Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.
Biosynthesis and secretion of procollagenase by rabbit synovial fibroblasts. Inhibition of procollagenase secretion by monensin and evidence for glycosylation of procollagenase.
复制标题
兔滑膜成纤维细胞原胶原酶的生物合成和分泌。
DOI：
10.1042/bj2140281
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
E. D. Harris
中科院分区：
文献类型：
--
作者：
H. Nagase;C. Brinckerhoff;C. Vater;E. D. Harris
用佛波醇肉豆蔻酸酯乙酸酯刺激以产生大量胶原酶的兔滑膜成纤维细胞的单层培养物（EC 3.4.24.7）用于研究该酶的生物合成和分泌。将[3 H]亮氨酸加入细胞培养物中进行脉冲追踪和连续标记实验。合成的标记的胶原原酶通过免疫沉淀，然后通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳和荧光法进行鉴定。通过测量掺入蛋白质的放射性来定量细胞内和细胞外酶原的量。前胶原酶合成为Mr 57000和Mr 61000的双联体蛋白。向细胞中加入[3 H]亮氨酸35分钟后，首次在培养基中检测到免疫沉淀的酶原蛋白。莫能菌素处理的细胞抑制胶原原酶的分泌，并导致细胞内积累的酶原。用衣霉素处理的细胞仅产生57 000-Mr形式，表明在兔滑膜细胞中，61 000-Mr形式通过向天冬酰胺残基添加寡糖进行后修饰。细胞裂解物和培养基中糖基化与非糖基化形式的比例分别为0.22：1和0.07：1。
Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of intracellular and extracellular proenzyme were quantified by measuring radioactivity incorporated into the proteins. procollagenase was synthesized as doublet proteins of Mr 57 000 and Mr 61 000. Immunoprecipitable proenzyme proteins were first detected in culture medium 35 min after [3H]leucine was added to the cells. Monensin treatment of the cells inhibited procollagenase secretion and led to intracellular accumulation of the proenzyme. Cells treated with tunicamycin produced only the 57 000-Mr form, indicating that in rabbit synovial cells the 61 000-Mr form was post-translationally modified by addition of oligosaccharides to asparagine residues. The ratios of glycosylated to unglycosylated forms in cell lysates and in culture medium were 0.22:1 and 0.07:1 respectively.