Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli.
Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.
D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; D000217:Acyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling; D008246:Lysophospholipids; D008565:Membrane Proteins / Q000378:metabolism; D010712:Phosphatidic Acids / Q000096:biosynthesis / Q000378:metabolism*; D012995:Solubility; D014316:Tritium
D002250:Carbon Radioisotopes; D029968:Escherichia coli Proteins; D008246:Lysophospholipids; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D014316:Tritium; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C495091:plsC protein, E coli