Inhibition of phospholipase D by a protein factor from bovine brain cytosol. Partial purification and characterization of the inhibition mechanism

Jae Ho Kim, Yoon Jung Suh, Taehoon G. Lee, Yong Kim, Sun Sik Bae, Myung Jong Kim, J. David Lambeth, Pann Ghill Suh, Sung Ho Ryu*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

A specific protein inhibitor of partially purified bovine brain phospholipase D (PLD) was identified from bovine brain cytosol. The PLD inhibitor has been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. The inhibitor showed an apparent molecular mass of 30 kDa by Superose 12 gel exclusion chromatography and inhibited PLD activity with an IC50 of 7 nM. The inhibitor had neither proteolytic activity nor phospholipid-hydrolyzing activity. Because phosphatidylinositol 4,5-bisphosphate (PIP2), which is included in substrate vesicles, is an essential cofactor for PLD, we examined whether the inhibition might be mediated by sequestration of PIP2. PIP2 hydrolysis by phospholipase C (PLC)-β1 was not affected by the inhibitor and the inhibitor did not bind to substrate vesicles containing PIP2. In contrast, a PH domain derived from PLC-δ1, which could bind to PIP2, showed a nearly identical inhibition of both PLC-β1 and PLD activities. Thus, the PLD inhibition by the inhibitor is due to the specific interaction with not PIP2 but PLD. The suppression of PLD activity by the inhibitor was largely eliminated by the addition of ADP-ribosylation factor (ARF) and GTPγS. We propose that the inhibitor plays a negative role in regulation of PLD activity by PIP2 and ARF.

Original languageEnglish (US)
Pages (from-to)25213-25219
Number of pages7
JournalJournal of Biological Chemistry
Volume271
Issue number41
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology

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