Ogier S A, Mitchell R, Bladon C M
MRC Brain Metabolism Unit, Edinburgh, Scotland.
Biochem J. 1989 Mar 15;258(3):881-8. doi: 10.1042/bj2580881.
A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.
已合成了多种新型促黄体生成激素释放激素（LHRH）类似物，这些类似物结合了生物素以及潜在的共价连接位点。基于去甘氨酸10-[D-赖氨酸6]-LHRH乙酰胺肽主链的类似物在与大鼠垂体前叶细胞膜上的LHRH受体结合方面表现出最有用的特性。其中，去甘氨酸10-[生物素基氨基乙基甘氨酰-D-赖氨酸6]-LHRH乙酰胺（XBAL）具有最佳的特异性：非特异性结合比，通过Scatchard分析测定，总结合的44±6%（±标准误）为特异性结合，解离常数Kd为131±16 pM（±标准误，n = 4）。已使用两种方法将这些类似物与LHRH受体共价交联；光亲和标记法和使用同双功能N-羟基琥珀酰亚胺酯交联剂。光亲和类似物的特异性：非特异性结合比很差。在所测试的化学交联剂中，发现乙二醇双（琥珀酰亚胺基琥珀酸酯）（EGS）在共价连接与LHRH受体位点结合的125I-XBAL方面效率最高。在EGS浓度为5 mM时，125I-XBAL特异性结合的23±3%（±标准误）被共价交联。
