Characterization of Fatty Acid Synthase Activity Using Scintillation Proximity

Abstract

FAS is a 544-kDa dimeric enzyme consisting of seven functional catalytic components that synthesize long-chain fatty acids from acetyl-CoA and malonyl-CoA using NADPH as a cofactor. We have developed a novel radiometric, homogeneous procedure that directly detects FAS activity. The assay determines incorporation of [³H]acetyl-CoA into palmitic acid as catalyzed by FAS from rat liver. Radiolabeled palmitic acid is captured on a 384-well phospholipid-coated microtiter plate and is brought into close proximity with embedded scintillant, stimulating the emission of photons. Because it uses acetyl-CoA and malonyl-CoA as substrates, the procedure mimics the classical reductase assay. However, this method eliminates such labor-intensive steps as organic extraction, aspirations and washes, phase separations, and sample transfers. Furthermore, it offers advantages over photometric and fluorometric methods that indirectly measure FAS activity via NADPH absorbance. We present here kinetic and inhibition data for FAS using scintillation proximity. The assay is shown to be robust and reproducible.