Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase
Fatty acyl ethyl esters arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of myocardium, as assayed by the rate of synthesis of ethyl (/sup 14/C)oleate from 0.4 mM (/sup 14/C)oleic acid and 0.2 M ethanol, was 31 nmol/ (g x h), and was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M0 separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was purified over 5000-fold to homogeneity. Gel electrophoresis showed a single polypeptide with M/sub r/ 26,000, and gel permeation chromatography under nondenaturing conditions indicated a M/sub r/ of 50,000 for the active enzyme. Kinetic analyses indicated that greatest rates of synthesis were observed with unsaturated octadecanoic fatty acid substrates. K/sub m/'s for these fatty acids were essentially identical and equal to 0.2 mM; substrate specificity resulted from varying K/sub m/'s for methanol, ethanol, 1-propanol, and 1-butanol, while V/sub max/ was constant at approximately 1.5 nmol/(mg x s). The amino acid analysis of this synthase distinguishes it from typical cholesterol esterases. When the enzyme is maximally active with respect to ethyl ester synthesis, it does not hydrolyze cholesterol oleate. Fatty acid ethyl esters are synthesized in myocardium primarily by a soluble dimeric enzyme comprised of two nearly identical subunits which esterifies free fatty acids with ethanol to produce a nonoxidative metabolite.
- Research Organization:
- Washington Univ. Medical School, St. Louis, MO
- OSTI ID:
- 5776314
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 23:18; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ALCOHOLS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACID ESTERS
CARDIOVASCULAR SYSTEM
DATA
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
ESTERS
ETHANOL
EXPERIMENTAL DATA
HEART
HOMOGENATES
HYDROXY COMPOUNDS
INFORMATION
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIGASES
MAMMALS
METABOLISM
MUSCLES
MYOCARDIUM
NUMERICAL DATA
ORGANIC COMPOUNDS
ORGANS
RABBITS
REACTION KINETICS
TRACER TECHNIQUES
VERTEBRATES