Studies on Cholesterol Esterase in the Rat Adrenal*

Abstract

We have investigated the characteristics and properties of cholesterol esterase (CEase) in the rat adrenal to clarify the mechanism of synthesis of free cholesterol from esterified cholesterol. CEase activity was estimated using substrate vesicles which were sonicated mixtures of cholesteryl oleate and phosphatidylcholine (PC). CEase showed two optima at around pH 4.5 and pH 8.25. The acid CEase was found mainly in the lysosomal fraction, and the alkaline CEase was found mainly in the microsomal fraction. Both acid and alkaline CEase activities were markedly enhanced by increasing the concentration of PC, but not by phospholipids. The activities were not increased by the addition of cAMP, ATP, and MgCl2, when the molar ratio of cholesteryl oleate to PC was 1:0.2, but were increased when the molar ratio was 1:2. Thus, it is suggested that free cholesterol for steroidogenesis may be supplied by two different organelles (lysosome and microsome), and that the composition of the substrate complex, such as the ratio of esterified cholesterol to phospholipids, may play a crucial role in the regulation of adrenal CEase. Increases in both acid and alkaline CEase activities were observed in adrenal homogenates prepared from hypophysectomized rats treated with an iv injection of ACTH when the molar ratio of cholesteryl oleate to PC used as a substrate vesicle was 1:0.2, but not when the ratio was 1:2. Therefore, the present observation indicate that activation of CEase by ACTH is dependent on the substrate state, especially that of the cholesterol ester droplet in vivo.

Regulation of Cholesterol Metabolism in Adrenal Cortex: Comparative Studies on Cholesterol Esterase in Human Adrenal Glands.

“…The postnuclear supernatant was used as an enzyme solution for estimating CEase activities. For studying subcellular distribution of CEase, adrenocortical tissues were also homogenized in 5 mM Tris-HC1 buffer, pH 7.4 containing 0.25M sucrose and 0.5 mM CaCl2, using a Potter-Elvejem type glass/Teflon homogenizer as previously reported [8,9]. The homogenate was centrifuged for 10 min to sediment the nuclei.…”

“…CEase activities were estimated as reported previously [8][9][10]. Acid phophatase and NADPH cytochrome C reductase were estimated for maker enzymes of lysosome and microsome, respectively, as previously reported [9].…”

“…4 [5][6][7], and adrenal CEase activity can be significantly enhanced by ACTH [8,11]. The nature and regulation of adrenal CEase had been well studied in rats [8], cattle [12], and guinea pigs [9], while those of CEase in human adrenals were still obscure. In this study, we used a homogenate of radioactive cholesteryl oleate and phosphatidylcholine in order to estimate the CEase activity in various human adrenals.…”

“…Adrenal CEase is an important enzyme, which can convert cholesterol esters into free cholesterol, thus providing it as a substrate for the cholesterol sidechain cleavage enzyme complex which is known to be the rate-limiting step for adrenal steroidogenesis. CEase has several unique biochemical charac-teristics as previously reported by authors using rat adrenal glands [8]. There exist two distinct CEases, acid CEase located in lysosomes and alkaline CEase in microsomes, and the regulation of these two enzymes is not only influenced by ACTH, but also by the physical state of the substrate such as phosphatidylcholine and apoproteins [l0].…”

“…-4]. It is well known that free cholesterol for adrenal steroidogenesis is mainly provided by hydrolysis of the deposited cholesterol esters by cholesterol esterase (CEase) [5][6][7][8]. Adrenal CEase is an important enzyme, which can convert cholesterol esters into free cholesterol, thus providing it as a substrate for the cholesterol sidechain cleavage enzyme complex which is known to be the rate-limiting step for adrenal steroidogenesis.…”