Turnover of two lysosomal enzymes in macrophages.
Turnover of two lysosomal enzymes in macrophages.
复制标题
巨噬细胞中两种溶酶体酶的周转。
DOI：
10.1016/s0021-9258(19)68754-5
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
R. Swank
中科院分区：
文献类型：
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作者：
M. Skudlarek;R. Swank
溶酶体水解酶# I-半乳糖苷酶（亚基 M,= 63,000）在硫代乙醇酸引发的小鼠腹膜巨噬细胞中合成为高分子量前体形式（M,= 82,000）（Skudlarek, M. D. 和 Swank, R. T.(1979) J. Biol. Chem 254， 9939-9942）。通过分析在十二烷基硫酸钠聚丙烯酰胺凝胶中电泳的 8-葡萄糖醛酸酶的免疫沉淀物，我们现在发现了成熟 8-葡萄糖醛酸酶 (Mr= 73,000) 的前体形式 (Mr= 75,000)。我们使用维持两种溶酶体酶和总蛋白稳态浓度的培养条件确定了两种酶的前体和成熟形式的周转动力学。免疫沉淀[36S]甲硫氨酸标记/3-葡萄糖醛酸酶的相对合成率比8-半乳糖苷酶高2倍（分别占总蛋白质合成的0.024%和0.01%）。对于任一酶，用[3H1甘露糖放射性标记的碳水化合物部分的相对合成率是总糖蛋白合成的0.04%。在放射性标记的第一小时内，仅通过放射线检测到前体形式。 1小时后，细胞相关的前体向成熟酶的转化开始。“其速度很快，并且遵循一级动力学，tIj2为1小时。8-半乳糖苷酶前体的加工包括富含甘露糖片段的裂解，因为与成熟酶相比，前体/3-半乳糖苷酶中甘露糖/蛋氨酸放射性标记的比率高出4倍。成熟酶的放射性标记丢失，半衰期为β-葡糖醛酸酶和β-半乳糖苷酶的肽基部分分别为1.8天和3.5天，在用甘露糖进行放射性标记后观察到β-葡糖醛酸酶的半衰期缩短了2倍，这表明每种酶内碳水化合物和肽基部分的协调周转导致了β-半乳糖苷酶和β-葡糖醛酸酶的成熟形式的损失的至少一半。在细胞外培养基中仅检测到成熟酶（细胞加培养基）的β-葡萄糖苷酸酶和10%。因此，蛋白质合成相对速率的2倍差异和放射性标记损失率的2倍差异表明巨噬细胞中两种溶酶体酶的单体存在非协调合成和损失。
The lysosomal hydrolase# I-galactosidase (subunit M,= 63,000) is synthesized as a high molecular weight precursor form (M,.= 82,000) in thioglycollate-elicited mouse peritoneal macrophages (Skudlarek, M. D., and Swank, R. T.(1979) J. Biol. Chem 254, 9939-9942). By analysis of immunoprecipitates of 8-glucuronidase electrophoresed in sodium dodecyl sulfate polyacrylamide gels, we now find a precursor form (Mr= 75,000) of mature 8-glucuronidase (Mr= 73,000). We have determined the turnover kinetics of precursor and mature forms of both enzymes using culture conditions which maintain steady state concentrations of the two lyso-somal enzymes and total protein. The relative rate of synthesis of immunoprecipitable [36S] methionine-labeled/3-glucuronidase was 2-fold higher than 8-galactosidase (0.024% and 0.01% of total protein synthesis, respectively). The relative rate of synthesis of the carbohydrate moiety radiolabeled with [3Hlmannose is 0.04% of total glycoprotein synthesis for either enzyme. Only precursor forms are detected radiographically during the 1st h of radiolabeling. After 1 h, a cellassociated conversion of precursor to mature enzyme begins.“his is rapid, and it follows first order kinetics with a tIj2 of 1 h. Processing of the 8-galactosidase precursor includes cleavage of a mannose-rich segment since ratios of mannose/methionine radiolabels are 4-fold greater in precursor/3-galactosidase compared to the mature enzyme.Radiolabel loss from mature enzyme occurred with a half-life of 1.8 days and 3.5 days for the peptidyl moieties of/?-glucuronidase and &galactosidase, respectively. A similar 2-fold shorter half-life of fl-glucuronidase was observed after radiolabeling with mannose suggesting coordinate turnover of carbohydrate and peptidyl portions within each enzyme. Secretion accounted for at least half of the loss of mature forms of &galactosidase and &glucuronidase. Daily enzyme secretion was 20% of total (cellular plus medium) for p-glucuronidase and 10% for &galactosidase. Only mature enzyme was detected in the extracellular medium. Therefore, the 2-fold difference in relative rates of protein synthesis and the 2-fold difference in rates of radiolabel loss indicate that there is both noncoordinate synthesis and loss of the monomers of two lysosomal enzymes in macrophages.