ARF1-regulated phospholipase D in human neutrophils is enhanced by PMA and MgATP.
Human neutrophil PLD activity stimulated with GTP-gamma-S was reconstituted with recombinant ARF1 in cytosol-depleted cells. PMA-pretreatment of intact cells greatly enhanced the subsequent reconstitution of the ARF1-regulated PLD activity. This enhancement was only observed provided that the intact cells were pretreated with PMA, suggesting the stable recruitment of a cytosolic component, presumably protein kinase C, to the membranes. rARF1-reconstituted PLD activity was not dependent on MgATP, but could be considerably enhanced by MgATP. Maximal effects of MgATP were seen at 1 mM. This enhancement by MgATP could not be attributed to protein kinase C. Neomycin was found to inhibit ARF1-regulated PLD activity suggesting the requirement for polyphosphoinositides. We conclude: (i) that many of the observed effects of PMA may be dependent on the presence of the small GTP-binding protein, ARF, and (ii) polyphosphoinositides are required for ARF1-stimulated PLD activity.
Whatmore
J
J
Department of Physiology, University College London, UK.
Cronin
P
P
Cockcroft
S
S
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D003600:Cytosol / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D009504:Neutrophils / Q000201:enzymology*; D018129:Phosphatidylinositol Phosphates / Q000502:physiology; D010739:Phospholipase D / Q000097:blood*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D018129:Phosphatidylinositol Phosphates; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D013755:Tetradecanoylphorbol Acetate