Enzymatic properties of human aminopeptidase A - Regulation of its enzymatic activity by calcium and angiotensin IV
Enzymatic properties of human aminopeptidase A - Regulation of its enzymatic activity by calcium and angiotensin IV
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DOI：
10.1074/jbc.m603191200
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发表时间：
2006-08-18
影响因子：
4.8
通讯作者：
Tsujimoto, Masafumi
中科院分区：
文献类型：
--
作者：
Goto, Yoshikuni;Hattori, Akira;Tsujimoto, Masafumi
氨肽酶A（阿帕）是一种II型膜结合蛋白，在脑肾素-血管紧张素系统中参与血压调节。在这项研究中，阿帕的重组可溶性形式在杆状病毒系统中表达，纯化至同质，并进行了表征。通过使用合成的底物，它表明，虽然酶具有相当广泛的底物特异性在Ca 2+的情况下，在Ca 2+的存在下，观察到的酸性氨基酸残基的优先释放。Ca ~（2+）对该酶活性的影响取决于底物。通过寻找阿帕的天然底物，我们发现在其N末端具有酸性氨基酸的肽（血管紧张素II、神经激肽B、胆囊收缩素-8和嗜铬粒蛋白A）在Ca 2+存在下被酶有效切割，但在Ca 2+不存在下不被酶有效切割。此外，激肽（赖氨酸缓激肽）转化为缓激肽有效地只有在Ca 2+的情况下。这些结果表明，Ca 2+增加了酶对具有N-末端酸性氨基酸的肽底物的偏好。此外，我们发现，血管紧张素IV可以结合阿帕在存在和不存在的Ca 2+和抑制阿帕的酶活性的竞争性，这表明，血管紧张素IV作为一个负调节剂的酶一旦产生的血管紧张素II的氨肽酶的系列行动。两者合计，这些结果表明，存在一个复杂的调节阿帕的酶活性，这可能有助于稳态，如调节血压，维持记忆，并通过控制肽底物的浓度正常怀孕。
Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up-or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.