Interaction of EcoP1 modification methylase with S-adenosyl-L-methionine: a UV-crosslinking study.
EcoP1 modification methylase was radioactively labeled when incubated with S-adenosyl-L-[methyl-3H]methionine in the presence of ultraviolet light. Crosslinking of the enzyme as detected by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel followed by fluorography and autoradiography, was shown to be specific by a number of criteria. More importantly, EcoP1 modification methylase was also radioactively labeled with S-adenosyl-L-[carboxyl-14C]methionine demonstrating that labeling involved binding of the entire AdoMet molecule rather than methylation of the protein. Further, c2 EcoP1 mutant DNA modification methylases which show negligible or very little methylation activity, correspondingly formed a weak or no adduct upon crosslinking. These results suggest that photolabeling of EcoP1 DNA modification methylase occurs at the AdoMet binding site.
Department of Biochemistry, Indian Institute of Science, Bangalore.
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Krishnamurthy
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Rao
D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D015263:Deoxyribonucleases, Type III Site-Specific / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D014316:Tritium; D014466:Ultraviolet Rays
D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D014316:Tritium; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C030432:endodeoxyribonuclease EcoP1; D015263:Deoxyribonucleases, Type III Site-Specific