Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.
Secretion and uptake of beta-N-acetylglucosaminidase by fibroblasts. Effect of chloroquine and mannose 6-phosphate.
复制标题
成纤维细胞分泌和摄取 β-N-乙酰氨基葡萄糖苷酶。
DOI：
10.1016/0304-4165(79)90034-5
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发表时间：
1979
期刊：
影响因子：
--
通讯作者：
S. Rattray
中科院分区：
文献类型：
--
作者：
P. Willcox;S. Rattray
比较了氯喹和 6-磷酸甘露糖对人成纤维细胞分泌和摄取溶酶体酶 ß-N-乙酰氨基葡萄糖苷酶 (EC 3.2.1.30) 的影响。在氯喹浓度范围为 5 μM 至 100 μM 时，细胞内酶的消耗与细胞外酶的积累之间存在相互关系。随着氯喹浓度的增加，系统中酶活性（细胞内活性和细胞外活性）的丧失是由于β-N-乙酰氨基葡萄糖苷酶的抑制所致。在浓度为50 μM时，氯喹在24小时内引起β-N-乙酰氨基葡萄糖苷酶的细胞外积累增加三倍，而添加5 mM甘露糖6-磷酸（受体介导的摄取的竞争性抑制剂）结果仅增加了13%。 5 mM 6-磷酸甘露糖完全抑制酶缺陷成纤维细胞对β-N-乙酰氨基葡萄糖苷酶的摄取。在氯喹存在的情况下，酶也没有被吸收，但细胞的残余活性显着下降。结果表明，氯喹对成纤维细胞的作用是刺激分泌，而不是像之前报道的那样抑制摄取。将正常培养基中β-N-乙酰氨基葡萄糖苷酶的同工酶模式与细胞暴露于50 μM氯喹后在培养基中积累的同工酶模式进行了比较。在氯喹存在的情况下，A 同工酶增加，但活性在宽峰中被洗脱，这可能代表该酶的几种密切相关的形式。在氯喹存在下培养的成纤维细胞中，β-N-乙酰氨基葡萄糖苷酶 A 同工酶几乎完全丧失。在氯喹处理的成纤维细胞的提取物中存在与分泌的As同工酶相似的位置洗脱的活性小峰，这表明Asiso酶在不同于细胞内同工酶的位点处形成和/或储存。
The effects of chloroquine and mannose 6-phosphate on the secretion and uptake of the lysosomal enzyme,ß-N-acetylglucosaminidase (EC 3.2.1.30), by human fibroblasts have been compared. There was a reciprocal relationship between intracellular depletion, and extracellular accumulation, of enzyme at chloroquine concentrations ranging from 5 μM to 100 μM. A loss of enzyme activity from the system (intra- plus extracellular activity) with increasing concentrations of chloroquine was due to inhibition of theß-N-acetylglucosaminidase.At a concentration of 50 μM, chloroquine elicited a three fold increase in the extracellular accumulation ofß-N-acetylglucosaminidase in 24 h wheras the addition of 5 mM mannose 6-phosphate (a competitive inhibitor of receptor-mediated uptake) resulted in only a 13% increase. Uptake ofß-N-acetylglucosaminidase by enzyme-deficient fibroblasts was completely inhibited by 5 mM mannose 6-phosphate. In the presence of chloroquine there was also no uptake on enzyme, however there was marked decrease in the residual activity of the cells. The results suggest that the effect of chloroquine on fibroblasts is to stimulate secretion rather than to inhibit uptake as previously reported.The isoenzyme pattern of theß-N-acetylglucosaminidase from normal culture medium was compared with that accumulating in the medium following exposure of the cells to 50 μM chloroquine. In the presence of chloroquine, there was an increase in the A isoenzyme, however the activity was eluted in a broad peak which probably represents several closely related forms of the enzyme. There was an almost total loss of the A isoenzyme ofß-N-acetylglucosaminidase from fibroblasts cultured in the presence of chloroquine. A small peak of activity eluting at a similar position to the secreted, As, isoenzyme was present in extracts of chloroquine-treated fibroblasts, suggesting that the Asisoenzyme is formed and/or stored at a site distinct from the intracellular isoenzyme.