The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture.
The proteinase inhibitors leupeptin, pepstatin A, and TLCK cause reduced collagen production in freshly isolated embryonic chick fibroblasts in suspension culture.
复制标题
蛋白酶抑制剂亮肽素、胃酶抑素 A 和 TLCK 会导致悬浮培养中新鲜分离的胚胎鸡成纤维细胞中胶原蛋白的产生减少。
DOI：
10.1016/0003-9861(84)90454-5
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发表时间：
1984
影响因子：
3.9
通讯作者：
Berg,RA
中科院分区：
文献类型：
--
作者：
Neblock,DS;Berg,RA
鸡胚腱细胞悬浮培养时，在溶酶促进剂NH4Cl或氯喹存在下孵育时，细胞产生的[14C]-脯氨酸标记的胶原减少。由于这些药物对成纤维细胞有多种作用，包括抑制胶原分泌，因此测试了特定的蛋白酶抑制剂对胶原产生的影响。本实验研究了半胱氨酸和丝氨酸蛋白酶专一性的N-对甲苯磺酰-L-赖氨酸氯甲基酮和亮氨酸蛋白酶抑制剂，以及天冬氨酸蛋白酶专一性的胃抑素A对胶原合成和分泌的影响。用蛋白水解酶抑制剂TLCK处理后，10μ组胶原蛋白合成百分率由对照的19.0±1.4%降至10.5±2.4%。100μ的mTLCK使胶原合成进一步减少，仅占总蛋白合成的1.2%。50μg/mlLeuPeptin和60μg/mlPepstatin A单独作用时，胶原酶可消化的多肽中[14C]Pro的掺入量仅略有下降，但两者共同作用时，胶原蛋白的合成减少到总蛋白合成的10.9±1.4%。新合成的[14C]-脯氨酸标记的胶原蛋白在细胞内的基础降解不受任何试剂的显著影响，所产生的胶原蛋白的分泌也不受影响，除非用100μ的mTLCK处理。这些数据符合这样的假设，即利用半胱氨酸、丝氨酸和天冬氨酸蛋白酶的某种组合的蛋白分解机制是新鲜分离的鸡腱成纤维细胞持续合成胶原所必需的，并表明迄今未知的调控系统可能在控制成纤维细胞中胶原的合成方面起作用。
The production of [14C]proline-labeled collagen by embryonic chick tendon cells in suspension culture is reduced when the cells are incubated in the presence of lysosomotropic agents NH4Cl or chloroquine. Since these agents have multiple effects on fibroblasts, including inhibition of collagen secretion, specific proteinase inhibitors were tested for their effect on collagen production. Here the proteinase inhibitorsN-p-tosyl-l-lysine chloromethylketone (TLCK) and leupeptin, specific for certain cysteine and serine proteinases, and pepstatin A, specific for aspartic proteinases, were tested for their effects on both the production and secretion of collagen. When treated with the proteinase inhibitor TLCK, the percentage of protein synthesis devoted to collagen decreased from control levels of 19.0 ± 1.4% to 10.5 ± 2.4% with 10 μmTLCK. Collagen synthesis was further reduced to only 1.2% of total protein synthesis with 100 μmTLCK. The incorporation of [14C]proline into collagenase-digestible peptides was only slightly decreased in the samples treated separately with 50 μg/ml leupeptin or 60 μg/ml pepstatin A. However, the production of collagen was reduced to 10.9 ± 1.4% of total protein synthesis in samples treated with leupeptin and pepstatin A together. The basal intracellular degradation of newly synthesized, [14C]proline-labeled collagen was not significantly altered by any of the reagents tested, and secretion of the collagen which was produced was not impaired except in samples treated with 100 μmTLCK. The data presented are consistent with the hypothesis that a proteolytic mechanism utilizing some combination of cysteine, serine, and aspartic proteinases is necessary for continued collagen synthesis in freshly isolated embryonic chick tendon fibroblasts, and suggests that a heretofore unknown regulatory system may be operative in controlling the synthesis of collagen in fibroblasts.