Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state.
ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins, which, like other members of the ras superfamily, are activated by exchanging bound GDP for GTP and inactivated through hydrolysis of the gamma-phosphate of bound GTP to form GDP in a highly regulated cycle. ARF 6, a class III ARF, was expressed in Escherichia coli with its amino terminus fused to maltose-binding protein. Following release from maltose-binding protein, recombinant ARF 6 (rARF 6) exhibited maximal activity with or without GTP. Such constitutive activation was due to the predominance of ARF-GTP over ARF-GDP, as demonstrated by nucleotide analysis. rARF 6 expressed in E. coli without amino-terminal extension was bound primarily to GDP and exhibited typical GTP-dependent activity. After release from maltose-binding protein, rARF 6-GTP was stable; only a fraction of the nucleotide was removed using EDTA, whereas urea denaturation restored complete GTP dependence. [alpha-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [alpha-32P]GTP, -GDP, and -GMP, whereas unbound nucleotide was completely hydrolyzed to guanosine. Thus, amino-terminal extension of rARF 6, by maltose-binding protein, promoted the formation of a constitutively activated GTP-bound species. By analysis of this species, we confirmed that rARF 6 lacks the intrinsic ability to hydrolyze bound GTP and speculate that maltose-binding protein may inhibit hydrolysis by extrinsic factors.
Welsh
C F
CF
Laboratory of Cellular Metabolism, NHLBI, National Institutes of Health, Bethesda, Maryland 20892.
Moss
J
J
Vaughan
M
M
D020727:ADP-Ribosylation Factors; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008322:Mammals; D037601:Mannose-Binding Lectin / Q000031:analogs & derivatives*; D037241:Mannose-Binding Lectins; D008970:Molecular Weight; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014508:Urea / Q000494:pharmacology
D002352:Carrier Proteins; D037601:Mannose-Binding Lectin; D037241:Mannose-Binding Lectins; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D014508:Urea; D004492:Edetic Acid; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D008274:Magnesium