SAPONIN-INDUCED RELEASE OF CELL-SURFACE-ANCHORED THY-1 BY SERUM GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-D

A glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was purified from human serum and used for studies on the release of GPI-a nchored Thy-1 glycoprotein from mouse T lymphoma cells Y191. Previous studies have shown that whereas GPI-PLD is highly active against deter gent-solubilized GPI-anchored proteins, it is normally unable to relea se GPI-containing proteins anchored in a lipid bilayer. Confirming the se findings, the addition of GPI-PLD to intact Y191 cells did not resu lt in cleavage of Thy-1. However, pretreatment of cells with saponin, a cholesterol-sequestering agent, rendered Thy-1 susceptible to hydrol ysis. Very little solubilization of GPI-containing Thy-1 occurred unde r these conditions. From experiments with reconstituted liposomes it w as inferred that the effect of saponin on cells was to aid in the pres entation of Thy-1 to GPI-PLD. Furthermore, it was concluded that chole sterol-saponin complexes formed in the membrane were not alone respons ible for the effect. Rather, additional molecules in the plasma membra ne are possibly involved in the presentation of Thy-1 on saponin-treat ed cells. This finding may have implications for a physiological role of circulating GPI-PLD in the regulation of GPI-anchored proteins on c ells.