Assay of cholesterol 7 alpha-hydroxylase activity in rat hepatocytes in primary monolayer culture.
A sensitive and precise method is described to assay cholesterol 7 alpha-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 7 alpha-[14C]-hydroxycholesterol. Since 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 7 alpha-hydroxylase determination was established. Exogenous cholesterol (105 microM), solubilized in Tween 80, was added to saturate the enzyme, giving an apparent Km of 56 microM. Under these conditions, 70% of the cholesterol present in the homogenates is directly accessible to the cholesterol 7 alpha-hydroxylase. The detection limit of the assay was found to be about 10 pmol per incubation. A time course of the cholesterol 7 alpha-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 7 alpha-hydroxylase activity was diminished by 94% after a 24-h incubation with 5 microM cycloheximide suggest that the enzyme activity is associated with de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Princen
H M
HM
Gaubius Institute TNO, Leiden, The Netherlands.
Meijer
P
P
Kwekkeboom
J
J
Kempen
H J
HJ
D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis; D002248:Carbon Monoxide / Q000494:pharmacology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis* / Q000037:antagonists & inhibitors; D002855:Chromatography, Thin Layer; D005459:Fluorides; D006888:Hydroxycholesterols / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology*; D011188:Potassium / Q000494:pharmacology; D017680:Potassium Compounds; D014176:Protein Biosynthesis; D051381:Rats; D013048:Specimen Handling; D013250:Steroid Hydroxylases / Q000032:analysis*; D013997:Time Factors
D001647:Bile Acids and Salts; D006888:Hydroxycholesterols; D017680:Potassium Compounds; D002248:Carbon Monoxide; C033320:potassium fluoride; D002784:Cholesterol; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase; D005459:Fluorides; D011188:Potassium