METHYL-ESTERIFIED PROTEINS IN A MAMMALIAN-CELL LINE
METHYL-ESTERIFIED PROTEINS IN A MAMMALIAN-CELL LINE
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DOI：
10.1021/bi00344a053
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
KOSHLAND, DE
中科院分区：
文献类型：
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作者：
CHELSKY, D;RUSKIN, B;KOSHLAND, DE
在完整的小鼠S49淋巴瘤细胞的羧酸残基的甲酯化进行了检查，并发现至少24个蛋白质被修改。细胞分级显示，这些蛋白质的一个独特的设置可以在四个馏分中的每一个被发现。细胞核含有12、15.5、18、19、39、41、45、70、90、105和130千道尔顿（kDa）的11种甲基酯化蛋白质。五种蛋白质与质膜/线粒体组分在13、24、25、27和28 kDa处共纯化。两种蛋白质在32和56 kDa的微粒体部分，和六个可溶性在16.5，21，24，26，34，和36 kDa。当细胞匀浆与S-腺苷-L-[甲基-3H]甲硫氨酸孵育时，这些蛋白质中的11个被[3 H]甲基酯化。在完整细胞中，甲基掺入蛋白质的稳态水平约为118 pmol/mg蛋白质。假设平均蛋白质为40 kDa，则每210个蛋白质中似乎有1个甲基。这与磷酸化相比，磷酸化为每四个蛋白质提供大约一个磷酰基。外源性添加的L-[甲基-3H]蛋氨酸在30 min内与细胞S-腺苷甲硫氨酸合并液平衡，该时间足够快，可测定甲基转换速率。大多数甲基化蛋白在脉冲追踪实验中发生去甲基化，半衰期为2.6至9.3 h。当用嘌呤霉素阻断蛋白质合成时，甲硫氨酸的氨基酸骨架掺入减少至对照的2%。然而，甲基基团的掺入是对照的39%。无论在加入嘌呤霉素后15或105分钟加入标记物，都可以达到这种甲基掺入水平，表明这些蛋白质正在连续甲基化或去甲基化。这种修饰的可逆性表明它可能在这些细胞中起调节作用。
Methyl esterification of carboxylic acid residues in intact mouse S49 lymphoma cells was examined, and at least 24 proteins were found to be modified. Cell fractionation revealed that a distinct set of these proteins could be found in each of the four fractions. Nuclei contained 11 methyl-esterified proteins at 12, 15.5, 18, 19, 39, 41, 45, 70, 90, 105, and 130 kilodaltons (kDa). Five proteins copurified with the plasma membrane/mitochondrial fraction at 13, 24, 25, 27, and 28 kDa. Two proteins at 32 and 56 kDa were in the microsomal fraction, and six were soluble at 16.5, 21, 24, 26, 34, and 36 kDa. Eleven of these proteins were [3H]methyl esterified when cell homogenates were incubated with S-adenosyl-L-[methyl-3H]methionine. The steady-state level of methyl group incorporation into protein in intact cells was approximately 118 pmol/mg of protein. Assuming the average protein is 40 kDa, there appears to be 1 methyl group per 210 proteins. This was compared to phosphorylation which gave approximately one phosphoryl group for every four proteins. Exogenously added L-[methyl-3H]methionine equilibrated with the cellular S-adenosylmethionine pool within 30 min which was sufficiently rapid to allow the rate of methyl group turnover to be determined. Most methyl-esterified proteins demethylated in a pulse-chase experiment with half-lives ranging from 2.6 to 9.3 h. When protein synthesis was blocked with puromycin, amino acid backbone incorporation of methionine was reduced to 2% of control. Methyl group incorporation, however, was 39% of the control. This level of methyl group incorporation could be attained whether label was added 15 or 105 min after the addition of puromycin, indicating that these proteins were being continuously methylated or demethylated. The reversible nature of this modification suggests that it may serve a regulatory function in these cells.