The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.
摘要
类风湿性滑膜组织外植体释放到培养基中的中性胶原酶已通过超滤和柱色谱法进行纯化，使用的是葡聚糖G - 200、季胺乙基葡聚糖A - 50和超细葡聚糖G - 100。2. 最终的胶原酶制剂对热重构胶原纤维的比活性为每分钟每毫克酶蛋白降解312微克胶原，比活性培养基提高了1000多倍。3. 在有无十二烷基硫酸钠的聚丙烯酰胺圆盘凝胶中进行电泳，结果显示该酶以单一蛋白带迁移。聚丙烯酰胺凝胶和色谱柱的洗脱实验没有提供存在多种胶原酶的证据。4. 通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，该酶的分子量为33000。5. 从离子交换树脂研究以及在酸性和碱性缓冲系统中的凝胶电泳获得的数据表明这是一种带基本电荷的酶。6. 它不水解合成胶原肽Pz - Pro - Leu - Gly - Pro - D - Arg，且不存在非特异性蛋白酶活性。7. 胶原酶在25℃时攻击溶液中的未变性胶原，导致粘度损失58%，并产生两种特征性产物TCA(3/4)和TCB(1/4)。8. 在37℃和pH 8.0时，重构胶原纤维和明胶均被降解为分子量小于10000的肽。9. 根据可溶性羟脯氨酸肽的释放和电子显微镜观察判断，该酶可降解来自肌腱和关节周围软组织的人不溶性胶原，尽管攻击速率低于重构纤维。10. 数据表明，在37℃和中性pH条件下，纯类风湿性滑膜胶原酶可在无非特异性蛋白酶干预的情况下降解明胶、重构纤维和不溶性胶原。11. 讨论了各种胶原底物对胶原酶攻击的不同敏感性。
