title: Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.
abstract: We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.
authors.affiliation: Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: H
authors.initials: H
authors.lastname: Möhn
authors.forename: V
authors.initials: V
authors.lastname: Chalifa
authors.forename: M
authors.initials: M
authors.lastname: Liscovitch
mesh_terms: D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism; D003065:Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013569:Synapses / Q000378:metabolism*
chemical_list: D008563:Membrane Lipids; D010743:Phospholipids; D000255:Adenosine Triphosphate; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D010739:Phospholipase D; D003066:Coenzyme A Ligases; D003065:Coenzyme A