PURIFICATION AND PHARMACOLOGICAL STUDY OF ENDOTHELIN CONVERTING ENZYME

1.测定内皮素-1(ET1)含量是评价内皮素转换酶(ECEs)活性的关键。我们建立了放射免疫法结合反相HPLC.2精确测定ET-1的方法。我们检测了ET-1和BIG ET-1对肾脏活动的影响，以研究BIG ET-1是否转化为ET-1。肾内注射ET-1可降低大鼠肾血流量(RBF)、尿量和肾小球滤过率(GFR)，而大ET-1对RBF、GFR和尿量无明显影响，提示大ET-1不能转化为成熟ET-1。我们观察到具有中性和酸性两种最适pH的粗膜组分中的ECEs活性。酸性酶活力可被天冬氨酸蛋白酶抑制剂胃抑素A抑制，中性酶活力可被EDTA、EGTA或磷酰胺阻断。进一步的鉴定和提纯工作正在进行中。

1. It is essential for the assessment of endothelin converting enzyme (ECE) activity to measure endothelin-1(ET1). We developed the precise measurement for ET-1 using radioimmunoassay combined with reverse phase HPLC.2. We examined the effects of ET-1 and big ET-1 on the renal action to study whether or not big ET-1 is converted to ET-1. Intrarenal infusion of ET-1 decreased renal blood flow(RBF), urine output and glomerular filtration rate (GFR), whereas infusion of big ET-1 did not significantly affect RBF, GFR, and urine output, suggesting that big ET-1 could not be converted to mature ET-1.1992To characterize the endothelin converting enzyme (ECE) in rat aorta, we prepared the crude membrane fractions obtained by homogenization and centrifugation of homogenate of rat aorta. We observed the ECE activities in the crude membrane fractions which had two optimal pH ; neutral and acidic. Acidic enzyme activity was inhibited by pepstatin A, aspartic protease inhibitor, whereas neutral enzyme was blocked by EDTA, EGTA or phosphoramidon. Further characterization and purification are under investigation.