Ghosh S K, Paik W K, Kim S
Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
J Biol Chem. 1988 Dec 15;263(35):19024-33.
Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other.
已从小牛脑中纯化出两种不同分子种类的蛋白甲基化酶I（S-腺苷甲硫氨酸：蛋白精氨酸N-甲基转移酶，EC 2.1.1.23），一种对髓鞘碱性蛋白（MBP）具有特异性，另一种对组蛋白具有特异性，通过非变性聚丙烯酰胺凝胶电泳可判断其已接近均一。尽管这两种甲基化酶具有一些共同特性，如利用S-腺苷-L-甲硫氨酸作为甲基供体以及对蛋白结合的精氨酸残基进行甲基化，但它们在分子量、催化特性以及免疫学特性方面明显不同。MBP特异性蛋白甲基化酶I（约500 kDa）优先使MBP甲基化（Km = 2×10⁻⁷ M），对组蛋白的甲基化程度则低得多（Km = 1×10⁻⁴ M），而组蛋白特异性甲基化酶I（约275 kDa）仅使组蛋白甲基化。两种甲基化酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上均显示出两条主要亚基带：MBP特异性的为100和72 kDa，组蛋白特异性的为110和75 kDa。在0.5 mM对氯汞苯甲酸存在下，约50%的MBP特异性酶仍保持活性，而大部分组蛋白特异性酶活性丧失。在2 mM盐酸胍中，前者约90%的酶活性得以保留，而后者几乎完全失活。这些酶对高温（45 - 65℃）也表现出截然不同的失活曲线；MBP酶在高达50℃时稳定，在更高温度下迅速失活，拐点约在57℃。然而，在相同条件下，组蛋白酶在相同温度范围内逐渐且线性地失活。最后，针对任一酶的多克隆抗体进行的蛋白质免疫印迹分析未显示与另一种酶有交叉反应。
