ADP-ribosylation factor-directed GTPase-activating protein. Purification and partial characterization.
The small GTP-binding protein ARF plays an established role in the control of vesicular traffic and in the regulation of phospholipase D activity. 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 suggested that GTP hydrolysis by ARFs is dependent on a GTPase-activating protein (GAP). Here we report the complete purification of an ARF GAP from rat liver cytosol. Advanced stages in the purification were carried out in the presence of denaturing agents, making use of an unusual conformational stability, or refolding capacity, of the GAP. The GAP 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 previously uncharacterized protein. Both crude and purified GAP migrated on a Superdex 200 column as a 200-kDa complex, suggesting a tetrameric structure. The purified ARF GAP was stimulated by phosphoinositides and was inhibited by phosphatidylcholine, similar to the results previously 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.
Makler
V
V
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Cukierman
E
E
Rotman
M
M
Admon
A
A
Cassel
D
D
D020727:ADP-Ribosylation Factors; D000818:Animals; D001921:Brain / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D020690:GTPase-Activating Proteins; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D011506:Proteins; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors