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  • Title: Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-L-methionine.
    Author: Ahmad I, Rao DN.
    Journal: Gene; 1994 May 03; 142(1):67-71. PubMed ID: 8181759.
    Abstract:
    Radioactivity from S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) was bound to the EcoP15 DNA methyltransferase (M.EcoP15) following short-wave ultraviolet (UV) irradiation. The labeled protein was subjected to polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and detected by fluorography and autoradiography. Labeling was found to be dependent on the concentration of AdoMet and time of UV irradiation. The photolabeling by [methyl-3H]AdoMet was specific and blocked by S-adenosyl-L-homocysteine (AdoHcy) and sinefungin which are known to function as competitive inhibitors. Limited digestion of the M.EcoP15-AdoMet adduct by Staphylococcus aureus protease V8 generated three peptides of approx. 50, 32 and 30 kDa. Interestingly, only the 30-kDa peptide fragment contained radioactivity, as detected by SDS-PAGE, followed by fluorography and autoradiography. Further, sequencing of a few amino acids at the N-terminus of these peptides showed that the 30-kDa fragment was the N-terminal portion of M.EcoP15. These results suggest that photolabeling is at the AdoMet-binding site and that the N-terminal half of M.EcoP15 may be involved in substrate binding.
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