title: Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose.
abstract: The membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5) from Escherichia coli has been solubilized wiTriton X-100 and purified 6000-fold to 85% of homogeneity. The major purification was attained using several modifications of the the CDP-diglyceride Sepharose affinity chromatography system described by Larson et al. (Larson, T.J., Hirabayashi, T., and Dowhan, W. (1976), Biochemistry 15, 974). The native enzyme in Triton X-100 had an apparent molecular weight of over 200 000, as judged by Sepharose 6B gel filtration. The apparent size of the native enzyme appeared to be due to its association with Triton X-100, as judged by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and the lack of affinity for ion-exchange resins. The minimum subunit molecular weight of the enzyme, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 24 000. This low molecular weight is consistent with the stability of enzyme to heat, urea, or sodium dodecyl sulfate denaturation. The purified enzyme had an absolute requirement for magnesium ion (KM = 50 mM) and Triton X-100 (0.5-6%) for activity when either CDP-diglyceride or dCDP-diglyceride was used as substrate. Kinetic analysis of the enzymatic reaction indicated an ordered sequential Bi-Bi reaction with the liponucleotide forming a dead-end complex at high concentration, which inhibited both the forward and reverse reactions. The enzyme would not hydrolyze the pyrophosphate bond of its lipid substrate or the phosphate esters of its lipid product but would catalyze a cytidine 5'-monophosphate dependent exchange reaction between glycero-3-phosphate and phosphatidylglycerophosphate.
mesh_terms: D002846:Chromatography, Affinity; D003567:Cytidine Diphosphate Diglycerides; D004926:Escherichia coli / Q000201:enzymology*; D005994:Glycerophosphates; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000201:enzymology; D008970:Molecular Weight; D010715:Phosphatidylglycerols / Q000096:biosynthesis*; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols; D017855:Transferases (Other Substituted Phosphate Groups)
chemical_list: D003567:Cytidine Diphosphate Diglycerides; D005994:Glycerophosphates; D010715:Phosphatidylglycerols; D011092:Polyethylene Glycols; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; D008274:Magnesium