Abstract
This chapter discusses the use of the enzyme glycosylphosphatidylinositol–phospholipase D (GPI-PLD) as a tool for structural analysis of GPI-anchored proteins. The GPI–PLD is a phospholipase D present in all mammalian plasma. Because it recognizes a common core element of the GPI anchor, consisting of glucosamine–phosphatidylinositol, the GPI–PLD is a useful analytical tool for characterizing the structure of GPI-anchored molecules. The GPI–PLD is purified to homogeneity from human serum using a four-step procedure involving fractionation first over an anion-exchange Mono Q column, followed by passage over a hydrophobic phenyl-5PW column. The GPI-PLD effectively hydrolyzes GPI-anchored proteins in detergent solutions. Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine–phosphatidylinositol. The cleavage by the GPI–PLD is unaffected by acylation of the inositol ring. Thus, the GPI–PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors.
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