title: Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli.
abstract: Membrane-associated enzymes are often solubilized with detergents, purified, and then reconstituted with phospholipid cofactors to regain function. Insofar as most purification and reconstitution procedures are not quantitative, the final reconstituted preparations could reflect a population of molecules ranging from fully functional to completely inactive. Quantitative studies on the efficiency of reconstitution of the Triton X-100-solubilized sn-glycerol 3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane were undertaken at each step of purification. Physical recovery of the 83,000 Mr polypeptide was quantitated in polyacrylamide gels using membranes from cells labeled with [3H]leucine. The 83,000 Mr polypeptide in such gels was demonstrated to consist exclusively of the glycerol-P acyltransferase peptide by V8 peptide mapping. Comparison between physical recovery of 83,000 Mr polypeptide and reconstituted activity allowed the efficiency of reconstitution to be determined. Unexpectedly, disproportionalities occurred during the purification. However, the final purification of reconstituted enzyme activity matched that of the 83,000 Mr polypeptide. This method also allowed measurement of the specific activities of the glycerol-P acyltransferase in membranes from a wild type E. coli strain and from plasmid-containing strains which express the plsB gene product to different extents. The physical amounts of the 83,000 Mr polypeptide and glycerol-P acyltransferase activity measured in membranes were not strictly proportional. In strains where the amount of 83,000 Mr polypeptide was enhanced, a larger proportion of latent activity was observed following solubilization and reconstitution. The results establish the suitability of the reconstituted preparations of glycerol-P acyltransferase for detailed kinetic analysis and permit inferences pertaining to regulation.
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012697:Serine Endopeptidases
chemical_list: D010446:Peptide Fragments; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase