Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1971 Sep 1;50(3):625–633. doi: 10.1083/jcb.50.3.625

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS

I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

Francis J Chlapowski 1, R Neal Band 1
PMCID: PMC2108296  PMID: 5098863

Abstract

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-14C and glycerol-3H were examined. Choline-14C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-14C in the reaction mixture and the choline-14C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-14C, the apparent turnover of glycerol-3H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-3H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-3H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (∼96%) of the glycerol-3H recovered from microsomal membranes was in phospholipids, whereas only a minor component (∼2%) of the glycerol-3H was in the phospholipids isolated from nonmembrane lipids, glycerol-3H was judged to be a specific marker for membrane phospholipids.

Full Text

The Full Text of this article is available as a PDF (593.9 KB).

Selected References

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

  1. BAND R. N. Nutritional and related biological studies on the free-living soil amoeba, Hartmannella rhysodes. J Gen Microbiol. 1959 Aug;21:80–95. doi: 10.1099/00221287-21-1-80. [DOI] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Bjørnstad P., Bremer J. In vivo studies on pathways for the biosynthesis of lecithin in the rat. J Lipid Res. 1966 Jan;7(1):38–45. [PubMed] [Google Scholar]
  4. DILS R. R., HUBSCHER G. Metabolism of phospholipids. III. The effect of calcium ions on the incorporation of labelled choline into rat-liver microsomes. Biochim Biophys Acta. 1961 Jan 29;46:505–513. doi: 10.1016/0006-3002(61)90581-9. [DOI] [PubMed] [Google Scholar]
  5. Dallner G., Siekevitz P., Palade G. E. Biogenesis of endoplasmic reticulum membranes. I. Structural and chemical differentiation in developing rat hepatocyte. J Cell Biol. 1966 Jul;30(1):73–96. doi: 10.1083/jcb.30.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. HOKIN L. E., HOKIN M. R. Phosphoinositides and protein secretion in pancreas slices. J Biol Chem. 1958 Oct;233(4):805–810. [PubMed] [Google Scholar]
  7. HUBSCHER G., DILS R. R., POVER W. F. Studies on the biosynthesis of phosphatidyl serine. Biochim Biophys Acta. 1959 Dec;36:518–528. doi: 10.1016/0006-3002(59)90194-5. [DOI] [PubMed] [Google Scholar]
  8. HUBSCHER G. Metabolism of phospholipids. VI. The effect of metal ions on the incorporation of L-serine into phosphatidylserine. Biochim Biophys Acta. 1962 Mar 12;57:555–561. doi: 10.1016/0006-3002(62)91163-0. [DOI] [PubMed] [Google Scholar]
  9. Nagley P., Hallinan T. The use of radioactive choline as a label for microsomal membranes. I. Selectivity of label for endoplasmic reticulum and specificity for lecithin. Biochim Biophys Acta. 1968 Sep 17;163(2):218–225. doi: 10.1016/0005-2736(68)90100-4. [DOI] [PubMed] [Google Scholar]
  10. Omura T., Siekevitz P., Palade G. E. Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J Biol Chem. 1967 May 25;242(10):2389–2396. [PubMed] [Google Scholar]
  11. PAULUS H., KENNEDY E. P. The enzymatic synthesis of inositol monophosphatide. J Biol Chem. 1960 May;235:1303–1311. [PubMed] [Google Scholar]
  12. Page F. C. Re-definition of the genus Acanthamoeba with descriptions of three species. J Protozool. 1967 Nov;14(4):709–724. doi: 10.1111/j.1550-7408.1967.tb02066.x. [DOI] [PubMed] [Google Scholar]
  13. Plagemann P. G. Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. I. Incorporation of choline into phosphatidylcholine of mitochondria and other membranous structures and effect of metabolic inhibitors. Arch Biochem Biophys. 1968 Oct;128(1):70–87. doi: 10.1016/0003-9861(68)90009-x. [DOI] [PubMed] [Google Scholar]
  14. Plagemann P. G. Choline metabolism and membrane formation in rat hepatoma cells grown in suspension culture. II. Phosphatidylcholine synthesis during growth cycle and fluctuation of mitochondrial density. J Cell Biol. 1969 Sep;42(3):766–781. doi: 10.1083/jcb.42.3.766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Plagemann P. G., Roth M. F. Permeation as the rate-limiting step in the phosphorylation of uridine and choline and their incorporation into macromolecules by Novikoff hepatoma cells. Competitive inhibition by phenethyl alcohol, persantin, and adenosine. Biochemistry. 1969 Dec;8(12):4782–4789. doi: 10.1021/bi00840a020. [DOI] [PubMed] [Google Scholar]
  16. Rytter D., Miller J. E., Cornatzer W. E. Specificity for incorporation of choline and ethanolamine into rat-liver microsomal lecithins. Biochim Biophys Acta. 1968 Mar 4;152(2):418–421. doi: 10.1016/0005-2760(68)90054-4. [DOI] [PubMed] [Google Scholar]
  17. Stein O., Stein Y. Lecithin synthesis, intracellular transport, and secretion in rat liver. IV. A radioautographic and biochemical study of choline-deficient rats injected with choline-3H. J Cell Biol. 1969 Feb;40(2):461–483. doi: 10.1083/jcb.40.2.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Treble D. H., Frumkin S., Balint J. A., Beeler D. A. The entry of choline into lecithin, in vivo, by base exchange. Biochim Biophys Acta. 1970 Feb 10;202(1):163–171. doi: 10.1016/0005-2760(70)90227-4. [DOI] [PubMed] [Google Scholar]
  19. Ulsamer A. G., Smith F. R., Korn E. D. Lipids of Acanthamoeba castellanii. Composition and effects of phagocytosis on incorporation of radioactive precursors. J Cell Biol. 1969 Oct;43(1):105–114. doi: 10.1083/jcb.43.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Vandor S. L., Richardson K. E. Incorporation of ethanolamine-1,2-14C into plant microsomal phospholipids. Can J Biochem. 1968 Oct;46(10):1309–1315. doi: 10.1139/o68-196. [DOI] [PubMed] [Google Scholar]
  21. Wetzel M. G., Korn E. D. Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes. J Cell Biol. 1969 Oct;43(1):90–104. doi: 10.1083/jcb.43.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. ZILVERSMIT D. B., DILUZIO N. R. The role of choline in the turnover of phospholipids. Am J Clin Nutr. 1958 May-Jun;6(3):235–241. doi: 10.1093/ajcn/6.3.235. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES