A specific inhibitor of vertebrate collagenase produced by human skin fibroblasts.
A specific inhibitor of vertebrate collagenase produced by human skin fibroblasts.
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DOI：
10.1016/s0021-9258(17)37747-5
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发表时间：
1979-03
期刊：
影响因子：
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通讯作者：
H. Welgus;G. Stricklin;A. Eisen;E. Bauer;R. Cooney;J. Jeffrey
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文献类型：
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作者：
H. Welgus;G. Stricklin;A. Eisen;E. Bauer;R. Cooney;J. Jeffrey
合成胶原原酶的正常人皮肤成纤维细胞也显示出产生这种酶原的活性形式的特异性抑制剂。该抑制剂来源于成纤维细胞本身，如以下标准所证明的：1）当成纤维细胞暴露于无血清培养基中的3 H-氨基酸混合物时，产生3 H-标记的抑制剂，2）胎牛血清，当通过用于抑制剂的基本纯化步骤处理时，不显示胶原酶抑制活性。通过阳离子交换和凝胶过滤层析的组合，从无血清培养基中广泛纯化抑制剂。聚丙烯酰胺凝胶的分光光度扫描表明，抑制剂的纯度大于95%。经十二烷基硫酸钠凝胶电泳测定，其表观分子量为31，000，并且具有显著的热稳定性，在90 ℃下20分钟后仍保持其活性的60%以上。抑制是化学计量的，完全抑制胶原酶活性所需的抑制剂与酶的摩尔比为1：1。试图证明酶-抑制剂复合物是不成功的;事实上，酶和抑制剂在各种色谱系统中彼此独立地表现。活性酶和抑制剂之间的紧密结合只发生在胶原底物的存在下。因此，推测该非活性物种为三元配合物E。1. S.人成纤维细胞抑制剂对所有测试的脊椎动物胶原酶都有效。非脊椎动物来源的非胶原蛋白溶解蛋白酶和胶原酶未受到抑制。不与胶原蛋白结合的前胶原酶也未能结合抑制剂。脊椎动物的胶原酶是一类能特异性降解动物体内天然胶原的酶。虽然现在已经在许多物种的许多组织中鉴定出了特异性的胶原蛋白溶解酶，但是调节脊椎动物胶原蛋白降解的过程的性质还没有明确定义。天然存在的胶原酶抑制剂的存在已经被认识了一段时间，并且已经被推测
Normal human skin fibroblasts which synthesize procollagenase were shown also to produce a specific inhibitor of the active form of this proenzyme. The inhibitor was derived from the flbroblasts themselves, as demonstrated by the following criteria: 1) 3H-labeled inhibitor was produced when flbroblasts were exposed to a 3H-amino-acid mixture in serum-free culture medium, 2) fetal calf serum, when processed through the basic purification steps employed for the inhibitor, displayed no collagenase inhibitory activity. Inhibitor was purified extensively from serum-free medium by a combination of cation exchange and gel filtration chromatography. Spectrophotometric scanning of polyacrylamide gels indicated that the inhibitor was greater than 95% pure. It had an apparent molecular weight of 31,000 as determined by sodium dodecyl sulfate-gel electrophoresis, and was remarkably heatstable, retaining more than 60% of its activity after 20 min at 90 C. Inhibition was stoichiometric, a 1: l molar ratio of inhibitor to enzyme being required for complete inhibition of collagenase activity. Attempts to demonstrate an enzyme-inhibitor complex were not successful; in fact, enzyme and inhibitor behaved independently of each other in a variety of chromatographic systems. Tight binding between active enzyme and inhibitor occurred only in the presence of the collagen substrate. It is therefore suggested that the inactive species is the ternary complex E. 1. S. Human fibroblast inhibitor was effective against all vertebrate collagenases tested. Noncollagenolytic proteases and collagenases of nonvertebrate origin were not inhibited. Procollagenase, which does not bind to collagen, also failed to bind the inhibitor. However, inhibitor itself was capable of binding to collagen.The vertebrate collagenases form a class of enzymes capable of initiating the specific degradation of native collagen in the animal organism. Although specific collagenolytic enzymes have now been identified in numerous tissues from a wide variety of species, the nature of the processes which regulate vertebrate collagen degradation have not been clearly defined. The existence of naturally occurring inhibitors of collagenase has been recognized for some time, and it has been speculated