7-ALPHA HYDROXYLATION OF 25-HYDROXYCHOLESTEROL IN LIVER-MICROSOMES - EVIDENCE THAT THE ENZYME INVOLVED IS DIFFERENT FROM CHOLESTEROL 7-ALPHA-HYDROXYLASE

Rat, pig and human liver microsomes were found to catalyze 7 alpha-hydroxylation of 25-hydroxycholesterol. In contrast to cholesterol 7 alpha-hydroxylase activity, the 7 alpha-hydroxylase activity towards 25-hydroxycholesterol in rat liver was not stimulated by cholestyramine treatment. After transfection with cDNA for human cholesterol 7 alpha-hydroxylase, COS cells showed a significant activity towards cholesterol but not towards 25-hydroxycholesterol. During purification of cholesterol 7 alpha-hydroxylase from pig liver microsomes, about 99% of the 7 alpha-hydroxylase activity towards 25-hydroxycholesterol and 27-hydroxycholesterol was clearly separated from 7 alpha-hydroxylase activity for cholesterol. The small amount of 25-hydroxycholesterol 7 alpha-hydroxylase activity retained in a partially purified preparation of cholesterol 7 alpha-hydroxylase was not inhibited by addition of cholesterol, indicating that the oxysterol binding site is different from the cholesterol binding site, presumely due to the presence of two different enzymes. It is concluded that different enzymes are involved in 7 alpha-hydroxylation of cholesterol and 7 alpha hydroxylation of side-chain-oxidized cholesterol in rat, pig and human liver. Inhibition experiments with a partially purified fraction of the oxysterol 7 alpha-hydroxylase from pig liver gave results consistent with the contention that the same enzyme is responsible for 7 alpha hydroxylation of both 25-hydroxycholesterol and 27-hydroxycholesterol. It has been suggested that cholesterol 7 alpha-hydroxylase can preferentially use oxysterols, in particular 25-hydroxycholesterol, as substrates and by this means inactivate important physiological regulators of cholesterol homeostasis. Such a mechanism would explain the unique property of the liver to resist down-regulation of the low-density-lipoprotein receptor [Dueland, S., Trawick, J. D., and Davies, R. A, (1993) J. Biol. Chern. 267, 22695-22698]. The present results do not support the contention that the important coupling between cholesterol 7 alpha-hydroxylase activity, the low-density-lipoprotein receptor activity and hydroxymethylglutaryl coenzyme A reductase activity in liver cells is due to inactivation of 25-hydroxycholesterol or 27-hydroxycholesterol by the action of cholesterol 7 alpha-hydroxylase.