A soluble protein negatively regulates phospholipase D activity. Partial purification and characterization

Area of Science:

• Cellular signaling
• Enzymology
• Molecular biology

Background:

• Phosphatidylcholine-specific phospholipase D (PLD) is a key signaling enzyme in mammalian cells.
• ARF (ADP-ribosylating factor) positively regulates PLD activity.
• Understanding PLD regulation is crucial for deciphering cellular processes.

Purpose of the Study:

• To identify and characterize factors that negatively regulate PLD activity.
• To investigate the mechanism of action of a novel PLD inhibitory factor.
• To explore the impact of this inhibitor on various PLD activation pathways.

Main Methods:

• Fractionation of bovine brain cytosol to isolate the inhibitory factor.
• Assay of PLD activity in permeabilized HL-60 cells.
• Investigation of inhibitor interaction with ARF and its regulators.
• Western blotting using specific antibodies.

Main Results:

• A large protein complex (>300 kDa) inhibiting PLD activity was identified in bovine brain cytosol.
• The inhibitor acts on GTP[S]-stimulated PLD, with immediate, persistent, and dose-dependent effects.
• The inhibitor does not prevent GTP[S] binding to ARF, ruling out a role as an ARF/guanine exchange factor inhibitor.
• The inhibitor affects PLD activated by ARF, PKC, and tyrosine kinases, and also decreases PLC activity.

Conclusions:

• A novel, high-molecular-weight inhibitor of PLD activity exists in bovine brain cytosol.
• This inhibitor modulates PLD signaling through multiple pathways, including ARF, PKC, and tyrosine kinases.
• The findings suggest a complex regulatory network for PLD, essential for membrane traffic and cellular communication.