ADP-RIBOSYLATION FACTOR-DIRECTED GTPASE-ACTIVATING PROTEIN - PURIFICATION AND PARTIAL CHARACTERIZATION

The small GTP-binding protein ARF plays an established role in the con trol of vesicular traffic and in the regulation of phospholipase D act ivity. Like other GTP binding proteins, ARF becomes activated upon the binding of GTP, whereas GTP hydrolysis acts as a turn-off signal. The fact that purified ARF proteins have negligible GTPase activity has s uggested that GTP hydrolysis by ARFs is dependent on a GTPase-activati ng protein (GAP). Here we report the complete purification of an ARF G AP from rat liver cytosol. Advanced stages in the purification were ca rried out in the presence of denaturing agents, making use of an unusu al conformational stability, or refolding capacity, of the GAP. The GA P was purified about 15,000-fold and was identified as a protein of 49 kDa. Partial amino acid sequence analysis showed that the GAP is a pr eviously uncharacterized protein Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tet rameri c structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previ ously reported for a preparation from brain (Randazzo, P. A., and Kahn , R. A. (1994) J. Biol. Chem. 269, 10758). The availability of the ARF GAP molecule will advance the understanding of the regulation of the cellular processes in which ARF proteins participate.