Zhou G X, Chao L, Chao J
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425.
J Biol Chem. 1992 Dec 25;267(36):25873-80.
A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.
一种名为激肽释放酶抑制蛋白的新型人体组织激肽释放酶抑制剂，已通过聚乙二醇分级分离以及在肝素 - 琼脂糖、二乙氨基乙基 - 琼脂糖、羟基磷灰石和苯基 - 超级琼脂糖柱上的连续层析，从血浆中纯化至表观均一状态。获得了4350的纯化因子，每升血浆的产量约为1.35毫克。在还原条件下进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析时，纯化后的抑制剂迁移为一条单一的条带，表观分子量为58 kDa。它是一种酸性蛋白，其等电点值在4.6至5.2之间。通过蛋白质免疫印迹分析发现，激肽释放酶抑制蛋白与其他丝氨酸蛋白酶抑制剂之间没有免疫交叉反应。激肽释放酶抑制蛋白抑制人体组织激肽释放酶对激肽原和三肽底物的活性。使用脯氨酸 - 苯丙氨酸 - 精氨酸 - 甲基香豆素酰胺测定的二级反应速率常数（ka）为2.6×10⁴ M⁻¹ s⁻¹。这种抑制伴随着组织激肽释放酶与激肽释放酶抑制蛋白之间形成等摩尔、热稳定且对十二烷基硫酸钠稳定的复合物，并且由于组织激肽释放酶在反应位点的切割，抑制剂产生了一个小的羧基末端片段。肝素可阻止激肽释放酶抑制蛋白与组织激肽释放酶形成复合物，并消除其对组织激肽释放酶活性的抑制作用。激肽释放酶抑制蛋白的氨基末端残基被封闭。对激肽释放酶抑制蛋白产生的羧基末端片段进行序列分析，揭示了从P1'到P15'的反应中心序列，该序列与包括蛋白C抑制剂、α1 - 抗胰蛋白酶和α1 - 抗糜蛋白酶在内的已知丝氨酸蛋白酶抑制剂具有序列相似性，但又有所不同。结果表明，激肽释放酶抑制蛋白是丝氨酸蛋白酶抑制剂超家族中抑制人体组织激肽释放酶的一个新成员。
