Mammalian fatty acid synthase activity from crude tissue lysates tracing ¹³C-labeled substrates using gas chromatography-mass spectrometry.

ABSTRACT: Fatty acid synthase (FASN or FAS, EC 2.3.1.85) is the sole mammalian enzyme to synthesize fatty acids de novo from acetyl- and malonyl-coenzyme A (CoA) esters. This article describes a new method that directly quantifies uniformly labeled (¹³C₁₆-labeled palmitate ([¹³C₁₆]palmitate) by tracing [¹³C₂]acetyl-CoA and [¹³C₃]malonyl-CoA using an in vitro FASN assay. This method used gas chromatography-mass spectrometry (GC-MS) to detect [¹³C₁₆]palmitate carboxylate anions (m/z 271) of pentafluorobenzyl (PFB) derivatives and was highly sensitive at femtomole quantities. Uniformly incorporated [¹³C₁₆]palmitate was the primary product of both recombinant and crude tissue lysate FASN. Quantification of FASN protein within crude tissue lysates ensured equal FASN amounts, preserved steady-state kineti

SUBMITTER: Rudolph MC 

PROVIDER: S-EPMC3415257 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature