Corder R
William Harvey Research Institute, St. Bartholomew's Hospital Medical College, Charterhouse Square, London, UK.
Biochem Pharmacol. 1996 Feb 9;51(3):259-66. doi: 10.1016/0006-2952(95)02164-7.
The importance of big endothelin-1 (big ET-1) retaining a specific conformation for its conversion to ET-1 has yet to be determined. As a prelude to developing affinity labels for studying the interaction between big ET-1 and endothelin converting enzyme (ECE), the effect on biological activity of modifying human big ET-1 with the N-hydroxysuccinimide esters of 3-(p-hydroxyphenyl)propionic acid (HPP) or S-acetylthioglycolic acid (ATG) was investigated. Mono-derivatized HPP-big-ET-1 and ATG-big-ET-1, and the corresponding ET-1 molecules, were purified by HPLC. The identity of the modified big ET-1 and ET-1 molecules were confirmed by mass spectrometry. Comparison of the pressor activities with big ET-1 (1 nmol/kg) in anaesthetized rats showed the responses to equivalent doses of HPP-big-ET-1 and ATG-big-ET-1 to be reduced by 67% and 73%, respectively. In contrast, the same modifications to ET-1 had no significant effect on blood pressure responses or vasoconstrictor activity on the isolated rat thoracic aorta. To evaluate the effect of these modifications on the conversion of big ET-1 to ET-1, cultured bovine aortic smooth muscle (BASMC) and endothelial (BAEC) cells were used as sources of endothelin converting enzyme activity. After a 4-hr incubation of the modified molecules with intact cells, the quantity of ET-1 immunoreactivity generated was compared to that from unmodified big ET-1. The amount of conversion, relative to big ET-1 (1 microM), for HPP-big-ET-1 was reduced by 21% for BAEC and by 50% for BASMC. The corresponding decreases for ATG-big-ET-1 were 79% and 82%. Because of the large decreases in the level of conversion, the linear big ET-1 molecule S-carboxy-amidomethylated big ET-1 (CM-big-ET-1) was prepared for comparison. Incubations of CM-big-ET-1 with BAEC and BASMC yielded only 53% and 23%, respectively, of the ET-1 immunoreactivity obtained with unmodified big ET-1. Thus, incorporation of the HPP or ATG groups, or removal of disulphide bridges decreases the ability of plasma membrane ectoenzyme ECE activities to hydrolyze the Trp21-Val22 bond of big ET-1. This indicates that the conformation of big ET-1 is important for obtaining an optimal rate of hydrolysis by ECE activities in vivo and in vitro. Further evidence of secondary structure was obtained from studies of the crossreactivity of big ET-1 in two RIAs recognising the ET-1 sequence.
大内皮素 -1（big ET-1）保持特定构象对于其转化为ET-1的重要性尚未确定。作为开发用于研究big ET-1与内皮素转化酶（ECE）相互作用的亲和标记的前奏，研究了用3 -（对羟基苯基）丙酸（HPP）或S -乙酰硫代乙醇酸（ATG）的N -羟基琥珀酰亚胺酯修饰人big ET-1对其生物活性的影响。通过高效液相色谱法（HPLC）纯化单衍生化的HPP - big - ET-1和ATG - big - ET-1以及相应的ET-1分子。通过质谱法确认修饰的big ET-1和ET-1分子的身份。在麻醉大鼠中比较big ET-1（1 nmol/kg）的升压活性表明，等效剂量的HPP - big - ET-1和ATG - big - ET-1的反应分别降低了67％和73％。相比之下，对ET-1进行相同的修饰对分离的大鼠胸主动脉的血压反应或血管收缩活性没有显著影响。为了评估这些修饰对big ET-1转化为ET-1的影响，使用培养的牛主动脉平滑肌（BASMC）和内皮（BAEC）细胞作为内皮素转化酶活性的来源。将修饰后的分子与完整细胞孵育4小时后，将产生的ET-1免疫反应性的量与未修饰的big ET-1产生的量进行比较。相对于big ET-1（1 microM），BAEC中HPP - big - ET-1的转化量降低了21％，BASMC中降低了50％。ATG - big - ET-1的相应降低分别为79％和82％。由于转化水平大幅下降，制备了线性big ET-1分子S -羧基酰胺甲基化big ET-1（CM - big - ET-1）用于比较。CM - big - ET-1与BAEC和BASMC孵育分别仅产生未修饰的big ET-1所获得的ET-1免疫反应性的53％和23％。因此，引入HPP或ATG基团或去除二硫键会降低质膜外切酶ECE活性水解big ET-1的Trp21 - Val22键的能力。这表明big ET-1的构象对于在体内和体外通过ECE活性获得最佳水解速率很重要。从big ET-1在两种识别ET-1序列的放射免疫分析（RIA）中的交叉反应性研究中获得了二级结构的进一步证据。
