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.
The incorporation of [3H]Ins into PtdIns by exchange of free and lipid-bound inositol moieties occurs via the action of at least two types of Mg2+/Mn(2+)-dependent enzymes in turkey erythrocytes. One is a nucleotide-independent PtdIns/Ins exchange enzyme and its function is, as yet, unknown, whereas the other is CMP-dependent and appears to be an exchange reaction catalysed by PtdIns synthase. The effects of analogues with modifications of the substituent at the 1-, 2-, 3-, 4- and 5-positions on the incorporation of [3H]Ins into PtdIns under both synthase and exchange reaction conditions were investigated in turkey erythrocytes. Analogues causing substantial inhibition of [3H]Ins incorporation were then used in kinetic experiments to determine the type of inhibition involved. The analogues 1-deoxy-1-fluoro-scyllo-inositol and 5-O-methyl-myo-inositol exhibited the greatest effects on the incorporation of [3H]Ins via both the synthase and exchange reactions, and the kinetic analysis indicated that they were competitive inhibitors of Ins. Ki values of 0.37 mM and 2.87 mM were observed for 1-deoxy-1-fluoro-scyllo-inositol under exchange and synthase reaction conditions respectively; similar Ki values of 0.26 mM and 2.80 mM were observed for 5-O-methyl-myo-inositol in the exchange and synthase reactions respectively. The ability of 1-deoxy-1-fluoro-scyllo-inositol and its diastereoisomer, 2-deoxy-2-fluoro-myo-inositol, to act as substrates for the synthase and exchange reactions in turkey erythrocytes was investigated. The radiolabelled derivative of the former analogue was not incorporated into phospholipids, whereas the radiolabelled derivative of the latter analogue was a poor substrate for the synthase and exchange enzymes. In the presence of ATP, the labelled analogue of PtdIns, derived from 2-deoxy-2-fluoro-myo-[2-3H]inositol, appeared to be converted into phosphorylated PtdIns analogues, presumably by the enzymes of the phosphoinositide pathway.
McPhee
F
F
Dyson Perrins Laboratory, Oxford University, U.K.
Downes
C P
CP
Lowe
G
G
D000818:Animals; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000378:metabolism*; D007294:Inositol / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000096:biosynthesis* / Q000097:blood*; D010743:Phospholipids / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D014422:Turkeys
D010716:Phosphatidylinositols; D010743:Phospholipids; C070062:2-deoxy-2-fluoroinositol; D007294:Inositol