Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C.
A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GP) anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysaPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca(2+)-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein.
Birrell
G B
GB
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Hedberg
K K
KK
Barklis
E
E
Griffith
O H
OH
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001360:Avidin; D006801:Humans; D051379:Mice; D008863:Microspheres; D008956:Models, Chemical; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D051381:Rats; D013499:Surface Properties
D001360:Avidin; D000469:Alkaline Phosphatase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C