Chauvet J, Dostal J P, Acher R
Int J Pept Protein Res. 1976;8(1):45-55. doi: 10.1111/j.1399-3011.1976.tb02480.x.
A trypsin-like enzyme has been isolated from the filtrate of a Streptomyces rimosus forma paromomycinus culture. Purification involves acetone fractionated precipitation, ultrafiltration on a Diaflo UM 10 membrane and affinity adsorption on to Kunitz pancreatic trypsin inhibitor linked to Sepharose. The trypsin-like enzyme (paromotrypsin) appears homogeneous by zone electrophoresis on gelatinized cellulose acetate. Specific activity toward Tos-Arg-OMe, calculated from amino acid analysis, is about 220 mu mg-1. The overall yield in activity is about 30%. The molecular weight of the trypsin-like enzyme, determined by gel filtration, is around 22,000-25,000 daltons. Electrophoretic migration on cellulose acetate strips indicates an isoelectric point around 8. Amino acid composition has been determined; the protein comprises about 210 residues on the basis of a single histidine residue per molecule. Paromotrypsin is unstable in acidic medium and is not stabilized by calcium ions. Enzymic activity towards Bz-Argo-OEt is not increased by the addition of calcium ion in contrast to the activating effect observed on bovine trypsin. Paromotrypsin is inhbited by TLCK and NPGB; it interacts with naturally occurring bovine trypsin inhibitors such as soya bean and Kunitz pancreatic inhibitors, but not with chicken ovomucoid. Proteolytic specificity, examined by hydrolysis of oxidized Kunitz pancreatic inhibitor and characterization of resulting peptides, seems similar to that of bovine trypsin.
已从龟裂链霉菌巴龙霉素变种培养物的滤液中分离出一种类胰蛋白酶。纯化过程包括丙酮分级沉淀、在Diaflo UM 10膜上进行超滤以及在与琼脂糖偶联的Kunitz胰蛋白酶抑制剂上进行亲和吸附。通过在凝胶化醋酸纤维素上进行区带电泳，类胰蛋白酶（巴龙胰蛋白酶）呈现均一性。根据氨基酸分析计算，其对甲苯磺酰精氨酸甲酯（Tos-Arg-OMe）的比活性约为220 μmg-1。活性的总收率约为30%。通过凝胶过滤法测定，类胰蛋白酶的分子量约为22,000 - 25,000道尔顿。在醋酸纤维素条上的电泳迁移表明其等电点约为8。已测定了氨基酸组成；基于每个分子有一个组氨酸残基，该蛋白质约由210个残基组成。巴龙胰蛋白酶在酸性介质中不稳定，且不受钙离子稳定。与在牛胰蛋白酶上观察到的激活作用相反，添加钙离子不会增加其对苯甲酰精氨酸乙酯（Bz-Arg-OEt）的酶活性。巴龙胰蛋白酶受N-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）和对硝基苯甲酰-L-精氨酸乙酯盐酸盐（NPGB）抑制；它与天然存在的牛胰蛋白酶抑制剂如大豆和Kunitz胰蛋白酶抑制剂相互作用，但不与鸡卵类粘蛋白相互作用。通过氧化的Kunitz胰蛋白酶抑制剂的水解及所得肽段的表征来研究其蛋白水解特异性，结果似乎与牛胰蛋白酶相似。
