Studies on Rat‐Liver Microsomal Cholesterol 7α‐Hydroxylase

Abstract

Cholesterol 7or-hydroxylase or cholesterol, reduced NADP : oxygen oxidoreductase (7a-hydroxylating), the initial and possibly rate-limiting step in bile acid formation is a mixed function oxidase of rat liver microsomes. This enzyme requires NADPH and oxygen. The enzyme system is induced by feeding the bile salt sequestering agent, cholestyramine, this compound effectively breaks the enterohepatic circulation of bile salts and cause about a five-fold increase in the activity of the enzyme within four days.The cholesterol 7a-hydroxylase enzyme has a K , for oxygen of approximately 20 pM. It is inhibited by carbon monoxide and this inhibition is released by irradiation with light of 450 nm. The enzyme is not inhibited by cyanide but is inhibited by cytochrome c and p-chloromercuribenzoate. The enzyme therefore appears to be a typical liver microsomal mixed function oxidase, involving NADPH, cytochrome c reductase and cytochrome P450.The cholesterol 7or-hydroxylase has an activation energy of 22 kcal/mol and is inhibited by the product of the reaction, namely, 7or-hydroxycholesterol and ah0 inhibited by bile acids and bile salts.

Bioconversion of 3β-hydroxy-5-cholenoic acid into chenodeoxycholic acid by rat brain enzyme systems

“…First, we incubated a 1:1 mixture of 18 O-labeled and unlabeled 3␤-hydroxy-5-cholenoic acid with rat brain microsomal preparations in the presence of NADPH at 37ЊC. Two new peaks were produced in the incubation mixture at m/z 389 and m/z 391, and their retention times coincided with the authentic specimens of 3␤,7␣-dihydroxy-5-cholenoic acid (Fig.…”

“…18 2 -CDCA and 12-oxolithocholic acid used as internal standards (ISs) were synthesized in our laboratory using previously reported methods (10). Sephadex LH-20 was obtained from Pharmacia (Uppsala, Sweden).…”

“…A tracer that can clearly distinguish target compounds from endogenous contaminants requires almost the same physicochemical properties as the corresponding endogenous molecule; among the possible stable isotopes, the 18 O atom is suitable for this purpose. The steroids used in this study have both of the hydroxyl groups on the steroid nucleus and have a carboxyl group at the side chain.…”

“…However, a hydroxyl group at the C-3 position is not suitable for labeling, because of the possibilities of elimination and isotope effects during enzymatic reactions. Therefore, we introduced an 18 O atom into the carboxyl group at the C-24 position according to the method previously reported by Strife and Murphy (13).…”

“…2A. The enzymatic formation of the product increased until 120 min, and the ratio of 18 O-labeled and unlabeled products was constant until the end of the reaction. This suggests that the products were produced from the substances added to the incubation mixture.…”