Isolation of an amino-terminal deleted recombinant ADP-ribosylation factor 1 in an activated nucleotide-free state.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (r delta 13ARF1-P) stimulated cholera toxin activity essentially equally with or without added GTP (and phospholipid or detergent), at least in part due to the presence of bound nucleotides, which later were identified as GTP and GDP. Nucleotide-free r delta 13ARF1 (r delta 13ARF1-F), prepared by dialysis against 7 M urea, was active without added GTP in the absence of SDS but inactive without added GTP in its presence. Renaturation of r delta 13ARF1-F in the presence of GTP, ITP, or GDP yielded, respectively, r delta 13ARF1-GTP and r delta 13ARF1-ITP, which were active, and r delta 13ARF1-GDP, which was inactive. Effects of phospholipids and detergents on nucleotide exchangeability evaluated as effects on activity of rARF1 and r delta 13ARF1-F differed. With r delta 13ARF1-F, 100 microM ITP and 100 microM GTP were essentially equally effective in the presence of cardiolipin or SDS. The finding that r delta 13ARF1 differs from rARF1 in the effects of phospholipids and detergents on nucleotide binding is consistent with the conclusion that the ARF amino terminus plays an important role in nucleotide binding and its specificity as well as the molecular conformation and associated activity.
Hong
J X
JX
Pulmonary-Critical Care Medicine Branch, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
X
X
Moss
J
J
Vaughan
M
M
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000032:analysis / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000032:analysis / Q000378:metabolism; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D010743:Phospholipids / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion
D002352:Carrier Proteins; D003902:Detergents; D010743:Phospholipids; D011994:Recombinant Proteins; D007293:Inosine Triphosphate; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors