The uptake of 3H-labelled monodeoxyfluoro-myo-inositols into thymocytes and their incorporation into phospholipid in permeabilized cells.
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.
Department of Biochemistry, University of Cambridge, U.K.
J
J
Offer
J C
JC
Metcalfe
G A
GA
Smith
D000818:Animals; D002463:Cell Membrane Permeability; D007294:Inositol / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D010716:Phosphatidylinositols / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism*; D014316:Tritium
C081001:6-deoxy-6-fluoroinositol; D010716:Phosphatidylinositols; D010743:Phospholipids; D014316:Tritium; C081000:5-deoxy-5-fluoroinositol; C080999:3-deoxy-3-fluoroinositol; D007294:Inositol