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)