Nucleotide binding and cofactor activities of purified bovine brain and bacterially expressed ADP-ribosylation factor.
The ADP-ribosylation factor (ARF) is a member of the small molecular weight GTP-binding protein family and serves as the cofactor in the cholera toxin-catalyzed activation of the stimulatory regulatory subunit (Gs) of adenylate cyclase. Bovine Arf1 has been expressed at high levels and purified from bacteria. The recombinant Arf1 was compared with purified bovine brain Arf and shown to be nearly identical with respect to immunoblotting, guanine nucleotide binding, GTP hydrolysis, and cholera toxin cofactor activities. The only known chemical difference between the recombinant and brain proteins is the lack of myristic acid at the amino terminus of the expressed protein. The preparation of nucleotide-free Arf1 has allowed a more accurate determination of the binding constants for guanine nucleotides and revealed a significantly higher affinity for GDP than was previously determined. The effect of magnesium ions on nucleotide affinities was also determined and found to be quite different for the different guanine nucleotides. We have shown that GDP binds to the protein in the absence of magnesium, while GTP or guanosine 5'-O-(thiotriphosphate) can only bind to Arf1 in the presence of nanomolar (or higher) levels of the free metal. This characterization of the nucleotide binding and the ability to produce large amounts of a single species of ARF with full retention of a range of activities should greatly facilitate subsequent studies on the structure and function of ARF.
Weiss
O
O
Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, Maryland 20892.
Holden
J
J
Rulka
C
C
Kahn
R A
RA
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D005796:Genes; D006150:Guanine Nucleotides / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D006150:Guanine Nucleotides; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors