Purification and characterization of canine urinary kallikrein.
Purification and characterization of canine urinary kallikrein.
复制标题
犬尿激肽释放酶的纯化和表征。
DOI：
10.1016/0003-9861(86)90624-7
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发表时间：
1986
影响因子：
3.9
通讯作者：
Scicli,AG
中科院分区：
文献类型：
--
作者：
Murthy,KK;Carretero,OA;Scicli,AG
肾脏激肽释放酶-激肽系统可能在钠水平衡的调节中起作用。虽然狗是肾激肽释放酶-激肽系统研究中常用的实验动物，但对狗尿激肽释放酶（DUKK）的研究却很少。我们已经通过一系列的色谱和电泳程序，包括阴离子交换色谱，过滤通过p-氨基苯脒-琼脂糖（以去除污染的非激肽释放酶酯酶），凝胶过滤，等电聚焦，和分子筛HPLC纯化DUKK。该DUKK制剂在聚丙烯酰胺凝胶电泳上给出三条蛋白带，每条蛋白带具有相似的酯酶和激肽原酶活性和免疫学特性。等电聚焦显示存在多种形式的激肽释放酶，pI为3.93、4.05、4.24和4.44，pI为4.24的物质构成主要组分。神经氨酸酶处理将所有形式转化为pI为4.44的组分，表明电荷异质性主要是由于唾液酸含量的差异。当部分纯化的狗激肽原用作底物时，DUKK具有3 mg缓激肽eq/min/mg蛋白的比活性。它是一种糖蛋白，分子量为40，500（氨基酸分析最佳拟合方法），最适碱性pH值（9.0-9.5）。DUKK对大豆胰蛋白酶抑制剂和利马豆胰蛋白酶抑制剂具有抗性，但被几种丝氨酸蛋白酶抑制剂如抗蛋白酶、亮抑酶肽和对氨基苯甲脒抑制。苯丙氨酸-苯丙氨酸-精氨酸-氯甲基酮是一种非常有效的DUKK抑制剂。与以前的报道相反，DUKK也被N-α-对甲苯磺酰-L-赖氨酸氯甲基酮和抑肽酶抑制，后者的抑制作用与培养基中NaCl的浓度成负相关。DUKK的酯解和酰胺解活性随培养基中NaCl浓度的增加而受到抑制。这种抑制作用可能与NaCl诱导的酶部分构象变化有关。
The renal kallikrein-kinin system may play a role in the regulation of sodium and water balance. Although the dog is a frequently used experimental animal in the study of the renal kallikrein-kinin system, dog urinary kallikrein (DUKK) has been poorly studied. We have purified DUKK by a series of chromatographic and electrophoretic procedures including anion-exchange chromatography, filtration throughp-aminobenz-amidine-Sepharose (to remove contaminating nonkallikrein esterases), gel filtration, isoelectric focusing, and molecular sieve HPLC. This DUKK preparation gave three protein bands on polyacrylamide gel electrophoresis, each having similar esterolytic and kininogenase activities and immunological identity. Preparative isoelectric focusing indicated the presence of multiple forms of kallikrein with pI's of 3.93, 4.05, 4.24, and 4.44, the species with a pIof 4.24 constituting the major component. Neuraminidase treatment converted all of the forms into the component with a pIof 4.44, suggesting the charge heterogeneity was due mainly to differences in sialic acid content. DUKK has a specific activity of 3 mg bradykinin eq/min/mg protein when partially purified dog kininogen is used as a substrate. It is a glycoprotein with a molecular weight of 40,500 (amino acid analysis best fit method) and an alkaline pH optimum (9.0–9.5). DUKK is resistant to soybean trypsin inhibitor and lima bean trypsin inhibitor but is inhibited by several serine protease inhibitors such as antipain, leupeptin, andp-aminobenzamidine. PhePheArgchloromethyl ketone is a very potent inhibitor of DUKK. Contrary to previous reports, DUKK is also inhibited byN-α-p-tosyl-l-lysine chloromethyl ketone and aprotinin, the inhibition by the latter being inversely related to the concentration of NaCl in the medium. The esterolytic and amidolytic activities of DUKK are inhibited by an increase in NaCl concentration of the medium. This inhibition may be related to a NaCl-induced conformational change in the enzyme moiety.