Investigation of kinin generating enzyme in the cardiovascular system.

有报道表明心内激肽释放具有抗缺血作用。本研究旨在纯化和表征狗心脏激肽形成酶。我们还研究了该酶从angi生成血管紧张素（Ang） II的能力，因为我们之前研究过组织激肽素可以从angi生成Ang II。心脏匀浆中的酶通过DEAE-Sepharose，抑肽蛋白亲和柱，小麦胚凝集素- sepharose 6MB柱层析分离。将样品与牛低分子量激肽原在37℃下孵育1小时后，用激肽放射免疫法测定激肽酶活性。用HPLC法测定样品与angi在37ºC孵育3小时后形成的Ang II的转化活性。酶在12.5% SDS-PAGE上电泳，然后进行考马斯亮蓝和PAS染色。纯化后的酶是一种糖蛋白，SDS-PAGE显示分子量为60 kDa。缓激肽原酶在最佳pH 8.0下的活性约为22杯缓激肽/小时/毫克蛋白质。在最佳pH为6.5的条件下，该酶将Ang I转化为Ang II，比活性约为2杯Ang II/小时/毫克蛋白质。这两种活性均被缓动因子抑制剂如抑肽酶、那莫他抑制，而不被凝乳抑素和胃抑素抑制。总之，目前的研究表明，狗心脏中的激肽形成酶也能够将Ang I转化为Ang II。它是一种类似钾化钾素的酶，不同于组织蛋白酶D、组织蛋白酶G和乳糜酶。该酶可能通过生成激肽和Ang II来调节心肌灌注。该酶的克隆及其定位有待进一步研究。

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.