Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C

G. Bruce Birrell, Karen K. Hedberg, Eric Barklis, O. Hayes Griffith

Research output: Contribution to journalArticle

4 Scopus citations

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 languageEnglish (US)
Pages (from-to)550-564
Number of pages15
JournalJournal of cellular biochemistry
Volume65
Issue number4
DOIs
StatePublished - 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

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