The polyphosphoinositide phosphodiesterase of erythrocyte membranes

A new assay procedure has been devised for measurement of the Ca²⁺-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [³²P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released ³²P-labeled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5 mM-Ca²⁺. For both esters, optimum rates of release were seen at pH 6.8-6.9. Mg²⁺ did not provoke release of either ester. Ca²⁺ provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca²⁺. In the presence of Mg²⁺, the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca²⁺ and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bis-phosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg²⁺-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.