EFFECTS OF NOVEL SYNTHETIC STEROL PROBES ON ENZYMES OF CHOLESTEROL-METABOLISM IN CELL-FREE AND CELLULAR-SYSTEMS

A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular chole sterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cho lesterol 7 alpha-hydroxylase. The compounds were 6-fluoro-5-cholesten- 3 beta-ol (6-fluorocholesterol), 7-cholesten-3 beta-ol (7-cholestenol) , 6 beta-fluorocholestan-3 beta-ol (6 beta-fluorocholestanol), 3-aceto xy-6-fluorocholestan-3 beta-ol (3-acetoxy-6-fluorocholestanol) and 7-m ethoxy-5-cholesten-3 beta-ol (7-methoxycholesterol). They were designe d to reveal the effect of small changes in sterol structure, particula rly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3 beta- hydroxy group was essential for interaction with both enzymes since 3- acetoxy-6-fluorocholestanol did not affect any of the enzyme-catalysed reactions. 6-Fluorocholesterol and 7-cholestenol had no effect on cho lesterol esterification but did inhibit the hydroxylation of cholester ol, as did the other compounds with groups that could influence the 7 position, namely 6 beta-fluorocholestanol and 7-methoxycholesterol. Th e fluorocholestanols were all competitive substrates for cholesterol e sterification in cell-free and cellular assays of ACAT activity. 7-Met hoxycholesterol was a surprisingly effective inhibitor of ACAT for a s imple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region a round C-6 and C-7 of the sterol are important. These results show that it is possible to dissect components of cholesterol metabolism using simple, specifically substituted sterols and thus define a new approac h to studying the relationships between the various enzymes that catal yse intracellular cholesterol metabolism.