Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells
Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells
复制标题
DOI：
10.1093/oxfordjournals.jbchem.a022812
复制
发表时间：
2000-11-01
影响因子：
2.7
通讯作者：
Tsujimoto, M
中科院分区：
文献类型：
--
作者：
Hattori, A;Kitatani, K;Tsujimoto, M
脂肪细胞来源的亮氨酸氨肽酶（A-A）是最近发现的锌金属肽酶M1家族的新成员。将A-LAP cDNA转染到COS-7细胞中导致该酶的分泌。本研究在中国仓鼠卵巢细胞中表达重组A-β，纯化至均一，并对其酶学性质进行了表征。纯化的酶对合成底物L-亮氨酰-对硝基苯胺有活性，产生的Vmax为3.55 μ mol/min/mg，K-m为1.28 mM，并且显示在溶液中是分子量为120 kDa的单体蛋白。通过监测N-末端氨基酸的顺序释放，发现该酶水解多种生物活性肽，包括血管紧张素II和胰激肽。免疫组织化学分析表明，这种酶在人类肾脏的皮质中表达，组织激肽释放酶位于那里。总之，这些结果表明，A-glutamate具有广泛的底物特异性对天然存在的肽激素，并表明，它在调节血压通过血管紧张素II的失活和/或缓激肽在肾脏中的产生的作用。
Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V-max of 3.55 mu mol/min/mg and a K-m of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution, By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.