Configuration of a scintillation proximity assay for the activity assessment of recombinant human adenine phosphoribosyltransferase.
Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.
Barrett
Cynthia
C
Discovery Technologies, Millennium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. cynthia.barrett@mpi.com
Alley
Jennifer
J
Pulido
Jacqueline C
JC
Spurling
Heidi
H
Li
Ping
P
Parsons
Thomas
T
Mallender
William D
WD
Bembenek
Michael E
ME
D000225:Adenine / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D011770:Pyruvate Kinase / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods*; D014316:Tritium
D011994:Recombinant Proteins; D009243:NAD; D014316:Tritium; D007770:L-Lactate Dehydrogenase; D000228:Adenine Phosphoribosyltransferase; D011770:Pyruvate Kinase; D000263:Adenylate Kinase; D000225:Adenine