Development and application of the first colorimetric poly(ADP-ribose)polymerase (PARP) substrate

BIOL 206

Amanda C. Nottbohm, boye@uiuc.edu1, Robin S. Dothager2, Karson S. Putt2, Mirth Hoyt1, and Paul J. Hergenrother, hergenro@uiuc.edu1. (1) Department of Chemistry, University of Illinois at Urbana-Champaign, Box 88-5, 600 S. Mathews, Urbana, IL 61801, (2) Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews, Urbana, IL 61801
Utilizing β-NAD+ to catalyze the synthesis of ADP-ribose polymers onto a variety of protein acceptors, the poly(ADP-ribose) polymerase (PARP) family of enzymes is involved in countless cellular functions. As PARPs play a critical role in cellular survival and maintenance of energy stores after genotoxic insult, small molecule inhibitors of PARP isozymes have been touted as possible therapies for neurodegeneration, ischemia, and as potentiators of anticancer therapies. Current methods for measuring PARP activity are inconvenient and low throughput; thus neither direct kinetic comparison of PARP isozyme activities nor IC50 comparison of known PARP inhibitors against the multitude of PARP isozymes has been offered in the literature. We have therefore developed an assay which utilizes a colorimetric PARP substrate to kinetically monitor PARP activity. By employing this new substrate, we have determined kinetic parameters of PARP-1, tankyrase, and VPARP, and these results offer insight into the isozyme specificity of known PARP inhibitors.
 

Enzymes
4:30 PM-6:30 PM, Wednesday, 13 September 2006 Moscone Center -- Hall D, Poster

Division of Biological Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006