The porcine LH/hCG receptor. Characterization and purification.
The porcine LH/hCG receptor. Characterization and purification.
复制标题
猪 LH/hCG 受体。
DOI：
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发表时间：
1985
影响因子：
4.8
通讯作者：
Thomas T. Chen
中科院分区：
文献类型：
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作者：
Jayantha Wimalasena;P. Moore;John P. Wiebef;John A. Abel;Thomas T. Chen
在25%甘油和蛋白酶抑制剂存在下，用TritonX-100溶解猪黄体LH/hCG受体（LH/hCG R），回收率为70-80%。溶解的受体在-60 ℃下保持90%的原始活性90天。观察到整个匀浆、质膜组分和溶解的LH/hCG R制剂的平衡结合常数（Ka）值分别为1.92、2.22和2.03 X 10（10）M-1。相同组分的特异性结合容量分别为49、70、55 fmol/mg蛋白。LH/hCG R和Triton X-100的复合物经Sepharose 6 B凝胶过滤分离为两个组分，其Mr分别约为2.7 × 10 ~（5）和5.4 × 10 ~（5）;通过在高度纯化的hCG-琼脂糖上进行两个循环的亲和层析来纯化溶解的猪LH/hCG R，在Triton提取物中的初始活性的总回收率为30-35%。纯化的猪LH/hCG R的特异性结合容量为2300 pmol/mg蛋白，Ka = 1.5 × 10（10）M-1。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳凝胶的银染色表明，猪LH/hCG R制剂中的主要蛋白质具有Mr = 68，000。在纯化的受体制剂中也观察到Mr = 45，000的弱染色条带。用放射自显影法分析碘化的纯化LH/hCG R证实了这些结果。猪LH/hCG R纯化40，000倍。
Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.