Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin.
Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin.
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人尿原激肽释放酶的纯化。
DOI：
10.1042/bj2320851
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Erdös,EG
中科院分区：
文献类型：
--
作者：
Takada,Y;Skidgel,RA;Erdös,EG
人尿活性激肽释放酶和前激肽释放酶在DEAE-纤维素和辛基-Sepharose柱上分离，并通过亲和层析、凝胶过滤和疏水h.p.l.c.纯化至均一。在纯化过程中通过胰蛋白酶活化监测激肽释放酶原，然后测定酰胺酶和激肽原酶活性。胰蛋白酶活化后，纯化的前激肽释放酶具有比激肽原酶活性为39.4微克缓激肽当量/分钟/mg，酰胺酶活性为16.5 μ mol/分钟/mg，具有D-Val-Leu-Arg-7-氨基-4-三氟甲基香豆素。纯化的活性激肽释放酶具有每毫克47微克缓激肽/分钟的比活性。前激肽释放酶的分子量在电泳上为48 kDa，在凝胶过滤上为53 kDa，而活性激肽释放酶的分子量分别为46 kDa和53 kDa。获得活性和前激肽释放酶的抗血清。在双向免疫扩散和免疫电泳中，活性激肽释放酶的抗血清与活性和前激肽释放酶反应。原激肽释放酶的抗血清含有活性激肽释放酶中未发现的决定簇的抗体，推测是由于酶原中存在活化肽。人激肽释放酶原可被嗜热菌蛋白酶、胰蛋白酶和人血浆激肽释放酶激活。用25 nM-嗜热菌蛋白酶、78 nM-胰蛋白酶或180 nM-人血浆激肽释放酶在30分钟内实现50%的激肽释放酶原（1.35 μ M）的活化。因此，嗜热菌蛋白酶是最有效的激活剂。嗜热菌蛋白酶通过释放具有N-末端Ile 1-Val 2的活性激肽释放酶激活前激肽释放酶。因此，人组织（腺）激肽释放酶原可以被两种类型的酶激活：丝氨酸蛋白酶，其在碱性氨基酸的C-末端裂解，以及金属蛋白酶，其在疏水氨基酸的N-末端裂解。
Human urinary active kallikrein and prokallikrein were separated on DEAE-cellulose and octyl-Sepharose columns and both purified to homogeneity by affinity chromatography, gel filtration and hydrophobic h.p.l.c. Prokallikrein was monitored during purification by trypsin activation followed by determination of both amidase and kininogenase activity. After trypsin activation, purified prokallikrein had a specific kininogenase activity of 39.4 micrograms of bradykinin equivalent/min per mg and amidase activity of 16.5 mumol/min per mg with D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin. Purified active kallikrein had a specific activity of 47 micrograms of bradykinin/min per mg. The molecular mass of prokallikrein was 48 kDa on electrophoresis and 53 kDa on gel filtration whereas active kallikrein gave values of 46 kDa and 53 kDa respectively. Antisera to active and prokallikrein were obtained. In double immunodiffusion and immunoelectrophoresis, antiserum to active kallikrein reacted with active and pro-kallikrein. Antiserum to prokallikrein contained antibodies to determinants not found in active kallikrein, presumably due to the presence of the activation peptide in the proenzyme. Human prokallikrein can be activated by thermolysin, trypsin and human plasma kallikrein. Activation of 50% of the prokallikrein (1.35 microM) was achieved in 30 min with 25 nM-thermolysin, 78 nM-trypsin or 180 nM-human plasma kallikrein. Thus thermolysin was the most effective activator. Thermolysin activated prokallikrein by releasing active kallikrein with N-terminal Ile1-Val2. Thus human tissue (glandular) prokallikrein can be activated by two types of enzymes: serine proteinases, which cleave at the C-terminus of basic amino acids, and by a metalloproteinase that cleaves at the N-terminus of hydrophobic amino acids.