Wenzel C, Moulard M, Løbner-Olesen A, Guschlbauer W
Département de Biologie Moleculaire et Cellulaire, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
FEBS Lett. 1991 Mar 11;280(1):147-51. doi: 10.1016/0014-5793(91)80224-q.
Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.
在冰浴中，使用传统的Mineralight紫外线灯，在不同浓度的放射性标记S-腺苷甲硫氨酸（AdoMet）存在下，对高度纯化的DNA-腺嘌呤甲基转移酶进行长达1小时的照射，照射时间从几分钟到1小时不等。通过在SDS-聚丙烯酰胺凝胶上对S-腺苷甲硫氨酸与Dam甲基化酶之间的交联物进行电泳，然后进行荧光自显影来监测放射性的掺入情况。在10 microM S-腺苷甲硫氨酸存在下，交联达到最大值；在能竞争性抑制Dam甲基化酶对DNA甲基化的物质（如杀稻瘟菌素或S-腺苷同型半胱氨酸）存在时，交联受到抑制，但在非抑制剂（如ATP或S-异丁基腺苷）存在时则不受抑制。所获得的交联物对蛋白质和肽化学中常用的各种甚至剧烈的条件都具有抗性。不结合S-腺苷甲硫氨酸的蛋白质以及热失活的Dam甲基化酶都没有被光标记。经过有限的蛋白酶解后，放射性标记仅出现在所获得的某些肽段中。用针对与Dam甲基化酶第92 - 106位氨基酸序列对应的合成肽产生的多克隆抗体进行的蛋白质印迹分析表明，AdoMet的交联位点可能位于第106位氨基酸之后。
