STREPTOLYSIN-O INDUCES RELEASE OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED ALKALINE-PHOSPHATASE FROM ROS CELLS BY VESICULATION INDEPENDENTLY OF PHOSPHOLIPASE ACTION

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studie s on the release of glycosylphosphatidylinositol (GPI)anchored alkalin e phosphatase (AP) from ROS eels. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extrace llular 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 Trito n X-114 phase separation. This result suggested that the released AP c ontained an intact GPI anchor, and thus both proteolysis and anchor de gradation by anchor-specific hydrolases, including GPI-PLD, as the pot ential mechanisms for SLO-mediated AP release were ruled out. The rele ased AP sedimented at 100 000 g. A substantial amount of lipids was de tected in the 100 000 g pellet. Cholesterol and sphingomyelin were enr iched in SLO-released material, compared with intact cells. These resu lts were consistent with vesiculation as the mechanism for SLO inducti on of AP release. Two other cholesterol-binding agents, saponin and di gitonin, were also able to release AP, possibly by a similar vesiculat ion mechanism, whereas others, including nystatin, filipin and beta-es cin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the v esicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored pro teins in the plasma membrane is responsible for their resistance to GP I-PLD cleavage.