Quantitation of the pool of cholesterol associated with acyl-CoA: Cholesterol acyltransferase in human fibroblasts
Quantitation of the pool of cholesterol associated with acyl-CoA: Cholesterol acyltransferase in human fibroblasts
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DOI：
10.1074/jbc.272.20.13103
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发表时间：
1997-05-16
影响因子：
4.8
通讯作者：
Steck, TL
中科院分区：
文献类型：
--
作者：
Lange, Y;Steck, TL
探讨了人成纤维细胞匀浆中胆固醇的酯化作用，作为估计体内内质网（ER）相关胆固醇池大小的一种手段。其基本原理是，酰基辅酶A：胆固醇酰基转移酶（ACAT）在匀浆中应该只有与胆固醇相关的（粗糙）ER膜片段，它驻留。将整个匀浆与过量的[C-14]油酰辅酶A反应至完成，将约0.1-2%的总细胞游离胆固醇转化为[C-14]胆固醇酯。对照研究表明，与ACAT无关的细胞膜对胆固醇的酯化反应没有贡献。暴露完整的细胞血清脂蛋白，氧化固醇，或鞘磷脂酶增加胆固醇酯化匀浆数倍;暴露细胞mevinolin或胆固醇氧化酶有相反的效果。胆固醇酯化的变化与细胞总胆固醇或ACAT的内在活性均不相关，两者均未被预处理显著改变。相反，在匀浆中酯化的胆固醇的总量超过相应完整细胞中胆固醇酯化的速率。因此，体外酯化的胆固醇库似乎反映了与体内ER相关的胆固醇库。由于保持细胞胆固醇处于严格反馈控制下的几种机制本身位于ER中，因此该池可能不仅在生理上受到调节，而且反过来有助于调节稳态效应子途径。
The esterification of cholesterol in homogenates of human fibroblasts was explored as a means of estimating the size of the pool of cholesterol associated with the endoplasmic reticulum (ER) in vivo. The rationale was that the acyl-coenzyme A:cholesterol acyltransferase (ACAT) in homogenates should have access only to cholesterol associated with the (rough) ER membrane fragments in which it resides. Reacting whole homogenates to completion with an excess of [C-14]oleoyl-CoA converted similar to 0.1-2% of total cell free cholesterol to [C-14]cholesteryl esters. Control studies indicated that membranes not associated with ACAT did not contribute cholesterol to this reaction.The extent of in vitro cholesterol esterification varied with pretreatment of the cells. Exposing intact cells to serum lipoproteins, oxysterols, or sphingomyelinase increased cholesterol esterification in homogenates severalfold; exposing the cells to mevinolin or cholesterol oxidase had the opposite effect. The variation in cholesterol esterification did not correlate with either the total cellular cholesterol or the intrinsic activity of ACAT, neither of which was changed significantly by the pretreatments. Rather, the total amount of cholesterol esterified in homogenates paralleled the rate of cholesterol esterification in the corresponding intact cells. The pool of cholesterol esterified in vitro therefore ap pears to reflect that associated with the ER in vivo. Since several of the mechanisms keeping cell cholesterol under tight feedback control are themselves located in the ER, this pool might not only be regulated physiologically, but could, in turn, help to regulate homeostatic effector pathways.