Lam Le Hoang, Shimamura Tomoko, Sakaguchi Ken, Noguchi Katsuya, Ishiyama Munetaka, Fujimura Yume, Ukeda Hiroyuki
Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan.
Anal Biochem. 2007 May 15;364(2):104-11. doi: 10.1016/j.ab.2007.02.017. Epub 2007 Feb 22.
Hypertension and related diseases afflict millions of individuals worldwide, and many investigations of angiotensin I-converting enzyme (ACE) activity have been carried out. Most of these have used hippuryl-histidyl-leucine (HHL) as a substrate for ACE reaction with considerable interferences. Here we propose the use of a new substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG) for the screening of ACE inhibitors. Under the actions of ACE and aminoacylase, 3HB-GGG is cleaved into amino acids (Gly and Gly-Gly) and 3-hydroxybutyric acid (3HB). The assay conditions were optimized and applied to monitor the ACE inhibitory activity in terms of 3HB measured using an F-kit. Under the optimum assay parameters-ACE (0.2 U/ml) and aminoacylase (172 kU/ml) incubated with 3HB-GGG (3.4 mg/ml) at 37 degrees C for 30 min-the Gly-Gly and Gly cleaved from 3HB-GGG by enzymes was able to be identified, affirming the feasibility of substituting 3HB-GGG for the conventional substrate HHL. In addition, the current method was more sensitive, accurate, rapid, and convenient than the conventional method.
高血压及相关疾病困扰着全球数百万人，人们已经对血管紧张素I转换酶（ACE）活性进行了许多研究。其中大多数研究使用马尿酰 - 组氨酰 - 亮氨酸（HHL）作为ACE反应的底物，但存在相当多的干扰。在此，我们提出使用一种新的底物3 - 羟基丁酰甘氨酰 - 甘氨酰 - 甘氨酸（3HB - GGG）来筛选ACE抑制剂。在ACE和氨基酰化酶的作用下，3HB - GGG被裂解为氨基酸（甘氨酸和甘氨酰 - 甘氨酸）和3 - 羟基丁酸（3HB）。对测定条件进行了优化，并应用于通过使用F试剂盒测量3HB来监测ACE抑制活性。在最佳测定参数下 - ACE（0.2 U/ml）和氨基酰化酶（172 kU/ml）与3HB - GGG（3.4 mg/ml）在37℃孵育30分钟 - 能够鉴定出被酶从3HB - GGG裂解出来的甘氨酰 - 甘氨酸和甘氨酸，证实了用3HB - GGG替代传统底物HHL的可行性。此外，当前方法比传统方法更灵敏、准确、快速且方便。
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