Taylor F R, Shown E P, Kandutsch A A
Jackson Laboratory, Bar Harbor, Maine 04609.
Arch Biochem Biophys. 1991 Aug 1;288(2):567-71. doi: 10.1016/0003-9861(91)90237-d.
The structural organization of the oxysterol receptor, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol biosynthesis in mammalian cells, has been explored by limited proteolysis with trypsin, alpha-chymotrypsin, and endoproteinase GluC. Treatment with each of these proteases converts the receptor from a homodimer of approximately 95 kDa subunits to a 44-kDa form, based on hydrodynamic measurements by sucrose density gradient centrifugation and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled preparations indicates that the oxysterol binding site is on a 28-kDa fragment within the 44-kDa limit form of the receptor. The limit proteolytic form exhibits the high affinity and structural specificity for oxysterols of the native dimeric receptor with an increase in the rate constant of association for 25-hydroxycholesterol. The proteolytic form also shows an increased binding affinity for nonspecific DNA, but no sequence specificity for the oxysterol regulatory element from the reductase gene was detected.
氧甾醇受体的结构组织被推测参与哺乳动物细胞中3-羟基-3-甲基戊二酰辅酶A还原酶的调节和胆固醇生物合成，已经通过用胰蛋白酶、α-胰凝乳蛋白酶和内蛋白酶GluC进行有限的蛋白水解来探索。根据蔗糖密度梯度离心和凝胶过滤色谱的流体动力学测量，用这些蛋白酶中的每一种处理都会将受体从大约95 kDa亚基的同二聚体转化为44 kDa的形式。光亲和标记制剂的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，氧甾醇结合位点位于受体44 kDa极限形式内的一个28 kDa片段上。极限蛋白水解形式对天然二聚体受体的氧甾醇表现出高亲和力和结构特异性，25-羟基胆固醇的缔合速率常数增加。蛋白水解形式对非特异性DNA的结合亲和力也增加，但未检测到对还原酶基因的氧甾醇调节元件的序列特异性。
