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Selective biotinylation of inosine 5′‐monophosphate dehydrogenase in cell extracts with a small molecule inhibitor
Author(s) -
Hoefler Chris,
Hedstrom Liz
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1058.4
Subject(s) - biotinylation , avidin , chemistry , biochemistry , small molecule , biotin , imp dehydrogenase , medicine , surgery , transplantation , mycophenolic acid
Recent investigations into the cellular functions of inosine 5′‐monophosphate dehydrogenase (IMPDH) have revealed roles for IMPDH in vivo that appear to be unrelated to its known catalytic activity. Of particular interest is its connection to the onset of autosomal dominant retinitis pigmentosa. To facilitate further investigation, it was necessary to design a system whereby IMPDH could be selectively labeled in cell extracts. A known small molecule inhibitor of IMPDH, 6‐chloropurine‐9‐β‐D‐ribofuranoside 5′‐monophosphate, was modified by introduction of an amino linker at the 2′‐OH. The modified inhibitor was then biotinylated using standard coupling chemistry. Studies with purified IMPDH were carried out to demonstrate the utility of the modified inhibitor. It was shown that IMPDH can be inhibited by the new compound in a time and concentration dependent manner. It was further shown that the inhibited IMPDH is also biotinylated by Western blot with avidin‐HRP conjugate. Finally, it was shown that IMPDH in bacterial cell extracts can be selectively labeled with the modified inhibitor and pulled out using avidin bound to sepharose beads. We can now use this compound to pull IMPDH out of native tissues and look for interactions with other proteins, nucleic acids, and macromolecular complexes. Supported by NIH EY017325 and GM054403.