STRUCTURE AND FUNCTION OF LATENT COLLAGENASE

潜在胶原酶的结构和功能

• 批准号: 4692750
• 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/4692750
• 关键词: calcium; chemical structure function; collagenase; enzyme structure; fibroblasts; human tissue; 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-sepharose 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/胶原酶分子和与胶原酶相关的变化。 激活过程。 人皮肤成纤维细胞分泌的胶原酶原 用连续亲和层析法将体外培养的细胞纯化至表观均一 在Zn++螯合物-琼脂糖和肝素-分离上层析， Ultrogel AcA 44分子筛色谱法。 所得制剂 由一个主要的前导（54 K）和一个次要的尾随（57 K）多肽组成 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。 的关系 使用多克隆抗体研究了各种分子形式之间的关系 (PAs)抗前胶原酶和单克隆抗体的补体 (MAs)针对前胶原酶的各种结构域 分子。 各种蛋白质印迹的免疫过氧化物酶染色 通过SDS-PAGE解析的胶原酶/前胶原酶的分子形式显示 两种额外的次要组分都存在于前胶原酶和胶原酶中， 胶原酶肽组。 过程中生成的每个组件 活化反应与α 2-巨球蛋白形成SDS稳定复合物 因此获得了催化活性。 单个MA（克隆X2 a） 仅与前胶原酶种类反应而不与活性形式反应 这使我们相信前胶原酶分子具有 在活化反应过程中丢失的结构域，可能是活化 肽。