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

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. 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 M ethanol, was 31 nmol/(g.h), and all of it 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 M) separated two 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 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr 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.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.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic bases for fatty acid ethyl ester synthase catalyzed esterification of free fatty acid with ethanol and accumulation of fatty acid ethyl esters.

Myocardial homogenates rapidly synthesize fatty acyl ethyl esters from nonesterified fatty acid and ethanol in the absence of coenzyme A or ATP, and the enzyme catalyzing this reaction, fatty acid ethyl ester synthase, has been purified 5400-fold to homogeneity [Mogelson, S., & Lange, L. G. (1984) Biochemistry (preceding paper in this issue)]. To define the factors permitting this de novo synthesis of ester bonds and the consequent accumulation of fatty acyl ethyl esters in myocardium, we determined thermodynamic parameters relevant to the kinetics and equilibria of this reaction and specifically characterized (1) the rates of synthesis of ethyl oleate, in both the presence and absence of purified enzyme catalyst, and (2) the physical properties of the product, ethyl oleate, in an aqueous milieu. Compared to the reaction of ethanol and oleate in the absence of catalyst, fatty acid ethyl ester synthase enhanced the rate of ethyl oleate synthesis by reducing the free energy of activation (delta G) from 32.5 to 19.9 kcal/mol, effected in large part by a positive entropy shift, delta Senz - delta S uncat = 23.9 cal/(mol.deg). Rate constants in the presence and absence of enzyme at 37 degrees C were 6 X 10(-2) s-1 and 7.8 X 10(-11) M-1 s-1, respectively, indicating a catalytic power of at least 10(8)M for this enzyme. Kinetic data indicated an enzymatic Vmax of 1.25 nmol/(mg.s) (37 degrees C). The equilibrium constant was calculated for the reaction oleate + ethanol in equilibrium ethyl oleate and was 0.095 M-1 at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Partial purification and product characterization of fatty acid ethyl ester synthases in rabbit myocardium.

Homogenates of rabbit ventricular myocardium synthesize fatty acid ethyl esters using as substrates nonesterified fatty acid and ethanol in the absence of coenzyme A and ATP. This catalytic activity resides in two soluble cytosolic enzymes accounting for 19 and 81% of total fatty acid ethyl ester synthetic capability. These enzymes have been separated and partially purified by anion exchange chromatography. Gas chromatographic/mass spectrometric analyses of the catalytic products formed by these enzymes from nonesterified fatty acid and ethanol confirm their identity as ethyl esters of fatty acids. Kinetic studies indicate apparent Km values for ethanol of 0.65 M and 0.75 M for the minor and major activities, respectively. These data confirm the presence of a myocardial pathway for nonoxidative ethanol metabolism and for a metabolism of fatty acids independent of coenzyme A.

Ethanolamine plasmalogen methylation by rabbit myocardial membranes.

Enzymatic methylation of alkenylacylglycerophosphoethanolamine to form alkenylacylglycerophosphocholine was observed in rabbit myocardial membranes, and was compared to the corresponding methylation sequence for diacyl substrates. Membranes were incubated with S-adenosyl-L-[methyl-3H]methionine and assayed for incorporation of radioactivity into selected lipids. The rate of incorporation of methyl groups into diacylglycerophosphocholine exceeded that for alkenylacylglycerophosphocholine, 12.0 +/- 3.6 vs. 3.9 +/- 0.7 pmol product formed/mg per h (mean +/- S.D.), even when normalized for ethanolamine substrate concentration (5.7 +/- 1.6 vs. 1.8 +/- 0.4 pmol CH3 incorporated/mumol diradylglycerophosphoethanolamine). Rabbit myocardial phospholipid methyltransferase activity is optimal at basic pH for each substrate, is moderately stimulated by added Ca2+ or Mg2+, and is completely inhibited by S-adenosylhomocysteine. An apparent Km of 0.2 mM for S-adenosylmethionine applies to diacyl- and alkenylacylglycerophosphocholine formation; at low concentrations of methyl donor (0.003 mM), the monomethylated products accumulate.

The effect of hyperbaric oxygen on infarct size in the conscious animal.

The effect of hyperbaric oxygen (HBO) on infarct size associated with myocardial infarction remains uncertain. Accordingly, the present study was performed in 46 conscious dogs with experimental infarction to determine the effect of HBO on enzymatic estimates of infarct size. Since HBO may affect plasma creatine kinase (CK) release or disappearance, parameters used to calculate enzymatic estimates of infarct size from plasma CK, we assessed infarct size by directly measuring myocardial CK depletion. Twenty-three animals were given HBO (2 atm of pressure) for 3 h immediately after coronary occlusion and results of infarct size compared to those in 23 dogs with occlusion who remained in room air. In 10 other animals CK release was measured after coronary occlusion in 5 controls and compared to 5 treated. In 5 normal animals the CK disappearance rate of purified canine CK was determined before and after HBO. Infarct size was determined 24 h after coronary occlusion and in the treated animals averaged 25.4 +/- 1.3% of LV (mean +/- SEM), and being similar to controls (26.7 +/- 1.4, P greater than 0.25). The plasma CK disappearance rate before and after HBO was the same being 0.0072 +/- 0.0022 (min-1) and 0.0073 +/- 0.0021, respectively. Total CK released into the plasma was also the same in treated and controls (2232 +/- 210 IU and 2011 +/- 232), as was the ratio of CK released to that depleted from the myocardium (0.15 +/- 2% vs 0.15 +/- 3%). Our results indicate: (1) HBO does not reduce infarct size produced experimentally in the conscious dog; (2) HBO does not affect CK release or disappearance; and (3) estimates of infarct size by plasma CK remain valid despite administration of HBO.

Characterization of rabbit myocardial phospholipids with 31P nuclear magnetic resonance.

To reduce chemical modifications of phospholipids during extraction and analysis, we developed and evaluated a method for analyzing tissue phospholipids with 31P NMR. Our results indicate: (1) 31P NMR of chloroform/methanol tissue extracts is a convenient and rapid technique for characterizing myocardial phospholipids. (2) Normal rabbit myocardium contains little, if any, lysoglycerophospholipid. (3) Conventional extraction and chromatography of phospholipids may convert choline and ethanolamine plasmalogens to lysoglycerophospholipids by acid-catalyzed hydrolysis.