Aswad D W, Deight E A
J Neurochem. 1983 Dec;41(6):1702-9. doi: 10.1111/j.1471-4159.1983.tb00883.x.
A method of polyacrylamide gel electrophoresis utilizing the discontinuous pH-stacking gel format, the cationic detergent cetylpyridinium chloride, and an acidic buffer system has been applied to detection of specific substrates for protein carboxyl methyltransferase (PCM, EC 2.1.1.24) in cytosol fractions of bovine cerebral cortex. This electrophoresis system produces a high-resolution separation of proteins while preventing spontaneous hydrolysis of protein carboxyl methyl esters. Separation occurs largely on the basis of molecular weight. By running polyacrylamide gels at 4 degrees C or 25 degrees C, it was possible to demonstrate that any specific methyl-accepting protein is modified to form a labile methyl ester rather than the more stable N-derivative. Using this system, we have found that partially purified fractions of PCM contain a variety of endogenous methyl-accepting proteins. The apparent specificity of these substrates varies widely; some apparently abundant proteins show little or no methylation, while other apparently less abundant proteins exhibit a relatively high degree of methylation. One protein, with an apparent Mr of 46,000, exhibited an exceptional degree of methylation. Two distinct classes of protein carboxyl methyl esters could be distinguished by their differing susceptibility to nonenzymatic hydrolysis. The possible relevance of our findings to the recent suggestion that PCM specifically methylates abnormal D-aspartyl residues in age-racemized proteins is considered.
一种利用不连续pH堆积凝胶形式、阳离子去污剂十六烷基氯化吡啶和酸性缓冲系统的聚丙烯酰胺凝胶电泳方法，已应用于检测牛大脑皮质胞质溶胶部分中蛋白质羧基甲基转移酶（PCM，EC 2.1.1.24）的特定底物。该电泳系统能实现蛋白质的高分辨率分离，同时防止蛋白质羧基甲酯的自发水解。分离主要基于分子量进行。通过在4℃或25℃下运行聚丙烯酰胺凝胶，有可能证明任何特定的甲基接受蛋白被修饰形成不稳定的甲酯，而不是更稳定的N-衍生物。使用该系统，我们发现PCM的部分纯化级分含有多种内源性甲基接受蛋白。这些底物的表观特异性差异很大；一些明显丰富的蛋白质几乎没有或没有甲基化，而其他明显不那么丰富的蛋白质则表现出相对较高程度的甲基化。一种表观分子量为46,000的蛋白质表现出异常程度的甲基化。两类不同的蛋白质羧基甲酯可根据它们对非酶水解的不同敏感性加以区分。我们还考虑了我们的发现与最近关于PCM特异性甲基化老化消旋蛋白中异常D-天冬氨酰残基这一观点的可能相关性。
