Brinckerhoff C E, Benoit M C, Culp W J
Proc Natl Acad Sci U S A. 1985 Apr;82(7):1916-20. doi: 10.1073/pnas.82.7.1916.
Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein that prevented the synthesis of the neutral proteinase collagenase. Conditioned medium was concentrated 10-fold and placed on cultures of rabbit synovial fibroblasts along with an inducer of collagenase (phorbol myristate acetate or latex particles) and [3H]leucine. Collagenase production was measured by immunoprecipitation of culture medium with monospecific antibody. Gel filtration showed that the inhibitory factor had MrS of 12,500, 25,000-50,000, and 150,000, suggesting that the protein may exist as aggregates. Activity was destroyed by boiling, by trypsin, and by dithiothreitol. Production of the inhibitory protein was prevented by cycloheximide. Isoelectric focusing purified the protein 100- to 150-fold and revealed pIs in the range of 3.2-3.7. Glycosylation was demonstrated by binding to Con A-Sepharose. Our data indicate that rabbit synovial fibroblasts autoregulate collagenase production and suggest that the low levels of collagenase seen in resting cultures result from an active suppression of collagenase synthesis.
取自静息兔滑膜成纤维细胞培养物的条件培养基含有一种可抑制中性蛋白酶胶原酶合成的蛋白质。将条件培养基浓缩10倍，与胶原酶诱导剂（佛波酯或乳胶颗粒）以及[3H]亮氨酸一起加入兔滑膜成纤维细胞培养物中。通过用单特异性抗体对培养基进行免疫沉淀来测定胶原酶的产生。凝胶过滤显示抑制因子的分子量分别为12,500、25,000 - 50,000和150,000，表明该蛋白质可能以聚集体形式存在。煮沸、胰蛋白酶和二硫苏糖醇可破坏其活性。放线菌酮可阻止抑制性蛋白的产生。等电聚焦将该蛋白质纯化了100至150倍，显示其等电点在3.2 - 3.7范围内。通过与伴刀豆球蛋白A - 琼脂糖结合证明了其糖基化。我们的数据表明兔滑膜成纤维细胞可自动调节胶原酶的产生，并提示在静息培养物中所见的低水平胶原酶是由于胶原酶合成受到主动抑制所致。
