PURIFICATION AND PHARMACOLOGICAL STUDY OF ENDOTHELIN CONVERTING ENZYME

内皮素转换酶的纯化及药理研究

• 批准号: 03670116
• 负责人: MATSUSHIMA Yohkazu
• 金额: $ 1.28万
• 依托单位: NATIONAL CARDIOVASCULAR CENTER RESEARCH INSTITUTE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670116/
• 关键词: ENDOTHELIN; BIG ENDOTHELIN; CONVERTING ENZYME; PHOSPHORAMIDON; EDTA; PEPSTATIN A; EDTA; phosphoramidon; エンドセリン; ビッグエンドセリン; 変換酵素

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.

1.内皮素-1（ET-1）的测定是评价内皮素转换酶（ECE）活性的重要指标。建立了ET-1的放射免疫分析法与反相高效液相色谱法相结合的精密测定方法.本实验通过检测ET-1和大ET-1对肾脏作用的影响，探讨大ET-1是否转化为ET-1。肾内注射ET-1可降低肾血流量（RBF）、尿量和肾小球滤过率（GFR），而注射大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。我们制备了通过匀浆和离心大鼠主动脉匀浆获得的粗膜级分。我们观察到ECE活性的粗膜组分，有两个最佳的pH值;中性和酸性。酸性酶活性被天冬氨酸蛋白酶抑制剂胃酶抑素A抑制，而中性酶则被EDTA、EGTA或磷酰胺阻断。进一步的表征和纯化正在研究中。