Investigation of kinin generating enzyme in the cardiovascular system.

心血管系统中激肽生成酶的研究。

• 批准号: 06670754
• 负责人: SASAGURI Manabu
• 金额: $ 1.28万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670754/
• 关键词: kinin; kallikrein; angiotensin; ischemic heart; kinin-tensin system

Reports have shown that intracardic kinin release exerts anti-ischemic effect. Our study aimed at purification and charaterizatiuon of the kinin-forming enzyme of the dog heart. The ability of the enzyme to generate angiotensin (Ang) II from Ang I was also examined since we previously tissue kallikrein could form Ang II from Ang I.The enzyme from cardiac homogenates has been isolated by a DEAE-Sepharose, aprotinin affinity column, wheat germ lectin-Sepharose 6MB column chromatography. Kininogenase activity was assessed by the measurement of generated kinins with a kinin radioimmunoassay after samples were incubated with bovine low molecular weight kininogen at 37ﾟC for 1 hr. Ang I converting activity was assessed by quantitation of Ang II formed on HPLC by the incubation of the sample with Ang I at 37ﾟC for 3 hrs. The enzyme was electrophoresed on a 12.5% SDS-PAGE followed by Coomassie Brilliant Blue and PAS staining. The purified enzyme is a glycoprotein with an apparent molecular weight of 60 kDa on SDS-PAGE.Kininogenase activity was approximately 22 mug of bradykinin/hr/mg protein at an optimal pH 8.0. The enzyme also converted Ang I to Ang II with a specific activity of approximately 2 mug of Ang II/hr/mg protein at an optimal pH 6.5. Both activity was inhibited by kallikrein inhibitors such as aprotinin, nafamostat, but not by chymostatin and pepstatin.In conclusion, the present study has shown that the kinin-forming enzyme in the dog heart is also able to convert Ang I to Ang II.It is kallikrein-like enzyme different from cathepsin D,cathepsin G,and chymase. This enzyme may play a role in regulating myocardial perfusion through generating kinins and Ang II.Cloning of this enzyme and its localization awaits further examination.

已有报道表明，心内释放激肽具有抗缺血作用。本研究旨在对犬心脏激动素形成酶进行纯化和性质研究。由于组织激肽释放酶可以从Ang I生成Ang II，因此我们还检测了该酶从Ang I产生血管紧张素(Ang II)的能力。心脏匀浆中的酶已通过DEAE-Sephose柱、抑肽酶亲和层析、小麦胚凝集素-Sepharose6MB柱层析分离。样品与牛低分子激肽原在37ﾟC孵育1小时后，用激动素放射免疫法测定激肽原酶活性。将样品与血管紧张素转换酶I在37ﾟC孵育3小时，通过在高效液相上生成血管紧张素转换酶II的定量来评估血管紧张素转换活性。该酶在12.5%的SDS-PAGE上进行电泳，考马斯亮蓝和PAS染色。纯化后的酶是一种表观分子量为60 kDa的糖蛋白，在最适pH为8.0时，激肽原酶活性约为22mug缓激肽/小时/mg蛋白。该酶还能将Ang I转化为Ang II，在最适pH为6.5时，其比活力约为2杯Ang II/hr/mg蛋白质。激肽释放酶抑制剂如抑肽酶、那福司他特可抑制这两种酶的活性，但不能被糜蛋白酶和胃抑素抑制。总之，本研究表明，狗心脏中的激动素形成酶也能将Ang I转化为Ang II。这是一种不同于组织蛋白酶D、组织蛋白酶G和糜酶的激肽释放酶。该酶可能通过产生激动素和Ang II发挥调节心肌血流的作用。该酶的克隆和定位有待进一步研究。

项目成果

Sasaguri M: "Purification and characterization of a kinin-and angiotensinII-forming enzyme in the dog heart" J Hypertens. (Abstract) (in press).

Sasaguri M：“狗心脏中激肽和血管紧张素 II 形成酶的纯化和表征”J Hypertens。

Sasaguri M: "Human urinary kallikrein can generate angiotensin II from homologous renin substrates" Hypertens Res. 18. 33-37 (1995)

Sasaguri M：“人尿激肽释放酶可以从同源肾素底物产生血管紧张素 II”Hypertens Res。

M Sasaguri, H Maeda, E Tsuji, A Kinoshita, S Miura, M Ideishi, K Arakawa.: "Charaterization of a kinin-tensin enzyme in the dog heart." Hypertens Res. (in press).

M Sasaguri、H Maeda、E Tsuji、A Kinoshita、S Miura、M Ideishi、K Arakawa.：“狗心脏中激肽-张力蛋白酶的表征。”

Sasaguri M: "Purification and characterization of a kinin-and angiotensinII-forming enzyme in the dog heart (Abstract)" J Hypertens. (in press).

Sasaguri M：“狗心脏中激肽和血管紧张素 II 形成酶的纯化和表征（摘要）”J Hypertens。

Sasaguri M: "Human Urinary Kallikrein can generate angiotensin II from hemologous renin substrates" Hypertens Res. 18. 33-37 (1995)

Sasaguri M：“人尿激肽释放酶可以从血源肾素底物产生血管紧张素 II”Hypertens Res。