The conformation of human big endothelin-1 favours endopeptidase hydrolysis of the TRP21-VAL22 bond.

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.