Isolation of recombinant ADP-ribosylation factor 6, a ∼20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state

Catherine Welsh, Joel Moss, Martha Vaughan

Research output: Contribution to journalArticle

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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 languageEnglish
Pages (from-to)15583-15587
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number22
StatePublished - Jun 3 1994

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Guanine Nucleotides
Guanosine Triphosphate
Carrier Proteins
Maltose-Binding Proteins
ADP-Ribosylation Factors
Hydrolysis
Nucleotides
Escherichia coli
ADP-ribosylation factor 6
Denaturation
Guanosine
Alkaline Phosphatase
Chemical activation
Phosphates

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; Moss, Joel; Vaughan, Martha.

In: Journal of Biological Chemistry, Vol. 269, No. 22, 03.06.1994, p. 15583-15587.

Research output: Contribution to journalArticle

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