Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells.
Overproduction of a Mr 92,000 protomer of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in compactin-resistant C100 cells.
复制标题
抗康克汀 C100 细胞中 3-羟基-3-甲基戊二酰辅酶 A 还原酶的 Mr 92,000 原体过量产生。
DOI：
10.1073/pnas.80.6.1516
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发表时间：
1983
影响因子：
11.1
通讯作者：
Simoni,RD
中科院分区：
文献类型：
--
作者：
Hardeman,EC;Jenke,HS;Simoni,RD
我们描述了一种命名为 C100 的细胞系，该细胞系的胆固醇生物合成途径的主要调节酶 3-羟基-3-甲基戊二酰辅酶 A 还原酶 [HMG-CoA；HMG-CoA；甲羟戊酸：NADP+氧化还原酶（CoA-酰化），EC 1.1.1.34]。在蛋白酶抑制剂苯甲基磺酰氟和亮肽素存在下，对来自 C100 微粒体膜的 [35S] 蛋氨酸标记的酶进行免疫沉淀，结果显示两种上调的蛋白质：主要条带为 Mr92,000，次要条带为 Mr63,000。根据以下标准，我们得出结论，Mr92,000 蛋白可能是 HMG-CoA 还原酶原聚体的完整形式。 (i) 它是一种高度上调的微粒体膜蛋白，与该细胞系中 HMG-CoA 还原酶比活性的增加相一致。 (ii) 在各种严格条件下，它可以被特定的 HMG-CoA 还原酶抗血清识别。 (iii)在不存在蛋白酶抑制剂的情况下，[35S]甲硫氨酸标记的C100微粒体膜的分离和溶解导致Mr92,000蛋白的消失以及Mr52,000和38,000两种蛋白的出现。 (iv)对用[35S]甲硫氨酸标记30分钟、远低于HMG-CoA还原酶半衰期的细胞进行分析，显示总细胞提取物中仅存在Mr92,000蛋白质。 (v)先前报道的用于Mr62,000的HMG-CoA还原酶的单一免疫沉淀多肽[Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982)Proc.国家。阿卡德。科学。 USA79, 1185-1189] 可以被分离出来，并且似乎是 NaDodSO4gel 电泳的蛋白水解和样品制备的结果。对用[35S]蛋氨酸标记的C100细胞进行24小时的分析表明，该酶的主要稳态形式是Mr92,000，而不是Mr63,000蛋白质，进一步表明这两种蛋白质不具有经典的前体-产物关系。
We describe a cell line, designated C100, that displays a 100-fold increase in the major regulatory enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase [HMG-CoA; mevalonate:NADP+oxido-reductase (CoA-acylating), EC 1.1.1.34]. Immunoprecipitation of [35S]methionine-labeled enzyme from C100 microsomal membranes prepared in the presence of the protease inhibitors phenyl-methylsulfonyl fluoride and leupeptin revealed two up regulated proteins: a major band ofMr92,000 and a minor band ofMr63,000. We conclude that theMr92,000 protein is probably the intact form of HMG-CoA reductase protomer based on the following criteria. (i) It is a highly up regulated microsomal membrane protein that coincides with the increase in HMG-CoA reductase specific activity in this cell line. (ii) It is recognized by a specific HMG-CoA reductase antiserum under a variety of stringencies. (iii) Isolation and solubilization of [35S]methionine-labeled C100 microsomal membranes in the absence of protease inhibitors resulted in the disappearance of theMr92,000 protein and the appearance of two proteins ofMr52,000 and 38,000. (iv) Analysis of cells labeled for 30 min with [35S]methionine, well under the half-life of HMG-CoA reductase, revealed only theMr92,000 protein to be present in total cell extract. (v) The previously reported single immunoprecipitation polypeptide for HMG-CoA reductase ofMr62,000 [Chin, D. J., Luskey, K. L., Anderson, R. G. W., Faust, J. R., Goldstein, J. L. & Brown, M. S. (1982)Proc. Natl. Acad. Sci. USA79, 1185-1189] can be isolated and appears to be the result of both proteolysis and sample preparation for NaDodSO4gel electrophoresis. Analysis of C100 cells labeled with [35S]methionine for 24 hr indicates that the predominant steady-state form of the enzyme is theMr92,000, rather than theMr63,000, protein, further suggesting that the two proteins do not have a classical precursor-product relationship.