STRUCTURE AND FUNCTION OF LATENT COLLAGENASE

潜在胶原酶的结构和功能

• 批准号: 3940063
• 负责人: HENNING BIRKEDAL-HANSEN
• 金额: --
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3940063
• 关键词: calcium; chemical structure function; collagenase; enzyme structure; fibroblasts; human tissue; monoclonal antibody; zinc; zymogens

The objective of this study is to investigate the structure of the procollagenase/collagenase molecule and the changes associated with the activation process. Procollagenase produced by human skin fibroblasts in vitro was purified to apparent homogenity by successive affinity chromatography on Zn++ chelate-separose and heparin-separose followed by Ultrogel AcA 44 molecular sieve chromatography. The resultant preparation consisted of a major lead (54K) and a minor trailing (57K) polypeptide chain. Conversion to active form either by trypsin or organomercurials resulted in generation of two new (44K/47K) components. The relationship between the various molecular forms was studied using polyclonal antibodies (PAs) against procollagenase and by a complement of monoclonal antibodies (MAs) directed against the various structural domains of the procollagenase molecule. Immunoperoxidase staining of Western blots of the various molecular forms of collagenase/procollagenase resolved by SDS- PAGE revealed two additional minor components both in the procollagenase and the collagenase peptide groups. Each of the components generated during the activation reaction formed SDS- stable complexes with Alpha2-macroglobulin and therefore had acquired catalytic activity. A single MA (Clone X2a) reacted only with the procollagenase species and not with the active forms which leads us to believe that the procollagenase molecule possesses a domain that is lost during the activation reaction, possibly an activation peptide.

本研究的目的是探讨 前胶原酶/胶原酶分子及其相关变化 激活过程。 人源胶原酶 体外皮肤成纤维细胞被纯化至表观均一， Zn ~（++）螯合分离连续亲和层析 肝素-分离，然后是Ultrogel AcA 44分子筛 层析 由此产生的准备工作包括一个主要的 前导（54 K）和次要拖尾（57 K）多肽链。 通过胰蛋白酶或有机汞转化为活性形式 导致产生两个新的（44 K/47 K）组件。 的 研究了各种分子形式之间的关系 使用抗前胶原酶的多克隆抗体（PA）， 单克隆抗体（MAs）的补体， 前胶原酶分子的各种结构域。 免疫过氧化物酶染色的各种蛋白质印迹 通过SDS-聚丙烯酰胺凝胶电泳分离的胶原酶/前胶原酶的分子形式 PAGE显示两个额外的次要组分， 前胶原酶和胶原酶肽组。 每个 在活化反应过程中产生的组分形成SDS- 与α 2-巨球蛋白形成稳定的复合物，因此 获得催化活性。 单个MA（克隆X2 a）仅反应 而不是活性形式 这让我们相信前胶原酶分子 具有在活化反应期间丢失的结构域， 可能是激活肽。