56] Glycosylphosphatidylinositol-specific phospholipase D

Abstract

The glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was first observed as a result of its ability to degrade the GPI anchor of alkaline phosphatase during extraction from mammalian tissues with butanol. The GPI-PLD has recently been purified from human and bovine serum. The physiological function of this enzyme has not been determined, but it is proposed to play a role in the regulation of cell surface expression of GPI-anchored proteins. The GPI-PLD hydrolyzes the phosphodiester linkage of the phosphatidylinositol moiety in the GPI anchor of several membrane proteins. The products of this reaction are phosphatidic acid and an inositol residue, which is attached to the C terminus of the protein through the glycan. The assay methods that have been used in the purification procedure described in the chapter monitor production of either of these products. In the assay where alkaline phosphatase is used as substrate, the loss of the hydrophobic part of the anchor from the protein is determined by partitioning in Triton X-114 H and measuring the distribution of the protein between the hydrophilic, detergent-poor phase and the hydrophobic, detergent-rich phase. The substrate is human placental alkaline phosphatase, and its distribution in Triton X-114 is determined by measuring its ability to hydrolyze p -nitrophenyl phosphate in a subsequent incubation. The major disadvantage of this type of assay is that it does not distinguish between GPI-PLD-mediated cleavage and those catalyzed by other hydrolases especially GPI-PLC. It is also more time consuming as it involves a second incubation to assay for the degraded alkaline phosphatase. However, an advantage over the other assay procedure is the ready availability of the starting material for preparation of the substrate.