Hydroxylation of cholesterol
Scholan, Norma A.
This investigation concerns the characteristics of the cholesterol 7a-hydroxylase enzyme which catalyses the initial reaction en route from cholesterol to bile acids. The reaction is considered to have rate-limiting properties since the sole fate of the product (7a-hydroxycholesterol) is its conversion to the two primary bile acids, cholic acid and chenodeoxycholic acid; such metabolic branch-points are targets for regulatory mechanisms. The enzyme system is located in the microsomal fraction of rat liver and requires molecular oxygen, reduced nicotinamide adenine dinucleotide phosphate (NADPH) and cell supernatant factors for optimal activity in vitro. Any study . of cholesterol oxidation is plagued by the ease with which the sterol is attacked non-enzymically by molecular oxygen to yield a variety of products. The microsomal fraction alone produces several oxygenated sterols from cholesterol (cholestan-3[3,5a ,6ptriol, 7p-hydroxycliolesterol, 7-ketocholesterol, s.nd possibly cholesterol-7a-hydroperoxide and cholesterol-5p ,6(3-epoxide) besides 7a-hydroxycholesterol; a similar spectrum of oxidation products results from the autoxidation of cholesterol. However, a selective assay was determined by introducing various physiological and non-physiological compounds into the incubation medium. This eliminated the possible non-enzymic formation of 7a-hydroxycholesterol since no "autoxidation" products were present. The physiological moderating species proved to be a sulfhydryl- containing protein present in the thermostable portion of the cell supernatant - the so-called Soluble Factor or S.F. The purification and characteristics of this cofactor are described; the mode of action of this thiol in controlling the aberrant attack on the substrate is suggested by analogy with the non-physiological moderators of cholesterol oxidation. Two routes of cholesterol oxidation from a possible common intermediate are implied, one to "autoxidation" products and the other to 7a-hydroxycholesterol. The enzyme proved to be released into a more soluble form by freeze-drying the microsomal fraction plus S.F. in the presence of the protective thiol, (3-me r cap toethylamine. Subsequent extraction of the lyophilised material with high molarity phosphate buffer liberated the enzymic components from the microsomal membranes and activity was present in the 105,000 g / 50 mins. supernatant fraction. Certain characteristics of the solubilised preparation were investigated, including the inhibition produced by carbon monoxide and the release of this inhibition by light. These observations suggested the participation of the cytochrome, P450, in the cholesterol 7a-hydroxylation reaction as the terminal electron acceptor and oxygen activator. The activity of the 7a-hydroxylase in the microsomal fraction was studied under various conditions; it was found to be increased by the regimen of cholesterol feeding and by the removal of the influence on the liver of the end products - bile acids - suggesting that a feed-back viicontrol mechanism was operative. The aclrninistration oi barbiturates failed, to affect significantly the activity oi the enzyme, though cytochrome P450 levels were considerably elevated. There was no apparent correlation between P450 levels and the amount of 7a-hydroxycholesterol formed in the microsomal fraction and it is therefore unlikely that this haeinoprotein is the rate-controlling species in the 7a-hydroxylation reaction.