O'Connor C M, Clarke S
Anal Biochem. 1985 Jul;148(1):79-86. doi: 10.1016/0003-2697(85)90630-x.
A two-dimensional polyacrylamide gel electrophoresis system which is suitable for the analysis of protein methylation reactions in cells incubated with L-[methyl-3H]methionine is described. The procedure separates proteins under primarily acidic conditions by isoelectric focusing in the first dimension and by sodium dodecyl sulfate electrophoresis at pH 2.4 in the second dimension. The low pH is essential for preserving protein [3H]methyl esters, but it limits the effective separating range of this system to proteins with isoelectric points between 4 and 8. With this system, we have shown that most, if not all, erythrocyte membrane and cytosolic proteins can act as substoichiometric methyl acceptors for an intracellular S-adenosylmethionine-dependent carboxyl methyltransferase and that protein carboxyl methylation reactions may be the major methyl transfer reaction in erythrocytes. These results are most consistent with the generation of protein substrate sites for the carboxyl methyltransferase by spontaneous deamidation and racemization reactions.
本文描述了一种二维聚丙烯酰胺凝胶电泳系统，该系统适用于分析用L-[甲基-³H]甲硫氨酸孵育的细胞中的蛋白质甲基化反应。该方法首先在酸性条件下通过等电聚焦在第一维分离蛋白质，然后在第二维通过pH 2.4的十二烷基硫酸钠电泳分离蛋白质。低pH对于保留蛋白质[³H]甲酯至关重要，但它将该系统的有效分离范围限制在等电点在4至8之间的蛋白质。使用该系统，我们已经表明，大多数（如果不是全部）红细胞膜和胞质蛋白可以作为细胞内S-腺苷甲硫氨酸依赖性羧基甲基转移酶的亚化学计量甲基受体，并且蛋白质羧基甲基化反应可能是红细胞中的主要甲基转移反应。这些结果与通过自发脱酰胺和消旋反应产生羧基甲基转移酶的蛋白质底物位点最为一致。
