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 reconst itute cell-free phospholipase D activity from phagocytes (Olson, S., B owman, E. P., and Lambeth, J. D. (1991) J. Biol. Chem. 266, 17236-1724 2); membrane factors include a small GTP-binding protein in the Rho fa mily (Bowman, E., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509-21512), ADP-ribosylation factor (ARF) was recently im plicated as the cytosolic factor, as it activates phospholipase D in H L-60 membranes, Herein, we show that ion exchange chromatography separ ates ARF from the major phospholipase D-stimulating cytosolic factor, Both bovine brain ARF and recombinant human ARF-1 stimulated a small a mount 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 cytoso l, ARF caused marked activation. Thus, ARF synergizes with the cytosol ic factor in phospholipase D activation.