Role of phosphatidylinositol in attachment of alkaline phosphatase to membranes.

The mechanism of release of alkaline phosphatase from membranes by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus was studied. Alkaline phosphatase was readily released from pig kidney microsomes by phospholipase C but not by a variety of other treatments, e.g., high ionic strength, extremes of pH, divalent cations, chelating agents, or analogues of the polar head group of phosphatidylinositol. Alkaline phosphatase released from microsomes by phospholipase C did not bind to phospholipid vesicles containing phosphatidylinositol. Alkaline phosphatase solubilized from microsomes by butanol extraction, however, was able to bind phospholipid vesicles even when they contained no phosphatidylinositol. The ability of butanol-extracted alkaline phosphatase to bind to phospholipid vesicles was destroyed by added phosphatidylinositol-specific phospholipase C. Hydrolysis of added phosphatidylinositol by endogenous phospholipase activity in butanol extracts was also accompanied by loss of binding ability. Loss of binding ability was paralleled by a decrease in the apparent molecular weight of alkaline phosphatase. These results indicate that alkaline phosphatase is attached to membranes by a strong interaction with phosphatidylinositol.