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

我们之前已经证明，从狗心脏纯化的激肽形成酶类似于组织激肽素，也能够将血管紧张素（Ang） I转化为Ang II。本研究的目的是阐明该酶的克隆、胞内定位和表达调控。Western blot分析表明，这种酶不仅存在于心脏（心房、心室、冠状动脉），还存在于其他组织，如主动脉、肾脏、胰腺、小肠和骨骼肌。在起源于犬远端小管的MDCK细胞中也证实了其存在。通过DEAE-Sepharose和抑肽蛋白亲和柱，从肾脏和主动脉的匀浆中纯化了钾化钾素样酶。从肾脏和主动脉纯化的酶在Western blotting上显示相对分子量为65 kDa，与从心脏纯化的钾化钾素样酶相同。对纯化的心脏酶进行氨基酸组成和序列分析。它有n端阻断氨基酸。从心脏中纯化的钾化钾素样酶的氨基酸组成与犬尿钾化钾素和胰钾化钾素不同。将该酶的自溶样品电印迹到膜上，切下含一条带的膜进行氨基酸序列分析。得到了三组部分氨基酸序列。这些序列不同于迄今为止已知的短链。新制备了混合引物，新制备了犬心脏cDNA文库。目前，DNA序列分析正在进行中。该酶可能通过生成激肽和Ang 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.