteinsRatsSpecimen HandlingSteroid HydroxylasesTime Factors
TNO Identifier
230558
Repository link
https://resolver.tno.nl/uuid:f3f580f7-739e-4788-a208-8424a3d3e01a
ISSN
0003-2697
Source
Analytical Biochemistry, 171(1), pp. 158-165.
Pages
158-165
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