Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae.
Purification and characterization of two trypsin inhibitors from the hemolymph of Manduca sexta larvae.
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DOI：
10.1016/s0021-9258(18)37989-4
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发表时间：
1988-08
期刊：
影响因子：
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通讯作者：
N. Ramesh;M. Sugumaran;J. E. Mole
中科院分区：
文献类型：
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作者：
N. Ramesh;M. Sugumaran;J. E. Mole
用固定化胰蛋白酶亲和层析法从烟草天蛾（Manducasexta）血淋巴中纯化胰蛋白酶抑制活性，并将其分离成分子量为14，000（M. sexta血淋巴胰蛋白酶抑制剂（HLTI）A）和8，000（HLTI B）。这些抑制剂在聚丙烯酰胺凝胶上在还原条件下的电泳得到HLTI A的分子量估计值为8，300，HLTI B的分子量估计值为9，100，表明HLTI A是二聚体，HLTI B是单体。在聚丙烯酰胺凝胶上的等电聚焦将HLTI A聚焦为pI为5.7的单一条带，而HLTI B分解为pI值为5.3和7.1的两个组分。这两种抑制剂在100 ℃和pH 1.0下稳定至少30 min。HLTI A和B抑制丝氨酸蛋白酶，如胰蛋白酶、胰凝乳蛋白酶和纤溶酶，但不抑制弹性蛋白酶、木瓜蛋白酶、胃蛋白酶、枯草杆菌蛋白酶BPN '和嗜热菌蛋白酶。事实上，枯草杆菌蛋白酶BPN完全灭活了这两种抑制剂。这两种抑制剂与胰蛋白酶以1：1的摩尔比形成低解离复合物。HLTI A对胰蛋白酶抑制的抑制常数估计为1.45 x 10（-8）M。HLTI A-胰凝乳蛋白酶复合物不抑制胰蛋白酶;类似地，HLTI A-胰凝乳蛋白酶复合物不抑制胰凝乳蛋白酶，表明HLTI A具有胰蛋白酶和胰凝乳蛋白酶两者的共同结合位点。HLTI A和B的氨基端氨基酸序列显示这两种抑制剂与牛胰蛋白酶抑制剂（Kunitz）同源。
Trypsin inhibitory activity from the hemolymph of the tobacco hornworm (Manduca sexta) was purified by affinity chromatography on immobilized trypsin and resolved into two fractions with molecular weights of 14,000 (M. sexta hemolymph trypsin inhibitor (HLTI) A) and 8,000 (HLTI B) by molecular sieve chromatography on Sephadex G-75. Electrophoresis of these inhibitors under reducing conditions on polyacrylamide gels gave molecular weight estimates of 8,300 for HLTI A and 9,100 for HLTI B, suggesting that HLTI A is a dimer and HLTI B is a monomer. Isoelectrofocusing on polyacrylamide gels focused HLTI A as a single band with pI 5.7, whereas HLTI B was resolved into two components with pI values of 5.3 and 7.1. Both inhibitors were stable at 100 degrees C and pH 1.0 for at least 30 min. HLTIs A and B inhibited serine proteases such as trypsin, chymotrypsin, and plasmin, but did not inhibit elastase, papain, pepsin, subtilisin BPN', and thermolysin. In fact, subtilisin BPN' completely inactivated both inhibitors. Both inhibitors formed low-dissociation complexes with trypsin in a 1:1 molar ratio. The inhibition constant for trypsin inhibition by HLTI A was estimated to be 1.45 x 10(-8) M. The HLTI A-chymotrypsin complex did not inhibit trypsin; similarly, the HLTI A-trypsin complex did not inhibit chymotrypsin, indicating that HLTI A has a common binding site for both trypsin and chymotrypsin. The amino-terminal amino acid sequences of HLTIs A and B revealed that both these inhibitors are homologous to bovine pancreatic trypsin inhibitor (Kunitz).