Fetters H A, Kelleher J, Duerre J A
Can J Biochem Cell Biol. 1985 Oct;63(10):1112-9. doi: 10.1139/o85-139.
The in vitro carboxyl methylation-demethylation of exogenous and endogenous proteins was investigated in rapidly proliferating thymocytes. Of all the cell fractions tested protein carboxyl methyltransferase activity was highest in the nucleoplasmic fraction (3.2 pmol/mg per minute with ACTH and 0.8 pmol/mg per minute with endogenous substrates). The only other fraction with significant activity was the cytosol (1.2 pmol/mg per minute with ACTH). The nuclei from thymocytes are extremely large; consequently some 70-80% of protein carboxyl methyltransferase in these cells is of nuclear origin. The cellular concentration of protein carboxyl methyltransferase and methyl acceptor proteins paralleled the development of the thymus. Mature lymphocytes contained about 25% of the activity of immature thymocytes. Failure to accumulate protein methyl esters, either in vitro in in vivo, was most likely due to the presence of a very active protein methylesterase. This rapid turnover of protein methyl esters was manifest by the continuous production of [3H]methanol when soluble fractions were incubated with S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet). [3H]Methanol production was enhanced upon the further addition of the particulate fractions, particularly chromatin. The turnover of protein methyl esters was primarily enzymatic, since no [3H]methanol was formed when ACTH was incubated with AdoMet and purified protein carboxyl methyltransferase. Utilizing [3H]methanol formation as an index of the rate of protein methylation-demethylation would yield minimal values, since this compound was oxidized via alcohol dehydrogenase or oxidase in these cells. The majority of the methyl acceptor proteins were located in the nuclei. The rapid methylation-demethylation of these proteins may play some role in development and (or) differentiation.
在快速增殖的胸腺细胞中研究了外源性和内源性蛋白质的体外羧基甲基化-去甲基化。在所有测试的细胞组分中，蛋白质羧基甲基转移酶活性在核质组分中最高（促肾上腺皮质激素存在时为3.2 pmol/mg每分钟，内源性底物存在时为0.8 pmol/mg每分钟）。唯一具有显著活性的其他组分是胞质溶胶（促肾上腺皮质激素存在时为1.2 pmol/mg每分钟）。胸腺细胞的细胞核极大；因此这些细胞中约70-80%的蛋白质羧基甲基转移酶起源于细胞核。蛋白质羧基甲基转移酶和甲基受体蛋白的细胞浓度与胸腺的发育平行。成熟淋巴细胞的活性约为未成熟胸腺细胞的25%。无论是在体外还是体内，未能积累蛋白质甲酯很可能是由于存在一种非常活跃的蛋白质甲酯酶。当可溶性组分与S-腺苷-L-[甲基-³H]甲硫氨酸（[甲基-³H]AdoMet）一起孵育时，[³H]甲醇的持续产生表明了蛋白质甲酯的这种快速周转。进一步添加颗粒组分，特别是染色质后，[³H]甲醇的产生增加。蛋白质甲酯的周转主要是酶促反应，因为当促肾上腺皮质激素与AdoMet和纯化的蛋白质羧基甲基转移酶一起孵育时，没有形成[³H]甲醇。利用[³H]甲醇的形成作为蛋白质甲基化-去甲基化速率的指标会得出最小值，因为这种化合物在这些细胞中会通过醇脱氢酶或氧化酶被氧化。大多数甲基受体蛋白位于细胞核中。这些蛋白质的快速甲基化-去甲基化可能在发育和（或）分化中起某种作用。
