ADP-ribosylation factor functions synergistically with a 50-kDa cytosolic factor in cell-free activation of human neutrophil phospholipase D.
Proteins in both the cytosol and plasma membrane are needed to reconstitute cell-free phospholipase D activity from phagocytes (Olson, S., Bowman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-17242); membrane factors include a small GTP-binding protein in the Rho family (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512). ADP-ribosylation factor (ARF) was recently implicated as the cytosolic factor, as it activates phospholipase D in HL-60 membranes. Herein, we show that ion exchange chromatography separates ARF from the major phospholipase D-stimulating cytosolic factor. Both bovine brain ARF and recombinant human ARF-1 stimulated a small amount of phospholipase D activity in the absence of cytosol (about 10% of the response seen with cytosol). With a high concentration of ARF-depleted cytosol, ARF did not further activate. However, at low cytosol, ARF caused marked activation. Thus, ARF synergizes with the cytosolic factor in phospholipase D activation.
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322.
J D
JD
Lambeth
J Y
JY
Kwak
E P
EP
Bowman
D
D
Perry
D J
DJ
Uhlinger
I
I
Lopez
D020727:ADP-Ribosylation Factors; D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology*; D010739:Phospholipase D / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D009419:Nerve Tissue Proteins; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors