UPTAKE AND INTRACELLULAR STABILITY OF GLYCOSYLPHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-D IN NEUROBLASTOMA-CELLS

Glycosylphosphatidylinositol-specific phospholipase D from mammalian s erum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may onl y be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following u ptake from body fluids. To test this hypothesis, we studied the possib le uptake and intracellular processing of purified glycosylphosphatidy linositol-specific phospholipase D into the mouse neuroblastoma cell l ine N2A. We found that after incubation of neuroblastoma cells with gl ycosylphosphatidylinositol-specific phospholipase D at 37 degrees C th e amount of cell-associated glycosylphosphatidylinositol-specific phos pholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with I-125-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phosphol ipase D. We found that the incorporated radiolabeled proteins were pro cessed intracellularly to distinct low molecular mass products, and th at this process was in part inhibited by the presence of chloroquine d uring incubation.