Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs)

Molecular basis for differences in requirements for activity of mammalian ARFs

S. Russ Price, Catherine Welsh, Randy S. Haun, Sally J. Stanley, Joel Moss, Martha Vaughan

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

ADP-ribosylation factors (ARFs) are highly conserved ∼20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of G and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/ detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5′-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required ∼4 μM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 μM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

Original languageEnglish
Pages (from-to)17766-17772
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number25
StatePublished - Sep 5 1992
Externally publishedYes

Fingerprint

ADP-Ribosylation Factors
Guanosine Triphosphate
Phospholipids
Cholera Toxin
Cholates
Dimyristoylphosphatidylcholine
Guanine Nucleotides
Detergents
ADP Ribose Transferases
Guanosine 5'-O-(3-Thiotriphosphate)
Molecular Cloning
Cloning
Transferases
Recombinant Proteins
Adenylyl Cyclases
Adenosine Diphosphate
Escherichia coli
Carrier Proteins
Assays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs) : Molecular basis for differences in requirements for activity of mammalian ARFs. / Price, S. Russ; Welsh, Catherine; Haun, Randy S.; Stanley, Sally J.; Moss, Joel; Vaughan, Martha.

In: Journal of Biological Chemistry, Vol. 267, No. 25, 05.09.1992, p. 17766-17772.

Research output: Contribution to journalArticle

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