Hwang P L
Department of Physiology, National University of Singapore.
Biochim Biophys Acta. 1990 Feb 26;1033(2):154-61. doi: 10.1016/0304-4165(90)90006-i.
Oxygenated derivatives of cholesterol are known to exhibit a number of biological activities including the inhibition of cholesterol biosynthesis and of cell proliferation, but their mechanism of action remains unclear. Previous studies have identified a cytosolic protein which binds 25-hydroxycholesterol, as well as several other oxysterols, with high affinity, possibly mediating some of their effects. We now report the existence of a high-affinity oxysterol binding site in rat liver microsomes which is distinct from the cytosolic binding protein. Among the oxygenated sterols examined, 5 alpha-cholestan-3 beta-ol-7-one (7-ketocholestanol) had the highest affinity for this microsomal binding site (Kd = 2.7 nM). Using 7-keto[3H]cholestanol as the radioactive ligand, we found that binding of this oxysterol to the microsomal binding site was saturable and reversible and was displaceable by the following oxysterols in descending order of potency: 7-ketocholestanol greater than 6-ketocholestanol greater than 7 beta-hydroxycholesterol = 7-ketocholesterol greater than cholesten-3 beta,5 alpha, 6 beta-triol = 7 alpha-hydroxycholesterol greater than 4-cholesten-3-one. All other sterols studied, including, notably, 25-hydroxycholesterol, had little or no inhibitory effect on 7-keto[3H]cholestanol binding. Additional studies revealed that the microsomal oxysterol binding site was probably identical to the antiestrogen binding site described by other workers. First, saturation analysis and kinetic studies demonstrated that the antiestrogen tamoxifen competed directly with 7-keto[3H]cholestanol for the same binding site. Second, the ability of different oxysterols and antiestrogens to inhibit 7-keto[3H]cholestanol binding to the microsomal binding site paralleled their ability to inhibit [3H]tamoxifen binding to the antiestrogen binding site. Third, the tissue distribution of binding sites for 7-keto[3H]cholestanol was similar to that of the antiestrogen binding site. We conclude that: (1) in rat liver microsomes there are high-affinity oxysterol binding sites whose ligand specificity is different from that of the cytosolic oxysterol binding protein; and (2) the microsomal oxysterol binding site is probably identical to the antiestrogen binding site. The biological significance of these observations remains to be explored.
已知胆固醇的氧化衍生物具有多种生物活性，包括抑制胆固醇生物合成和细胞增殖，但其作用机制仍不清楚。先前的研究已鉴定出一种胞质蛋白，它能与25-羟基胆固醇以及其他几种氧化甾醇高亲和力结合，可能介导了它们的一些效应。我们现在报告在大鼠肝微粒体中存在一个高亲和力的氧化甾醇结合位点，它与胞质结合蛋白不同。在所检测的氧化甾醇中，5α-胆甾烷-3β-醇-7-酮（7-酮胆甾醇）对该微粒体结合位点具有最高的亲和力（解离常数Kd = 2.7 nM）。以7-酮[³H]胆甾醇作为放射性配体，我们发现这种氧化甾醇与微粒体结合位点的结合是可饱和且可逆的，并且能被以下氧化甾醇以效力递减的顺序取代：7-酮胆甾醇＞6-酮胆甾醇＞7β-羟基胆固醇 = 7-酮胆固醇＞胆甾-3β,5α,6β-三醇 = 7α-羟基胆固醇＞4-胆甾烯-3-酮。所有其他研究的甾醇，尤其是25-羟基胆固醇，对7-酮[³H]胆甾醇的结合几乎没有抑制作用。进一步的研究表明微粒体氧化甾醇结合位点可能与其他研究者描述的抗雌激素结合位点相同。首先，饱和分析和动力学研究表明抗雌激素他莫昔芬与7-酮[³H]胆甾醇直接竞争同一个结合位点。其次，不同氧化甾醇和抗雌激素抑制7-酮[³H]胆甾醇与微粒体结合位点结合的能力与其抑制[³H]他莫昔芬与抗雌激素结合位点结合的能力平行。第三，7-酮[³H]胆甾醇结合位点的组织分布与抗雌激素结合位点的相似。我们得出以下结论：（1）在大鼠肝微粒体中存在高亲和力的氧化甾醇结合位点，其配体特异性与胞质氧化甾醇结合蛋白不同；（2）微粒体氧化甾醇结合位点可能与抗雌激素结合位点相同。这些观察结果的生物学意义仍有待探索。
