Cloning of a kinin-tensin enzyme and kinin-destruction enzyme in the dog heart

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kalikrein，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结构和组织定位。在家兔中培养了针对纯化的心脏酶的抗血清。Western blot分析表明，这种酶不仅存在于心脏（心房、脉室、冠状动脉），还存在于其他组织，如肝、肾、胰腺、小肠和骨骼肌。它也存在于起源于犬远端小管的MDCK细胞中。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。从心脏中提取的酶进行氨基酸组成和序列分析。用赖氨酸内肽酶在37℃的温度下裂解14小时，在C18柱上以线性（0-90%）乙腈梯度在0.1%的三氟乙酸中分离。分馏后用于序列分析。然而，纯化的心脏钾化钾素样酶的氨基酸组成与犬尿钾化钾素不同。它有n端阻断氨基酸。目前的研究表明，钾化钾素样酶不仅分布于心脏，还分布于其他组织，如主动脉、肾脏、胰腺、小肠和骨骼肌。该酶可能通过生成激肽和Ang II发挥病理生理作用。这种酶的克隆目前正在进行中，使用的是针对这种酶的抗体。

We have previously shown that the purified kinin-forming enzyme from the dog heart is similar to tissue kalikrein and is also able to convert angiotensin (Ang) I to Ang II.The aim of the present study is to clarify the structure and tissue localization of this enzyme. Antiserum against the purified enzyme from the heart was raised in rabbits. Western blot analysis indicated the presence of this enzyme not only the heart (atria, ventiricule, coronary artery) but also other tissues such as sorta, kidney, pancreas, small intestine, and skeletal muscle. Its presence was also demonstrated in MDCK cell originated fron canine distal tubules. The kallikrein-like enzyme has been purified from homogenates of the kidney and aorta by a DEAE-Sepharose and aprotinin affinity columnn. 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 porified enzyme from the heart was proceeded to amino acid composition and sequence analysis. It was cleaved by lysyl-endopeptidase at 37ﾟC for 14 hrs and fractionated on a C18 column with a linear (0-90%) acetonitrile gradient in 0.1% trigluoroacetic acid. After fractionation, it was used for sequence analysis. However, the amino acid composition of purified kallikrein-like enzeme from the heart was different from that of dog urinary kallikrein. It has N-terminal bloked amino acid. the present study has shown that kallikrein-like enzyme is distributed not only in the heart, but also in the other tissues such as aorta, kidney, pancreas, small intestine, and skeletal muscle. This enzyme may exert pathophysiological effects through generating kinins and Ang II.The cloning og this enzyme iks now ongoing using antibody against the enzyme.