title: Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.
abstract: Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.
authors.affiliation: Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland.
authors.forename: T
authors.initials: T
authors.lastname: Hari
authors.forename: P
authors.initials: P
authors.lastname: Bütikofer
authors.forename: U N
authors.initials: UN
authors.lastname: Wiesmann
authors.forename: U
authors.initials: U
authors.lastname: Brodbeck
mesh_terms: D000818:Animals; D002417:Cattle; D002738:Chloroquine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D051379:Mice; D009447:Neuroblastoma / Q000201:enzymology*; D010739:Phospholipase D / Q000097:blood / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D017261:Glycosylphosphatidylinositols; D007457:Iodine Radioisotopes; D002738:Chloroquine; D010739:Phospholipase D; D010450:Endopeptidases