TY - JOUR
T1 - Effects of novel synthetic sterol probes on enzymes of cholesterol metabolism in cell-free and cellular systems
AU - Harte, Rachel A.
AU - Yeaman, Stephen J.
AU - McElhinney, John
AU - Suckling, Colin J.
AU - Jackson, Brian
AU - Suckling, Keith E.
N1 - Funding Information:
RAH was supported by a collaborative studentship from the Medical Research Council (UK). JMcE was supported by a grant from the Science and Engineering Research Council.
PY - 1996/9/16
Y1 - 1996/9/16
N2 - A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7α-hydroxylase. The compounds were 6-fluoro-5-cholesten-3β-ol (6-fluorocholesterol), 7-cholesten-3β-ol (7-cholestenol), 6β-fluorocholestan-3β-ol (6β-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3β-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3β-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3β-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 cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6β-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around 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 approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.
AB - A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7α-hydroxylase. The compounds were 6-fluoro-5-cholesten-3β-ol (6-fluorocholesterol), 7-cholesten-3β-ol (7-cholestenol), 6β-fluorocholestan-3β-ol (6β-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3β-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3β-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3β-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 cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6β-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around 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 approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.
KW - novel synthetic sterol probes
KW - enzymes of cholesterol
KW - novel sterols
KW - intracellular cholesterol metabolism
UR - http://www.scopus.com/inward/record.url?scp=0030590507&partnerID=8YFLogxK
U2 - 10.1016/0009-3084(96)02593-5
DO - 10.1016/0009-3084(96)02593-5
M3 - Article
C2 - 8858832
AN - SCOPUS:0030590507
SN - 0009-3084
VL - 83
SP - 45
EP - 59
JO - Chemistry and Physics of Lipids
JF - Chemistry and Physics of Lipids
IS - 1
ER -