Evidence for the Compartmentation of Cholesterol in Rat‐Liver Microsomes

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Abstract
The formation of 7α‐hydroxycholesterol from cholesterol by membrane‐bound cholesterol 7α‐hydroxylase in rat‐liver microsomes was investigated in vitro. When [14C]cholesterol of known specific radioactivity was incubated in suspensions of rat‐liver microsomes, the specific radioactivity of the total cholesterol in the suspension compared with that of the 7α‐hydroxychol‐esterol formed during the incubation showed that [14C]cholesterol entered a pool of cholesterol in the microsomes (substrate pool) accessible to the enzyme. Estimates of the maximal size of the substrate pool suggested that not more than 70% of the cholesterol in rat‐liver microsomes is immediately accessible to cholesterol 7α‐hydroxylase. The extent to which [14C]cholesterol entered the substrate pool was influenced by the form in which the [14C]cholesterol was added to the suspension. The specific radioactivity of 7α‐hydroxycholesterol reached a maximum value early during the incubation and remained constant thereafter. When nonradioactive cholesterol was added to a microsomal suspension pre‐incubated with a trace of [14C]cholesterol, the proportion of the nonradioactive cholesterol that entered the substrate pool was smaller than that of the [14C]cholesterol. When [14C]cholesterol was injected intravenously into rats, the [14C]cholesterol that entered the liver microsomes was found to be predominantly in the portion of microsomal cholesterol that is not accessible to cholesterol 7α‐hydroxylase.