The submicrosomal localization of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase, cholesterol 7alpha-hydroxylase and cholesterol in rat liver.

Abstract

In order to determine the submicrosomal distribution of cholesterol 7α‐hydroxylase, 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase and of free cholesterol, the microsomal fraction and the digitonin‐treated microsomal preparation of rat liver were subjected to analytical isopycnic centrifugation on sucrose density gradients.Cholesterol 7α‐hydroxylase, its substrate and hydroxymethylglutaryl‐CoA reductase are present in similar membranes originating from the endoplasmic reticulum with low density of ribosomes. Smooth membranes originating from Golgi apparatus and plasma membranes do not contain appreciable amounts of cholesterol 7α‐hydroxylase and of hydroxymethylglutaryl‐CoA reductase, whilst they contain a considerable portion of the free cholesterol present in microsomal preparations.The addition of trace amounts of [14C]cholesterol to untreated and digitonin‐treated microsomal preparations resulted in heterogeneous labelling of cholesterol as seen from the variation of the specific radioactivity of free cholesterol in the microsomal subfractions. Both with untreated and with digitonin‐treated microsomes the specific radioactivity of cholesterol was minimal in the fractions of maximal contamination with plasma membranes and in those of maximal contamination with smooth membranes from Golgi apparatus. Therefore, the [14C]cholesterol added to the microsomal preparations did not label the plasma membrane and the Golgi membrane cholesterol pools as heavily as it labelled other pools of microsomal cholesterol. The ‘substrate pool’ for cholesterol 7α‐hydroxylase was heavily labelled by [14C]cholesterol added to untreated or digitonin‐treated microsomes and, since the specific radioactivity of 7α‐hydroxycholesterol obtained with various microsomal subfractions from the same preparation was very similar, the ‘substrate pool’ was uniformly labelled.