ABSTRACT
Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipase. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5'nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was approximately 6.5. Over 80% of 5'-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5'-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5'-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.
