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

狗心脏中激肽-张力蛋白酶和激肽破坏酶的克隆

基本信息

批准号：
08670838
负责人：
SASAGURI Manabu
金额：
$ 1.41万
依托单位：
Fukuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670838/
关键词：
kallikrein angiotensin kinin kinin-tensin enzyme

项目摘要

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.

我们之前已经证明，从狗心脏中纯化的激肽形成酶与组织激肽释放酶相似，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。在兔子体内制备了针对来自心脏的纯化酶的抗血清。蛋白质印迹分析表明，这种酶不仅存在于心脏（心房、心室、冠状动脉），而且还存在于其他组织，如各种组织、肾脏、胰腺、小肠和骨骼肌。它的存在也在源自犬远端小管的 MDCK 细胞中得到证实。激肽释放酶样酶已通过 DEAE-Sepharose 和抑肽酶亲和柱从肾脏和主动脉的匀浆中纯化。从肾脏和主动脉纯化的酶在蛋白质印迹中显示为 65 kDa 的相对分子量，这与从心脏纯化的激肽释放酶样酶的分子量相同。对来自心脏的多孔酶进行氨基酸组成和序列分析。它被赖氨酰内肽酶在 37°C 下裂解 14 小时，并在 C18 柱上用 0.1% 三氯乙酸中的线性 (0-90%) 乙腈梯度进行分级。分级分离后，用于序列分析。然而，从心脏纯化的激肽释放酶样酶的氨基酸组成与狗尿激肽释放酶的氨基酸组成不同。它具有 N 末端封闭的氨基酸。目前的研究表明，激肽释放酶样酶不仅分布在心脏中，还分布在主动脉、肾、胰腺、小肠和骨骼肌等其他组织中。这种酶可能通过产生激肽和血管紧张素II来发挥病理生理学作用。目前正在使用针对该酶的抗体对该酶进行克隆。