Hosoi T, Sawyer S T, Krantz S B
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Biochemistry. 1991 Jan 15;30(2):329-35. doi: 10.1021/bi00216a004.
Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.
纯人重组促红细胞生成素（EP）通过一个伯氨基残基与一种交联试剂N-[[3-[[4-[(对叠氮间-[¹²⁵I]碘苯基)偶氮]苯甲酰基]氨基]丙酰基]氧基]-琥珀酰亚胺（丹尼-贾菲试剂）进行酰化反应，该试剂具有光反应性且在偶氮残基处可裂解。通过反相高压液相色谱从未修饰的EP中纯化得到所得的共轭激素（DJ-EP），并且它保持了与红系祖细胞上EP受体结合的能力。通过与¹²⁵I-EP进行化学交联，EP受体先前被鉴定为分子量分别为100 kDa和85 kDa的两种相关蛋白。最近，D'Andrea及其同事[（1989年）《细胞》57卷，277 - 285页]克隆了一个编码55 - 66 kDa蛋白的cDNA，该蛋白被认为是EP受体。在本报告中，通过单功能DJ-EP与受体交联标记出了与¹²⁵I-EP和辛二酸二琥珀酰亚胺酯交联的相同的140 kDa和125 kDa分子量条带（100 kDa和85 kDa蛋白）。此外，连二亚硫酸钠（80℃，5分钟）对DJ-EP受体复合物偶氮键的裂解表明，105 kDa和90 kDa的蛋白以无配体形式被DJ-EP标记。该结果表明，多种蛋白的人为交联或其他交联假象并不能解释通过交联鉴定的EP受体与通过表达克隆鉴定的受体在分子量上的差异。
