BIOSYNTHESIS OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED HUMAN PLACENTALALKALINE-PHOSPHATASE - EVIDENCE FOR A PHOSPHOLIPASE C-SENSITIVE PRECURSOR AND ITS POST-ATTACHMENT CONVERSION INTO A PHOSPHOLIPASE C-RESISTANT FORM

Previous studies have shown that some cells (e.g. SKG3a) express human placental alkaline phosphatase (AP) in a form which can be released f rom the membrane by bacterial PtdIns-specific phospholipase C (PI-PLC) while others (e.g. HeLa) are relatively resistant to this enzyme. Che mical and enzymic degradation studies have suggested that the PI-PLC r esistance of AP is due to inositol acylation of its glycosylphosphatid ylinositol (GPI) anchor. In order to identify the biosynthetic origin of PI-PLC resistance we determined the PI-PLC sensitivity of AP in S-3 5-labelled cells (10 min pulse; 0-60 min chase) by Triton X-114 phase separation. At the beginning of the chase period, the majority of the AP synthesized was hydrophilic, indicating that it had not acquired a GPI anchor. The concentration of hydrophilic AP species decreased with a t(1/2) of 30-60 min but was not processed to an endoglycosidase fi- resistant species or secreted into the medium. In both SKG3a and HeLa cells all of the hydrophobic, GPI-anchored AP detectable at the beginn ing of the chase was PI-PLC sensitive. PI-PLC-resistant species of AP were only observed in HeLa cells and these only appeared after about 3 0 min. The delayed appearance of PI-PLC resistance was unexpected as p revious studies have suggested that candidate GPI-anchor precursors ar e PI-PLC-resistant as a result of inositol acylation. This work reveal s unanticipated complexities in the biosynthesis of AP and its GPI anc hor.