RESOLUTION OF 2 ADP-RIBOSYLATION FACTOR-1 GTPASE-ACTIVATING PROTEINS FROM RAT-LIVER

ADP-ribosylation factor 1 (ARF1) is a 21 kDa GTP-binding protein that regulates multiple steps in membrane traffic. Here, two ARF1 GTPase-ac tivating proteins (GAPs) from rat liver were resolved. The GAPs were a ntigenically distinct. One reacted with a polyclonal antibody raised a gainst the GAP catalytic peptide previously purified by Makler et al. [Makler, Cukierman, Rotman, Admon and Cassel (1995) J. Biol. Chem. 270 , 5232-5237], and here is referred to as GAP1. The other GAP (GAP2) di d not react with the antibody. These GAPs differed in phospholipid dep endencies. GAP1 was activated 3-7-fold by the acid phospholipids phosp hatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) and ph osphatidylserine (PS). In contrast, GAP2 was stimulated 20-40-fold by PIP2. PA and PS had no effect by themselves but PA increased GAP2 acti vity in the presence of PIP2. The GAPs were otherwise similar in activ ity. In the presence of phosphoinositides, the K-m of GAP1 for ARF1-GT P was estimated to be 8.1 +/- 1.6 mu M and the dissociation constant f or ARF1-guanosine 5',3-O-(thio)triphosphate (GTP[SI) was 7.4 +/- 2.2 m u M. GAP2 was similar with a K-m for ARF1-GTP of 5.4 +/- 1.2 mu M and a dissociation constant for ARF1-GTP[S] of 4.8 +/- 0.3 mu M. Similarly , no differences were found in substrate preferences. Both GAP1 and GA P2 used ARF1 and ARF5 as substrates but not ARF6 or ARF-like protein-2 . The potential role of multiple ARF GAPs in the independent regulatio n of ARF at specific steps in membrane traffic is discussed.