Michalak M, Wandler E L, Strynadka K, Catena R, Liu H J, Olley P M
Department of Pediatrics, University of Alberta, Edmonton, Canada.
Biochim Biophys Acta. 1992 Nov 9;1111(2):247-55. doi: 10.1016/0005-2736(92)90317-f.
A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.
一种前列腺素E2（PGE2）受体从心肌肌膜中被溶解并分离出来。其结合特性与膜结合受体几乎相同。[3H]PGE2与溶解的和膜结合的受体的结合对温度升高敏感，且在无NaCl时未观察到结合。未观察到二硫苏糖醇（DTT）、三磷酸腺苷（ATP）、镁离子（Mg2+）、钙离子（Ca2+）或缓冲液pH变化对[3H]PGE2与溶解的或膜结合的受体结合有显著影响。未标记的PGE1从CHAPS溶解的受体上取代了超过90%的[3H]PGE2。前列腺素D2（PGD2）、前列环素（PGI2）、前列腺素F2α（PGF2α）和6-酮-前列腺素F1α（6-keto-PGF1α）不能有效地从受体上取代[3H]PGE2。对[3H]PGE2与CHAPS溶解的受体结合进行Scatchard分析，发现存在两种类型的PGE2结合位点，解离常数（Kd）分别为0.33±0.05 nM和3.00±0.27 nM，最大结合量（Bmax）分别为0.5±0.04和2.0±0.1 pmol/mg蛋白质。功能性PGE2受体通过两种独立方法从CHAPS溶解的肌膜中分离出来：第一种是通过麦胚凝集素（WGA）-琼脂糖凝胶色谱法，第二种是通过蔗糖梯度密度离心法。通过这两种方法分离的受体都能结合[3H]PGE2。未标记的PGE1和PGE2从纯化的受体上取代了[3H]PGE2。对[3H]PGE2与纯化受体结合进行Scatchard分析，发现与膜结合的和CHAPS溶解的受体一样存在两种结合位点。纯化受体组分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示存在一条分子量（M(r)）约为100,000的蛋白带。这条100 kDa的蛋白用[3H]叠氮基-PGE2（PGE2的一种光活性衍生物）进行了光标记。我们认为这条100 kDa的蛋白是心脏PGE2受体。
