Abstract
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 (GPI)-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 (lysoPI) 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 Ca2+ -dependent release of overexpressed PLAP from ecto PLC-positive cells was observed. To investigate the cell surface linkage of the ecru 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.
Original language | English (US) |
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Pages (from-to) | 550-564 |
Number of pages | 15 |
Journal | Journal of cellular biochemistry |
Volume | 65 |
Issue number | 4 |
DOIs | |
State | Published - Jun 15 1997 |
Keywords
- cell surface enzyme
- ecto-PLC
- ecto-enzyme
- lyso-Pl-cleaving PLC
- phosphoinositide-specific phospholipase C
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology