Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

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Abstract
Non‐oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE‐cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5,7 and 11 mS, respectively. The major synthase was purified 8900‐fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti‐human albumin affinity‐chromatographies with an overall yield of 2596. SDS‐PAOE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates (V max = 100 and 65 nmol/mg/h for oleate and linoleate, respectively). K m values for oleate, linoleate, arachidonate, palmitate and stéarate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised of two identical, or nearly identical, subunits (M r = 26000).