摘要
目的探讨不同BigET 1类似物对内皮素转换酶 (ECE)活性的影响、ECE对底物的作用机制 ,以寻找ECE的抑制物。方法克隆人ECE的cDNA ,将其亚克隆至表达载体 ,然后把该表达载体导入CHO K1细胞内使其表达。将该ECE分别加入不同浓度的多种BigET 1类似物中 ,无BigET 1类似物的作为对照。ECE的活性则通过放免法直接测定ET 1的生成量而反映的。结果在多种BigET 1类似物中 [Phe2 1]BigET 1(18- 34)和 [Ala3 1]BigET 1(18- 34)以不同的形式抑制了ECE的活性 ,前者为竞争性抑制 ,其抑制常数Ki为 (2 0 .6± 3.8) μmol/L ;后者为非竞争性抑制 ,其抑制常数Ki为 (35 .6± 8.1) μmol/L。 结论ECE能够识别其底物BigET 1的活性部位和C末端的 2个区域 ,此 2个区域也以不同的形式被该酶所识别。
Objective To study the effects of variously substituted analogues of big endothelin-1(BigET-1) on ECE-1 activity and the mechanism of ECE active and to identify the inhibitors of ECE. Methods cDNA encoding ECE was cloned and then it was subcloned to the expressed vector. The resultant expression plasmid constructs were then transfected into CHO-K1 cells. The expressed protein contain the activity of native ECE. Then solubilized CHO-K1 membranes were incubated in Tris-HCL containing varied concentrations of BigET-1 analogues, none as control.The effects of analogues at various concentration on ECE activity were examind by a specific radioimmunoassay to measure the amout of ET-1. Results Among the BigET-1 analigues tested, [Phe21]BigET-1(18-34) and [Ala31]BigET-1(18-34) exhibited a significant inhibition of ECE. The former was a competitive inhibitor (Ki=20.6 3.8 μmol/L); while the latter was a non-compectitive inhibitor (Ki=35.6±8.1 μmol/L). Conclusion ECE-1 recognizes BigET-1 both at the substrate active position and the C-terminal region and two regions are recognized by this enzyme in a different manner.
出处
《上海第二医科大学学报》
CSCD
2004年第B11期37-39,共3页
Acta Universitatis Medicinalis Secondae Shanghai
