Abstract
ADP-ribosylation factors (ARFs) are ∼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 γ-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. [α-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [α-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.
| Original language | English |
|---|---|
| Pages (from-to) | 15583-15587 |
| Number of pages | 5 |
| Journal | Journal of Biological Chemistry |
| Volume | 269 |
| Issue number | 22 |
| State | Published - Jun 3 1994 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Isolation of recombinant ADP-ribosylation factor 6, a ∼20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state. / Welsh, Catherine F.; Moss, Joel; Vaughan, Martha.
In: Journal of Biological Chemistry, Vol. 269, No. 22, 03.06.1994, p. 15583-15587.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Isolation of recombinant ADP-ribosylation factor 6, a ∼20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state
AU - Welsh, Catherine F.
AU - Moss, Joel
AU - Vaughan, Martha
PY - 1994/6/3
Y1 - 1994/6/3
N2 - ADP-ribosylation factors (ARFs) are ∼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 γ-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. [α-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [α-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.
AB - ADP-ribosylation factors (ARFs) are ∼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 γ-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. [α-32P]GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of [α-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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0028275353&partnerID=8YFLogxK
M3 - Article
C2 - 8195204
AN - SCOPUS:0028275353
VL - 269
SP - 15583
EP - 15587
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 22
ER -