Koike S, Nii A, Sakai M, Muramatsu M
Biochemistry. 1987 May 5;26(9):2563-8. doi: 10.1021/bi00383a023.
For the purpose of characterizing the estrogen binding domain of porcine estrogen receptor (ER), we have made use of affinity labeling of partially purified ER with [3H]tamoxifen aziridine. The labeling is very efficient and selective particularly after partial purification of ER. A 65,000-dalton (65-kDa) band was detected on the fluorogram of a sodium dodecyl sulfate-polyacrylamide gel, together with a 50-kDa band and a few more smaller bands. The 50-kDa protein appears to be a degradation product of the 65-kDa protein in view of the similar peptide map. ER was affinity labeled before or after controlled limited proteolysis with either trypsin, papain, or alpha-chymotrypsin. The labeling patterns of limited digests indicate that a fragment of about 30 kDa is relatively resistant to proteases and has a full and specific binding activity to estrogen, whereas smaller fragments have lost much of the binding activity. This fragment is very hydrophobic and probably corresponds to the carboxy half of ER.
为了表征猪雌激素受体（ER）的雌激素结合结构域，我们利用[3H]他莫昔芬氮丙啶对部分纯化的ER进行亲和标记。这种标记非常高效且具有选择性，尤其是在ER部分纯化之后。在十二烷基硫酸钠-聚丙烯酰胺凝胶的荧光图谱上检测到一条65,000道尔顿（65 kDa）的条带，以及一条50 kDa的条带和几条更小的条带。鉴于相似的肽图，50 kDa的蛋白质似乎是65 kDa蛋白质的降解产物。在用胰蛋白酶、木瓜蛋白酶或α-胰凝乳蛋白酶进行可控的有限蛋白酶解之前或之后，对ER进行亲和标记。有限酶解的标记模式表明，一个约30 kDa的片段相对抗蛋白酶，并且对雌激素具有完全和特异性的结合活性，而较小的片段则失去了大部分结合活性。这个片段非常疏水，可能对应于ER的羧基末端一半。
