Polyphosphoinositide synthesis in rabbit erythrocyte membranes

Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm γ-³²P]ATP and MgCl₂ results in incorporation of ³²P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol ³²P/mg/h, respectively. Incorporation of ³²P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with ³²P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of ³²P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl₂ and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.