Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase

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Abstract
Fatty acyl ethyl esters, previously identified as metabolities of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and CoA. The enzyme in rabbit myocardium that catalyzes this reaction was isolated and purified. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 .MU. ethanol, was 31 nmol/(g .cntdot. h), and all of it was recovered in the 48,400 g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated 2 enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 .mu.g of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with MW 26,000 and gel permeation chromatography under nondenaturing conditions indicated a MW of 50,000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg .cntdot. s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate and arachidonate substrates [0.14, 0.03 and 0.35 nmol/(mg .cntdot. s), respectively]. Km for these fatty acids were essentially identical and equal to 0.2 mM; substrate specificity with respect to alcohol chain length, however, resulted from varying Km for methanol, ethanol, 1-propanol and 1-butanol, i.e., > 1.4, 1.10, 0.53 and 0.07 M, respectively, while Vmax was constant at .apprx. 1.5 nmol/(mg .cntdot. s). The amino acid analysis of this synthase is not unusual and serves to distinguish it from typical cholesterol esterases. Under conditions in which the enzyme is maximally active with respect to ethyl ester synthesis, it did not hydrolyze cholesterol oleate. Thus, fatty acid ethyl esters are synthesized in myocardium primarily by a soluble dimeric enzyme comprised of 2 identical or nearly identical subunits (MW 26,000). This enzyme, not described previously, esterifies free fatty acids with ethanol to produce a nonoxidative metabolite of ethanol that accumulates in vivo with potentially toxic effects.