Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1978 Oct;136(1):85–95. doi: 10.1128/jb.136.1.85-95.1978

Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli.

K E Langley, E P Kennedy
PMCID: PMC218635  PMID: 30751

Abstract

The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli (C. R. H. Raetz and E. P. Kennedy, J. Biol. Chem. 248:1098--1105, 1973). The enzyme responsible for their synthesis, CTP:phosphatidic acid cytidylytransferase, was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin and was purified about 70-fold (relative to cell-free extracts) in the presence of detergent. In studies of the heat stability of the enzyme, activity decayed slowly at 63 degrees C. Initial velocity kinetic experiments suggested a sequential, rather than ping-pong, reaction mechanism; isotopic exchange reaction studies supported this conclusion and indicated that inorganic pyrophosphate is released before CDP-diglyceride in the reaction sequence. The enzyme utilized both CTP and dCTP as nucleotide substrate for the synthesis of CDP-diglyceride and dCDP-diglyceride, respectively. No distinction was observed between CTP and dCTP utilization in any of the purification, heat stability, and reaction mechanism studies. In addition, CTP and dCTP were competitive substrates for the partially purified enzyme. It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride, i.e., the reverse of its physiologically important catalysis.

Full text

PDF
95

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AGRANOFF B. W., BRADLEY R. M., BRADY R. O. The enzymatic synthesis of inositol phosphatide. J Biol Chem. 1958 Nov;233(5):1077–1083. [PubMed] [Google Scholar]
  2. BAER E. Synthesis of enantiomeric alpha-phosphatidic acids. J Biol Chem. 1951 Mar;189(1):235–247. [PubMed] [Google Scholar]
  3. Bell R. M., Mavis R. D., Osborn M. J., Vagelos P. R. Enzymes of phospholipid metabolism: localization in the cytoplasmic and outer membrane of the cell envelope of Escherichia coli and Salmonella typhimurium. Biochim Biophys Acta. 1971 Dec 3;249(2):628–635. doi: 10.1016/0005-2736(71)90144-1. [DOI] [PubMed] [Google Scholar]
  4. COHEN G. N., RICKENBERG H. V. Concentration spécifique réversible des amino acides chez Escherichia coli. Ann Inst Pasteur (Paris) 1956 Nov;91(5):693–720. [PubMed] [Google Scholar]
  5. Carter J. R., Jr Cytidine triphosphate: phosphatidic acid cytidyltransferase in Escherichia coli. J Lipid Res. 1968 Nov;9(6):748–754. [PubMed] [Google Scholar]
  6. Carter J. R., Kennedy E. P. Enzymatic synthesis of cytidine diphosphate diglyceride. J Lipid Res. 1966 Sep;7(5):678–683. [PubMed] [Google Scholar]
  7. Chang Y. Y., Kennedy E. P. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli. J Lipid Res. 1967 Sep;8(5):447–455. [PubMed] [Google Scholar]
  8. DITTMER J. C., LESTER R. L. A SIMPLE, SPECIFIC SPRAY FOR THE DETECTION OF PHOSPHOLIPIDS ON THIN-LAYER CHROMATOGRAMS. J Lipid Res. 1964 Jan;5:126–127. [PubMed] [Google Scholar]
  9. Dowhan W., Wickner W. T., Kennedy E. P. Purification and properties of phosphatidylserine decarboxylase from Escherichia coli. J Biol Chem. 1974 May 25;249(10):3079–3084. [PubMed] [Google Scholar]
  10. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  11. Hirabayashi T., Larson T. J., Dowhan W. Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose. Biochemistry. 1976 Nov 30;15(24):5205–5211. doi: 10.1021/bi00669a002. [DOI] [PubMed] [Google Scholar]
  12. Hutchison H. T., Cronan J. E., Jr The synthesis of cytidine diphosphate diglyceride by cell-free extracts of yeast. Biochim Biophys Acta. 1968 Dec 18;164(3):606–608. doi: 10.1016/0005-2760(68)90193-8. [DOI] [PubMed] [Google Scholar]
  13. Koes M. T., Stasiw R. O., Forrester L. J., Chattopadhyay S. K., Bartling G. J., Cowan D., Brown H. D. Characterization of solubilized cytochrome P-450. Int J Pept Protein Res. 1973;5(5):345–351. doi: 10.1111/j.1399-3011.1973.tb02338.x. [DOI] [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. Larson T. J., Dowhan W. Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol. Biochemistry. 1976 Nov 30;15(24):5212–5218. doi: 10.1021/bi00669a003. [DOI] [PubMed] [Google Scholar]
  16. McCaman R. E., Finnerty W. R. Biosynthesis of cytidine diphosphate-diglyceride by a particulate fracgion from Micrococcus cerificans. J Biol Chem. 1968 Oct 10;243(19):5074–5080. [PubMed] [Google Scholar]
  17. Moore R. J., Wilson J. D. Extraction of the reduced nicotinamide adenine dinucleotide phosphate:delta 4-3-ketosteroid-5-alpha oxidoreductase of rat prostate with digitonin and potassium chloride. Biochemistry. 1974 Jan 29;13(3):450–456. doi: 10.1021/bi00700a009. [DOI] [PubMed] [Google Scholar]
  18. Nunn W. D., Cronan J. E., Jr Regulation of membrane phospholipid synthesis by the relA gene: dependence on ppGpp levels. Biochemistry. 1976 Jun 15;15(12):2546–2550. doi: 10.1021/bi00657a009. [DOI] [PubMed] [Google Scholar]
  19. PATTERSON M. S., GREENE R. C. MEASUREMENT OF LOW ENERGY BETA-EMITTERS IN AQUEOUS SOLUTION BY LIQUID SCINTILLATION COUNTING OF EMULSIONS. Anal Chem. 1965 Jun;37:854–857. doi: 10.1021/ac60226a017. [DOI] [PubMed] [Google Scholar]
  20. Patterson P. H., Lennarz W. J. Studies on the membranes of bacilli. I. Phospholipid biosynthesis. J Biol Chem. 1971 Feb 25;246(4):1062–1072. [PubMed] [Google Scholar]
  21. Petzold G. L., Agranoff B. W. The biosynthesis of cytidine diphosphate diglyceride by embryonic chick brain. J Biol Chem. 1967 Mar 25;242(6):1187–1191. [PubMed] [Google Scholar]
  22. Raetz C. R., Dowhan W., Kennedy E. P. Partial purification and characterization of cytidine 5'-diphosphate-diglyceride hydrolase from membranes of Escherichia coli. J Bacteriol. 1976 Mar;125(3):855–863. doi: 10.1128/jb.125.3.855-863.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Raetz C. R., Kennedy E. P. Function of cytidine diphosphate-diglyceride and deoxycytidine diphosphate-diglyceride in the biogenesis of membrane lipids in Escherichia coli. J Biol Chem. 1973 Feb 10;248(3):1098–1105. [PubMed] [Google Scholar]
  24. Snider M. D., Kennedy E. P. Partial purification of glycerophosphate acyltransferase from Escherichia coli. J Bacteriol. 1977 Jun;130(3):1072–1083. doi: 10.1128/jb.130.3.1072-1083.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sokawa Y., Nakao E., Kaziro Y. On the nature of the control by RC gene in e. coli: amino acid-dependent control of lipid synthesis. Biochem Biophys Res Commun. 1968 Oct 10;33(1):108–112. doi: 10.1016/0006-291x(68)90263-5. [DOI] [PubMed] [Google Scholar]
  26. Wang C., Smith R. L. Lowry determination of protein in the presence of Triton X-100. Anal Biochem. 1975 Feb;63(2):414–417. doi: 10.1016/0003-2697(75)90363-2. [DOI] [PubMed] [Google Scholar]
  27. White D. A., Albright F. R., Lennarz W. J., Schnaitman C. A. Distribution of phospholipid-synthesizing enzymes in the wall and membrane subfractions of the envelope of Escherichia coli. Biochim Biophys Acta. 1971 Dec 3;249(2):636–642. doi: 10.1016/0005-2736(71)90145-3. [DOI] [PubMed] [Google Scholar]
  28. van den Bosch H., van Golde L. M., van Deenen L. L. Dynamics of phosphoglycerides. Ergeb Physiol. 1972;66:13–145. doi: 10.1007/3-540-05882-6_2. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES