The collagenase inhibitor from human polymorphonuclear leukocytes. Isolation, purification and characterisation.
The collagenase inhibitor from human polymorphonuclear leukocytes. Isolation, purification and characterisation.
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来自人多形核白细胞的胶原酶抑制剂。
DOI：
10.1111/j.1432-1033.1983.tb07119.x
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
H. Tschesche
中科院分区：
文献类型：
--
作者：
H. W. Macartney;H. Tschesche
一种新的潜伏性人多形性胶原酶的胶原酶抑制剂，最初由Macartney和Tschesche报道[Hoppe-Seyler's Z. 361，298-299（1980）和FEBS Lett. 119，327-332（1980）]，已纯化至表观均一性。我们描述了几种纯化这种抑制剂的方法，但最快和最具重现性的方法是将纯化的潜伏性人多形核白细胞胶原酶在活化的硫醇-琼脂糖4-B上传代（本杂志的前一篇论文）。活性胶原酶从柱中洗脱，胶原酶抑制剂被阻滞。通过用平衡缓冲液中的硫醇化合物洗脱柱来实现抑制剂的洗脱。通过Sephacryl S-300上的凝胶过滤和DEAE-Sephacel上的离子交换层析获得抑制剂的进一步纯化。
该抑制剂对哺乳动物胶原酶具有严格的特异性。它抑制从人滑液、人正常和糖尿病皮肤成纤维细胞以及人胚胎皮肤成纤维细胞中分离的胶原酶。无活性的复合物都受到几种二硫化物化合物的活化，例如通过已经描述的硫醇/二硫化物交换反应的氧化谷胱甘肽。
十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（10%凝胶）显示纯化的抑制剂是均匀的，并显示表观分子量为24500。用Ellmans试剂滴定该抑制剂，发现它含有一个游离巯基，这是其抑制活性的先决条件。该抑制剂以严格的1：1化学计量反应抑制人多形核白细胞胶原酶。由抑制剂和活性酶形成的复合物的氨基酸组成再现了潜在胶原酶的氨基酸组成，从而证实潜在酶是1：1复合物。
该抑制剂是一种酸性蛋白质，其等电点为pH 5.5，这是氨基酸分析所预期的。该组合物揭示了约240个残基的糖蛋白，其含有由N-乙酰葡糖胺、甘露糖、半乳糖和葡萄糖组成的碳水化合物部分。该抑制剂的氨基酸组成与人血浆中β1-抗胶原酶的氨基酸组成不同。
A new collagenase inhibitor of latent human polymorphonuclear collagenase, initially reported by Macartney and Tschesche [Hoppe-Seyler's Z. Physiol. Chem. 361, 298–299 (1980) and FEBS Lett. 119, 327–332 (1980)], has been purified to apparent homogeneity. We described several methods of purification of this inhibitor, but the fastest and most reproducible method is by passage of purified latent human polymorphonuclear leukocyte collagenase on activated thiol-Sepharose 4-B (preceding paper in this journal). Active collagenase is eluted from the column and the collagenase inhibitor is retarded. Elution of the inhibitor is achieved by eluting the column with thiol compounds in the equilibration buffer. Further purification of the inhibitor is obtained by gel filtration on Sephacryl S-300 and ion-exchange chromatography on DEAE-Sephacel.
The inhibitor exhibits a strict specificity for mammalian collagenases. It inhibits collagenases isolated from human synovial fluid, human normal and diabetic skin fibroblasts and human embryonic skin fibroblasts. The inactive complexes are all subject to activation by several disulfide compounds, such as oxidised glutathione by the thiol/disulfide interchange reaction already described.
The purified inhibitor was shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (10% gel) to be homogeneous and displayed an apparent molecular weight of 24500. Titration of the inhibitor with Ellmans reagent revealed that it contains one free sulfhydryl group, a prerequisite for its inhibitory activity. The inhibitor inhibits human polymorphonuclear leukocyte collagenase in a strict 1:1 stoichiometric reaction. The amino acid composition of the complex formed from the inhibitor and active enzyme reproduces that of the latent collagenase thus confirming that the latent enzyme is a 1:1 complex.
The inhibitor is an acidic protein having an isoelectric point at pH 5.5, which would be expected from the amino acid analysis. The composition reveals a glycoprotein of about 240 residues containing a carbohydrate moiety composed of N-acetylglucosamine, mannose, galactose and glucose. The amino acid composition of the inhibitor is not identical to that of β1-anticollagenase from human plasma.