CHARACTERIZATION OF NEUTROPHIL COLLAGENASE

胶原蛋白溶解是急性和慢性炎症的一个显著特征。 胶原蛋白的丢失，无论是在数量上还是在质量上，都可能导致 各种组织的功能异常。胶原酶、金属蛋白酶 主要负责自然胶原蛋白的降解，是 由多种细胞类型合成。而间隙组织 成纤维细胞和滑膜细胞的胶原酶 相同的，中性粒细胞的胶原酶显示出不同的，尽管 有争议的，合成和储存以及生化方面的差异 和免疫学的差异。 该项目将利用抗中性粒细胞胶原酶的单抗。 由研究人员开发和表征，以帮助提纯 中性粒细胞上清液中该酶的活性(见所附转载和 手稿)。对纯化的酶进行了定量表征。 氨基酸分析、氨基端氨基酸序列分析、多肽 测绘和中性糖分析。底物的专一性 纯化的胶原酶将被评估，以及活化能和 氚同位素效应。降解产物的特定裂解位点 通过氨基酸序列分析测定胶原蛋白。的程度 人中性粒细胞与皮肤的免疫交叉反应 胶原酶将在竞争抑制酶联免疫吸附试验中使用 抗HNC的多克隆抗体。 拟议的研究将加深对 中性粒细胞胶原酶的结构和功能生化 以及进一步定义这种胶原酶之间的区别 以及先前鉴定的胶原酶。这一基本原则 生化和免疫学特征应该提供更好的 评估中性粒细胞胶原酶的潜力以及促进 该酶的实际参与作用的进一步研究 炎症时结缔组织的破坏。

Collagenolysis is a prominent feature in acute and chronic inflammation. The loss of collagen, either quantitatively or qualitatively, may lead to abnormal function of various tissues. Collagenases, metalloproteinases primarily responsible for the degradation of native collagen, are synthesized by a variety of cell types. While the interstitial collagenases of the fibroblast and synovial cell apppear virtually identical, the collagenase of the neutrophil shows distinct, although controversial, differences in synthesis and storage as well as biochemical and immunological differences. This project will utilize a monoclonal antibody to neutrophil collagenase developed and characterized by the investigator to aid in the purification of this enzyme from neutrophil supernatants (see appended reprint and manuscript). The purified enzyme will be characterized by quantitative amino acid analysis, amino-terminal amino acid sequence analysis, peptide mapping and neutral sugar analysis. The substrate specificity of the purified collagenase will be assessed as well as the activation energy and deuterium isotope effect. The specific cleavage site of the degraded collagen will be determined by amino acid sequence analysis. The degree of immunological cross-reactivity between human neutrophil and skin collagenase will be evaluated in a competitive inhibition ELISA using a polyclonal antibody to HNC. The proposed studies will provide an increased understanding of the biochemistry of neutrophil collagenase from both structural and functional aspects as well as further define the differences between this collagenase and the previously characterized collagenases. This fundamental biochemical and immunologic characterization should provide a better assessment of the potential of neutrophil collagenase as well as facilitate further studies on the actual participation of this proteinase in connective tissue destruction during inflammation.