Crosslinking of Dam methyltransferase with S‐adenosyl‐methionine

FEBS Letters

Guschlbauer, Wilhelm²  Wenzel, Caroline²  Moulard, Maxime²  Lobner-Olesen, Anders¹ 

[1] Department of Microbiology, The Technical University of Denmark, DK-2800 Lyngby, Denmark;Service de Biochimie et de Gènètique Molèculaire, Bât. 142. Department de Biologie Moleculaire et Cellulaire Centre d'Etudes Nuclèaires de Saclay, F-91191 Gif-sur-Yvette Cedex, France

关键词: Dam methylase;    S-Adenosyl-methionine;    Photocrosslinking;    Sinefungin;    Proteolysis;    AdoMet;    S-adenosyl-methionine;    AdoHcy;    S-adenosyl-homocysteine;    SIBA;    S-isobutyl-adenosine;    [3H]AdoMet;    3H3C-S-adenosyl-methionine;    [14C]AdoMet;    S-adenosyl[3;    414C]methionine;    Dam methylase;    DNA-adenine-methyltransferase;    DTT;    dithiothreitol;   

DOI  :  10.1016/0014-5793(91)80224-Q

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 μM S-adenosyl-methionine; it was inhibited in the presence of substates 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 activated 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.

【 授权许可】

Unknown