Cholesterol 7alpha-hydroxylase. 2. Biochemical properties and participation of endogenous cholesterol in the assay in vitro.

70% of the microsome-bound cholesterol is directly accessible to cholesterol 7alpha-hydroxylase in an assay in vitro. After 5 min of incubation this endogenous cholesterol makes a single pool with the exogenously added substrate and modifies its specific radioactivity. Thus, an accurate estimation of the enzymic activity should take the participation of endogenous cholesterol into account. Cholesterol 7alpha-hydroxylase activity is enhanced in vitro by thiol-containing substances like mercaptoethanol, dithiothreitol, or cysteamine. On the contrary, the enzymic activity is inhibited by heavy cations (Hg2+, Pb2+, Cu2+, Zn2+), or --SH-blocking agents (mersalic acid p-chloro-mercuribenzoic acid). Several steroids are potent inhibitors (Ki less than Km) of the enzyme, among them pregnenolone and its acetate derivative and the cholesterol closely related 7-oxocholesterol and 7-dehydrocholesterol. The cholesterol esters are neither substrates nor inhibitors of cholesterol 7alpha-hydroxylase. Only a high concentration (1 mM) of biliary acids or of their glyco or tauro derivatives inhibits cholesterol 7alpha-hydroxylase. The quantitatively less important lithocholic acid and deoxycholic acid are the strongest inhibitors; the most common cholic acid does not affect the enzymic activity even at 1 mM.