Division of Nephrology and Hypertension; The Center for Kidney Research and Therapeutics at the Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Sci Rep. 2017 Apr 5;7:45473. doi: 10.1038/srep45473.
Degradation of the biologically potent octapeptide angiotensin Ang II-(1-8) is mediated by the activities of several peptidases. The conversion of Ang II to the septapeptide Ang-(1-7) is of particular interest as the latter also confers organ protection. The conversion is catalyzed by angiotensin-converting enzyme 2 and other enzymes that selectively cleave the peptide bond between the proline and the phenylalanine at the carboxyl terminus of Ang II. The contribution of various enzyme activities that collectively lead to the formation of Ang-(1-7) from Ang II, in both normal conditions and in disease states, remains only partially understood. This is largely due to the lack of a reliable and sensitive method to detect these converting activities in complex samples, such as blood and tissues. Here, we report a fluorometric method to measure carboxypeptidase activities that cleave the proline-phenylalanine dipeptide bond in Ang II. This method is also suitable for measuring the conversion of apelin-13. The assay detects the release of phenylalanine amino acid in a reaction with the yeast enzyme of phenylalanine ammonia lyase (PAL). When used in cell and mouse organs, the assay can robustly measure endogenous Ang II and apelin-13-converting activities involved in the renin-angiotensin and the apelinergic systems, respectively.
生物活性八肽血管紧张素 Ang II-(1-8) 的降解由多种肽酶的活性介导。Ang II 转化为七肽 Ang-(1-7) 特别有趣，因为后者也赋予了器官保护作用。这种转化由血管紧张素转化酶 2 和其他选择性切割 Ang II 羧基末端脯氨酸和苯丙氨酸之间肽键的酶催化。在正常条件和疾病状态下，共同导致 Ang-(1-7) 从 Ang II 形成的各种酶活性的贡献仅部分被理解。这主要是由于缺乏一种可靠和敏感的方法来检测血液和组织等复杂样本中的这些转化活性。在这里，我们报告了一种荧光法来测量羧肽酶活性，该活性可切割 Ang II 中的脯氨酸-苯丙氨酸二肽键。该方法也适用于测量阿肽素 13 的转化。该测定法通过与酵母苯丙氨酸氨裂解酶 (PAL) 反应检测苯丙氨酸氨基酸的释放。当在细胞和小鼠器官中使用时，该测定法可以稳健地测量分别涉及肾素-血管紧张素和阿肽素能系统的内源性 Ang II 和阿肽素 13 转化活性。
