Masferrer J, Albertini R, Croxatto H R, García P, Pinto I
Biochem Pharmacol. 1985 Jan 1;34(1):51-6. doi: 10.1016/0006-2952(85)90099-1.
A method has been developed to purify glandular kallikrein present in rat plasma by using Sepharose-Aprotinin affinity chromatography and elution of the enzyme with p-aminobenzamidine. The isolated enzyme liberated kinins from kininogen II of low molecular weight (sp. act. 14 ng kinins/min X mg) and p-nitroaniline (pNA) from the substrate S-2266 (sp. act. 1.23 nmoles pNA/min X mg); it was inhibited by aprotinin, benzamidine and rat urinary antikallikrein antibody but not by ovomucoid. In polyacrylamide gel electrophoresis, the enzymatic activities of the preparation were associated with two light protein bands of molecular weights equal to that of urinary kallikrein (35,000 daltons). Using this method, the recovery of [125I]kallikrein added to the plasma was 82-88%. The concentration of the enzyme in normal rat plasma was equivalent to 6.1 +/- 2.1 (S.D.) ng kallikrein/ml. The mean value found in nephrectomized rats was 20.0 +/- 6.3 (S.D.) ng kallikrein/ml. This increment was highly significant (P less than 0.001). Our results confirm the presence of glandular kallikrein in plasma which had been detected by other methods; they also demonstrate that the material purified from plasma is enzymatically active, suggesting that kallikrein may play a biological role in the control of blood circulation.
已开发出一种方法，通过使用琼脂糖-抑肽酶亲和色谱法并用对氨基苯甲脒洗脱酶来纯化大鼠血浆中存在的腺体激肽释放酶。分离出的酶能从低分子量激肽原II释放激肽（比活性为14 ng激肽/分钟×毫克），并从底物S-2266释放对硝基苯胺（pNA）（比活性为1.23纳摩尔pNA/分钟×毫克）；它被抑肽酶、苯甲脒和大鼠尿抗激肽释放酶抗体抑制，但不被卵类粘蛋白抑制。在聚丙烯酰胺凝胶电泳中，该制剂的酶活性与两条分子量与尿激肽释放酶（35,000道尔顿）相等的轻蛋白带相关。使用这种方法，添加到血浆中的[125I]激肽释放酶的回收率为82 - 88%。正常大鼠血浆中该酶的浓度相当于6.1±2.1（标准差）纳克激肽释放酶/毫升。在肾切除大鼠中发现的平均值为20.0±6.3（标准差）纳克激肽释放酶/毫升。这种增加非常显著（P小于0.001）。我们的结果证实了通过其他方法检测到的血浆中存在腺体激肽释放酶；它们还表明从血浆中纯化的物质具有酶活性，提示激肽释放酶可能在血液循环控制中发挥生物学作用。
