A High-throughput two-phase partition assay to measure the activity of lipid-metabolizing enzymes.
A new method to measure the activity of lipid-metabolizing enzymes is described. Subsequent to an enzymatic reaction, a two-phase system (organic/aqueous) is established by the addition of a phase partition scintillation fluid (PPSF). The PPSF serves as a scintillation fluid, a phase partition agent, and a carrier/separator of an organic-soluble radiolabeled reaction substrate or product. Applying an empirically derived set of conditions typically enhances the separation of substrate from product whereby one species is effectively solubilized in the PPSF. In situ partitioning of the radionuclide-containing organic/lipid phase from the aqueous phase occurs within individual wells of 96-well or 384-well density PPSF-resistant microtiter plates without the requirement for multiple organic solvent extractions and aspirations, making this method applicable to high-throughput screening. The utility of this method for both kinetic characterization and high-throughput screening is demonstrated with acetyl-CoA carboxylase and fatty acid synthase.
Kristie
James
J
Bayer Healthcare, Pharmaceuticals Corp., 400 Morgan Lane, West Haven, CT 06516, USA.
Toth
Joshuaine G
JG
Silverstrim
Christine
C
Pickett
Walter
W
Landro
James A
JA
D000106:Acetate-CoA Ligase / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D013058:Mass Spectrometry
D064429:Fatty Acid Synthases; D000106:Acetate-CoA Ligase