Cloning of a kallikrein-like enzyme originated from dog heart and investigation of its intracellular localization and expression regulation.

我们先前已经证明，从狗心脏中纯化的激动素形成酶类似于组织激肽释放酶，并且能够将血管紧张素(Ang)I转化为Ang II。本研究的目的是阐明该酶的克隆、细胞内定位和表达调控。Western印迹分析表明，该酶不仅在心脏(心房、心室、冠状动脉)中存在，而且在其他组织中也存在，如主动脉、肾脏、胰腺、小肠和骨骼肌。在犬远端小管来源的MDCK细胞中也有表达。用DEAE-Sepharose和抑肽酶亲和层析柱从肾和主动脉匀浆中纯化了激肽释放酶样酶。Western blotting显示肾脏和主动脉中的纯化酶的相对分子质量为65 kDa，这与从心脏中纯化的激肽释放酶的相对分子质量相同。对纯化的心脏酶进行氨基酸组成和序列分析。它含有N端封闭的氨基酸。纯化的心脏激肽释放酶的氨基酸组成不同于狗的尿激肽释放酶和胰腺激肽释放酶。将该酶的自溶样品电印迹到膜上，切割成一条带的膜进行氨基酸序列分析。获得了三组部分氨基酸序列。这些序列与迄今已知的激肽释放酶不同。设计了新的混合引物，并新制备了犬心脏的cDNA库。目前，DNA序列分析正在进行中。这种酶可能通过产生激动素和血管紧张素II在心血管系统中发挥重要作用。

We have previously shown that the purified kinin-forming enzyme from the dog heart is similar to tissue kallikrein and is also able to convert angiotensin (Ang) I to Ang II. The aim of the present study is to clarify the cloning, intracellular localization and expression regulation of this enzyme. Western blot analysis indicated the presence of this enzyme not only the heart (atria, ventricle, coronary artery) but also other tissues such as aorta, kidney, pancreas, small intestine, and skeletal muscle. Its presence was also demonstrated in MDCK cell originated from canine distal tubules. The kallikrein-like enzyme has been purified from homogenates of the kidney and aorta by a DEAE-Sepharose and aprotinin affinity column. Purified enzyme from the kidney and aorta was visualized at the relative molecular weight of 65 kDa on Western blotting which is identical to that of the kallikrein-like enzyme purified from the heart. The purified enzyme from the heart was proceeded to amino acid composition and sequence analysis. It has N-terminal blocked amino acid. The amino acid composition of purified kallikrein-like enzyme from the heart was different from that of dog urinary and pancreatic kallikrein. Autolysed samples of this enzyme were electroblotted to the membrane, and cutted membrane including one band was proceeded to amino acid sequence analysis. Three sets of partial amino acid sequence were obtained. These sequences are different to the hitherto known kallikrein. Mixed primer was newly made, and cDNA library from the dog heart was also newly prepared. At the present, DNA sequence analysis is now ongoing. This enzyme may play an important role in cardiovascular systems through generating kinins and Ang II.