INDUCTION OF PYRUVATE-CARBOXYLASE APOENZYME AND HOLOENZYME IN 3T3-L1 CELLS DURING DIFFERENTIATION
INDUCTION OF PYRUVATE-CARBOXYLASE APOENZYME AND HOLOENZYME IN 3T3-L1 CELLS DURING DIFFERENTIATION
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DOI：
10.1073/pnas.77.3.1321
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发表时间：
1980-01-01
期刊：
影响因子：
--
通讯作者：
UTTER, MF
中科院分区：
文献类型：
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作者：
FREYTAG, SO;UTTER, MF
当3 T3-L1 [小鼠成纤维细胞]细胞分化为脂肪细胞样形式时，这些细胞中丙酮酸羧化酶[丙酮酸：二氧化碳连接酶（ADP形成）; EC 6.4.1.1]的比活性增加约20倍。纯化的大鼠肝酶的特异性抗体从3 T3-L1粗匀浆中定量沉淀丙酮酸羧化酶。使用这种免疫学技术可以证明丙酮酸羧化酶活性的增加是由于该酶的细胞内浓度的增加。在分化的3 T3-L1细胞中丙酮酸羧化酶的含量足够高（占总蛋白的1-2%），当3 T3-L1粗提物在十二烷基硫酸钠/聚丙烯酰胺凝胶上进行电泳时，可以观察到这种大蛋白（亚基MW = 130，000）的增加。当3 T3-L1细胞在抗生物素蛋白存在下分化时，它们含有少于5%的丙酮酸羧化酶活性的细胞，在抗生物素蛋白不存在下分化。抗生物素蛋白处理的细胞的免疫沉淀的丙酮酸羧化酶的含量基本上是相同的细胞，没有抗生物素蛋白分化。在加入过量生物素后，抗生物素蛋白处理的细胞中酶的全部活性迅速恢复。活性的恢复与[14 C]生物素掺入免疫沉淀丙酮酸羧化酶密切相关。恢复活性的速度和该过程对蛋白质合成抑制剂的不敏感性强烈表明，在抗生物素蛋白存在下，丙酮酸羧化酶的脱辅基酶在分化过程中积累。
The specific activity of pyruvate carboxylase [pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.1] in 3T3-L1 [mouse fibroblast] cells increases approximately 20-fold when these cells differentiate to an adipocyte-like form. A specific antibody to the purified rat liver enzyme quantitatively precipitated pyruvate carboxylase from 3T3-L1 crude homogenates. Use of this immunological technique permitted a demonstration of an increase in pyruvate carboxylase activity due to an increase in the intracellular concentration of the enzyme. The content of pyruvate carboxylase in differentiated 3T3-L1 cells is sufficiently high (1-2% of total protein) that the increase in this large protein (subunit MW = 130,000) can be visualized when 3T3-L1 crude extracts are subjected to electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. When 3T3-L1 cells differentiated in the presence of avidin, they contained less than 5% of the pyruvate carboxylase activity of cells that differentiated in the absence of avidin. The immunoprecipitable pyruvate carboxylase content of the avidin-treated cells was essentially the same as that of cells that differentiated without avidin. Full activity of the enzyme was rapidly restored in the avidin-treated cells upon the addition of excess biotin. The recovery of activity was closely correlated with the incorporation of [14C]biotin into immunoprecipitable pyruvate carboxylase. The rapidity with which the activity was restored and the insensitivity of the process to inhibitors of protein synthesis strongly suggest that the apoenzyme of pyruvate carboxylase accumulates during differentiation in the presence of avidin.