Functional heterogeneity of polyphosphoinositides in human erythrocytes.
After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of labelled PtdIns(4,5)P2 molecules accessible to PIC after a prelabelling period of 5 min and different times of reincubation. Hydrolysis by PIC was also measured in erythrocytes in which the phosphoinositide content had been modified by activation (Mg2+-enriched cells) or inhibition (ATP-depleted cells) of the phosphoinositide kinases. The sizes of the PIC-resistant pools of polyphosphoinositides were not affected by these treatments, indicating that the kinases (and the phosphatases) act on the PIC-sensitive pools. This was also shown by the decrease in the production of Ins(1,4,5)P3 upon PIC activation in ATP-depleted erythrocytes. A model is presented in which the PIC-sensitive pools of polyphosphoinositides are those which are accessible to the kinases and the phosphatases and are rapidly turned over.
Gascard
P
P
URA CNRS D.0646, Université Paris XI, Orsay, France.
Journet
E
E
Sulpice
J C
JC
Giraud
F
F
D000255:Adenosine Triphosphate / Q000097:blood; D002118:Calcium / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008563:Membrane Lipids / Q000097:blood* / Q000302:isolation & purification; D008954:Models, Biological; D010710:Phosphates / Q000097:blood; D010716:Phosphatidylinositols / Q000097:blood* / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes
D008563:Membrane Lipids; D010710:Phosphates; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium