Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Apr 15;291(Pt 2):553–560. doi: 10.1042/bj2910553

The uptake of 3H-labelled monodeoxyfluoro-myo-inositols into thymocytes and their incorporation into phospholipid in permeabilized cells.

J Offer 1, J C Metcalfe 1, G A Smith 1
PMCID: PMC1132560  PMID: 8387272

Abstract

Monodeoxyfluoro-myo-inositols were applied to electropermeabilized and intact thymocyte preparations to study their metabolism and uptake in order to investigate their suitability as potential inhibitors of phosphoinositide-mediated cellular responses. Only three of the monodeoxyfluoro-myo-inositols were incorporated into the phospholipids of thymocytes: 1D-3-deoxy-3-fluoro-myo-inositol, 5-deoxy-5-fluoro-myo-inositol and 1D-6-deoxy-6-fluoro-myo-inositol, all of which were weaker substrates for phosphatidylinositol synthase than was myo-inositol. The 3-, 5- and 6-fluoro analogues also behaved as competitive inhibitors, with K1 values of 350 +/- 5 microM, 350 +/- 5 microM and 2.9 +/- 2 mM respectively, compared with a Km for myo-inositol of 31 +/- 4 microM. When incubated with electropermeabilized thymocyte preparations, these three analogues of myo-inositol all formed phospholipids with chromatographic properties which corresponded to those of substituted phosphatidylinositol and phosphatidylinositol monophosphate. The uptake of myo-inositol and of the monodeoxyfluoro-myo-inositols into intact thymocytes was studied by a dual-label technique. All the monodeoxyfluoro-myo-inositols were taken up to some extent, but only 2-deoxy-2-fluoro-myo-inositol and 1D-3-deoxy-3-fluoro-myo-inositol were actively concentrated. The monodeoxyfluoro-myo-inositols were also assayed for their ability to inhibit the uptake of myo-inositol into cells. Both 2-deoxy-2-fluoro-myo-inositol and 1D-3-deoxy-3-fluoro-myo-inositol were effective inhibitors of myo-inositol uptake. Furthermore, 1D-1-deoxy-1-fluoro-myo-inositol, which was not taken up actively, was an effective inhibitor of myo-inositol uptake. The three effective inhibitors all showed Ki values of approximately 150 microM, close to the apparent Km for inositol uptake of 180 microM, and the 4-, 5- and 6-fluoro analogues had Ki values in excess of 10 mM.

Full text

PDF
553

Selected References

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

  1. Alcover A., Alberini C., Acuto O., Clayton L. K., Transy C., Spagnoli G. C., Moingeon P., Lopez P., Reinherz E. L. Interdependence of CD3-Ti and CD2 activation pathways in human T lymphocytes. EMBO J. 1988 Jul;7(7):1973–1977. doi: 10.1002/j.1460-2075.1988.tb03035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Auchus R. J., Wilson D. B., Covey D. F., Majerus P. W. The 5-hydroxyl of myo-inositol is essential for uptake into HSDM1C1 mouse fibrosarcoma cells. Biochem Biophys Res Commun. 1985 Aug 15;130(3):1139–1146. doi: 10.1016/0006-291x(85)91734-6. [DOI] [PubMed] [Google Scholar]
  3. Authi K. S., Hughes K., Crawford N. High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation. FEBS Lett. 1989 Aug 28;254(1-2):52–58. doi: 10.1016/0014-5793(89)81008-7. [DOI] [PubMed] [Google Scholar]
  4. Benjamins J. A., Agranoff B. W. Distribution and properties of CDP-diglyceride:inositol transferase from brain. J Neurochem. 1969 Apr;16(4):513–527. doi: 10.1111/j.1471-4159.1969.tb06850.x. [DOI] [PubMed] [Google Scholar]
  5. Brand M. D., Felber S. M. The intracellular calcium antagonist TMB-8 [8-(NN-diethylamino)octyl-3,4,5-trimethoxybenzoate] inhibits mitochondrial ATP production in rat thymocytes. Biochem J. 1984 Dec 15;224(3):1027–1030. doi: 10.1042/bj2241027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Desai D. M., Newton M. E., Kadlecek T., Weiss A. Stimulation of the phosphatidylinositol pathway can induce T-cell activation. Nature. 1990 Nov 1;348(6296):66–69. doi: 10.1038/348066a0. [DOI] [PubMed] [Google Scholar]
  7. Fischl A. S., Homann M. J., Poole M. A., Carman G. M. Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity. J Biol Chem. 1986 Mar 5;261(7):3178–3183. [PubMed] [Google Scholar]
  8. Ghalayini A., Eichberg J. Purification of phosphatidylinositol synthetase from rat brain by CDP-diacylglycerol affinity chromatography and properties of the purified enzyme. J Neurochem. 1985 Jan;44(1):175–182. doi: 10.1111/j.1471-4159.1985.tb07128.x. [DOI] [PubMed] [Google Scholar]
  9. Levis G. M., Evangelatos G. P., Crumpton M. J. Lipid composition of lymphocyte plasma membrane from pig mesenteric lymph node. Biochem J. 1976 Apr 15;156(1):103–110. doi: 10.1042/bj1560103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McPhee F., Downes C. P., Lowe G. Studies of inositol analogues as inhibitors of the phosphoinositide pathway, and incorporation of 2-deoxy-2-fluoro-myo-inositol to give analogues of phosphatidylinositol intermediates. Biochem J. 1991 Jul 15;277(Pt 2):407–412. doi: 10.1042/bj2770407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McPhee F., Lowe G., Vaziri C., Downes C. P. Phosphatidylinositol synthase and phosphatidylinositol/inositol exchange reactions in turkey erythrocyte membranes. Biochem J. 1991 Apr 1;275(Pt 1):187–192. doi: 10.1042/bj2750187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moore J. P., Smith G. A., Hesketh T. R., Metcalfe J. C. The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes. Biochem J. 1983 Jun 15;212(3):691–697. doi: 10.1042/bj2120691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moyer J. D., Malinowski N., Napier E. A., Strong J. Uptake and metabolism of myo-inositol by L1210 leukaemia cells. Biochem J. 1988 Aug 15;254(1):95–100. doi: 10.1042/bj2540095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moyer J. D., Reizes O., Ahir S., Jiang C., Malinowski N., Baker D. C. Substrate properties of analogs of myo-inositol. Mol Pharmacol. 1988 Jun;33(6):683–689. [PubMed] [Google Scholar]
  15. PAULUS H., KENNEDY E. P. The enzymatic synthesis of inositol monophosphatide. J Biol Chem. 1960 May;235:1303–1311. [PubMed] [Google Scholar]
  16. Stanley P. E., Williams S. G. Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem. 1969 Jun;29(3):381–392. doi: 10.1016/0003-2697(69)90323-6. [DOI] [PubMed] [Google Scholar]
  17. Sussman J. J., Bonifacino J. S., Lippincott-Schwartz J., Weissman A. M., Saito T., Klausner R. D., Ashwell J. D. Failure to synthesize the T cell CD3-zeta chain: structure and function of a partial T cell receptor complex. Cell. 1988 Jan 15;52(1):85–95. doi: 10.1016/0092-8674(88)90533-8. [DOI] [PubMed] [Google Scholar]
  18. Takenawa T., Egawa K. Phosphatidyl inositol: myo-inositol exchange enzyme from rat liver: partial purification and characterization. Arch Biochem Biophys. 1980 Jul;202(2):601–607. doi: 10.1016/0003-9861(80)90467-1. [DOI] [PubMed] [Google Scholar]
  19. Taylor M. V., Metcalfe J. C., Hesketh T. R., Smith G. A., Moore J. P. Mitogens increase phosphorylation of phosphoinositides in thymocytes. 1984 Nov 29-Dec 5Nature. 312(5993):462–465. doi: 10.1038/312462a0. [DOI] [PubMed] [Google Scholar]
  20. Van Haastert P. J., De Vries M. J., Penning L. C., Roovers E., Van der Kaay J., Erneux C., Van Lookeren Campagne M. M. Chemoattractant and guanosine 5'-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation. Biochem J. 1989 Mar 1;258(2):577–586. doi: 10.1042/bj2580577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Whitman M., Cantley L. Phosphoinositide metabolism and the control of cell proliferation. Biochim Biophys Acta. 1989 Feb;948(3):327–344. doi: 10.1016/0304-419x(89)90005-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES