24-, 25- and 27-hydroxylation of cholesterol by a purified preparation of 27-hydroxylase from pig liver.

Pig liver mitochondria were found to catalyze 27-, 25- and 24-hydroxylation of cholesterol at relative rates of about 1:0.2:0.04. An apparently homogeneous preparation of pig liver mitochondrial cytochrome P-450-27 was found to catalyze the same three hydroxylations at about the same relative rates when reconstituted with adrenodoxin and adrenodoxin reductase. The 24-hydroxycholesterol formed was shown to consist of one of the two possible stereoisomers. When using specifically deuterium-labeled substrates a significant isotope effect was observed in the case of 24-hydroxylation (KH/KD > 10), but not 25-hydroxylation (KH/KD = 1.1), or 27-hydroxylation (KH/KD = 1.1). The difference between the 24-hydroxylation and the other two hydroxylations may be due to different interactions between cholesterol and the same enzyme, with a resulting difference with respect to the rate-limiting step in the reaction. The physiological significance of the mitochondrial 24-hydroxylation is discussed.