AN ASSAY FOR GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHOR DEGRADING PHOSPHOLIPASES

This paper describes a new approach to assay phospholipases which clea ve glycosylphosphatidylinositol using a biotinylated protein substrate coupled to I-125-streptavidin and Triton X-114 phase separation. Subs trate preparation with variant surface glycoprotein of Trypamosoma bru cei, its characterization and solubilization by glycosylphosphatidylin ositol-specific phospholipase C and D are reported. Hydrolysis of subs trate exhibited first-order kinetics with respect to enzyme concentrat ion, and the rate constant of the reaction is independent both from su bstrate concentration and reaction time. This assay was compared with the one using H-3-myristoylated variant surface glycoprotein and prove d to be equally suitable to quantitate glycosylphosphatidylinositol-sp ecific phospholipases, with the advantage that avoids biosynthetic lab eling. Furthermore, it introduces a basic methodology which can be eas ily adapted to use other glycosylphosphatidylinositol-anchored protein s as substrates.