Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine

Ahmad, Ishtiyaque ; Rao, Desirazu N. (1994) Photolabeling of the EcoP15 DNA methyltransferase with S-adenosyl-l-methionine Gene, 142 (1). pp. 67-71. ISSN 0378-1119

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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 [methyl3H]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 autoradiog- raphy. 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.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:DNA Methylation; Sinefungin; Restriction-modification; UV Crosslinking
ID Code:51211
Deposited On:28 Jul 2011 07:23
Last Modified:28 Jul 2011 07:23

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