Abstract
Plasmenylethanolamines represent the major endogenous phospholipid storage depot of arachidonic acid in many mammalian cells. To elucidate the biochemical mechanisms contributing to the high plasmalogen content and arachidonic acid enrichment present in myocardial ethanolamine glycerophospholipids, the substrate specificity of rabbit myocardial ethanolamine phosphotransferase (EPT) was quantified utilizing multiple molecular species of each subclass of diradyl glycerol substrate. Myocardial EPT demonstrated over a 16-fold selectivity for 1-O-alk-1'-enyl-2-acyl-sn-glycerol (AAG) compared to 1,2-diacyl-sn-glycerol (DAG) substrate utilizing individual molecular species of each subclass dispersed in Tween 20. The selective utilization of AAG by EPT was substantiated utilizing two independent assay systems which employed either the presentation of substrate to enzyme as a substitutional impurity in Triton X-100 mixed micelles or the obligatory utilization of endogenously generated diradyl glycerol substrates. Although rabbit myocardial microsomes contained over a 20-fold molar excess of endogenous DAG to AAG mass, incubation of rabbit myocardial microsomes with CDP-ethanolamine resulted in the highly selective synthesis of plasmenylethanolamines which were predominantly comprised of molecular species containing arachidonic acid at the sn-2 position (greater than 75%). Endogenous AAG molecular species in rabbit myocardial microsomes were similarly enriched in arachidonic acid, and the distribution of AAG molecular species closely paralleled the distribution of plasmenylethanolamine (but not plasmenylcholine) molecular species. Thus, the subclass and molecular species distribution of the ethanolamine glycerophospholipids synthesized by rabbit myocardial EPT reflects independent contributions from the subclass selectivity of EPT for AAG substrate in conjunction with the enrichment of arachidonic acid in microsomal AAG molecular species.
Highlights
The Primary Determinant of Rabbit Myocardial Ethanolamine Phosphotransferase SubstrateSelectivity Is the Covalent Nature of the sn-1 Aliphatic Groupof Diradyl Glycerol Acceptors*
Microsomes contained over a 20-fold molar excess of The only documented biosynthetic pathway for the introendogenous DAG to AAG mass, incubation of rabbit duction of the vinyl ether bond into cellular lipids is mediated myocardial microsomes with CDP-ethanolamine re- by the action of a NADH-dependent alkyl ether sulted in the highly selective synthesis of plasmenyl- desaturase whichspecificallyoxidizes 1-0-alkyl-2-acyl-snethanolamines which were predominantly comprised of molecular species containing arachidonic aciadt the sn-2 position (>75%).Endogenous AAG molecular species in rabbit myocardial microsomes were enriched in arachidonic acid, and the distribution of glycero-3-phosphoethanolaminesubstrate resulting in the generation of plasmenylethanolamine [12, 13]
Incubation of rabbit myocardial microsomes with DAG containing oleic or arachidonic acid at the sn-2 position did not result in substantial amounts of phosphatidylethanolamine synthesis (V, I 0.1 nmol/mghmin)
Summary
Biosynthesis purchased from Boehringer Mannheim and Pierce Chemical Co., respectively. Oleoyl chloride and arachidonoyl chloride were purchased from Nu Chek Prep. Magnesium chloride was greater than 99.99% pure and was obtained from Aldrich. Diazomethane was prepared from iV-methyl-N-nitroso-p-toluene (Aldrich) according to the- instructions supplied by the manufacturer. Most other unspecified reagent grade chemicals including 1-O-hexadecyl-2-octadec-9’-enoyl-GPC were purchased from Sigma.
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