MICROPLATE CHROMATOGRAPHY ASSAY FOR ACETYL-COA - LYSOPLATELET-ACTIVATING FACTOR ACETYLTRANSFERASE

Acetyl-CoA:lysoplatelet-activating factor (1-O-alkyl-sn-glycero-3-phos phocholine) acetyltransferase (lysoPAF-AT) (EC 2.3.1.67) is a key enzy me in the biosynthesis of platelet-activating factor (PAF) and has bee n shown to be activated by various extracellular stimuli. A novel meth od to determine the enzyme activity is described here, which enables 9 6 simultaneous assays in a standard 96-well microplate format. The ass ay is based on the quantification of the incorporation of [H-3]acetyl- CoA into PAF in the presence of lysoPAF. The radioactive products are separated from the substrate with a 96-well-formatted chromatography d evice using a Multiscreen plate (Millipore) prefilled with octyl-silic a gel. As little as 1 mg octyl-silica gel was sufficient for the effic ient recovery of the radioactive product, resulting in the very low ba ckground and thus high sensitivity. The enzyme activity could be measu red directly with whole cell lysates from various cells cultured in 96 -well microplate scale, This tailor-made microplate chromatography sep aration step is readily applicable for other kinds of enzyme assays. ( C) 1997 Academic Press.