Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis.
A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP-diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity.
D001407:Bacillus / Q000201:enzymology*; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002712:Chlorides; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D008970:Molecular Weight; D017830:Octoxynol; D010770:Phosphotransferases / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000494:pharmacology
D002712:Chlorides; D017895:Manganese Compounds; D011092:Polyethylene Glycols; D008345:Manganese; D017830:Octoxynol; D010770:Phosphotransferases; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase; C025340:manganese chloride