Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)anchored alkaline phosphatase (AP) from ROS eels. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100 000 g. A substantial amount of lipids was detected in the 100 000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.
