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 Gsα 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 language | English |
|---|---|
| Pages (from-to) | 17766-17772 |
| Number of pages | 7 |
| Journal | Journal of Biological Chemistry |
| Volume | 267 |
| Issue number | 25 |
| State | Published - Sep 5 1992 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs)
T2 - Molecular basis for differences in requirements for activity of mammalian ARFs
AU - Price, S. Russ
AU - Welsh, Catherine
AU - Haun, Randy S.
AU - Stanley, Sally J.
AU - Moss, Joel
AU - Vaughan, Martha
PY - 1992/9/5
Y1 - 1992/9/5
N2 - 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 Gsα 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.
AB - 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 Gsα 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.
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M3 - Article
C2 - 1517219
AN - SCOPUS:0026666365
VL - 267
SP - 17766
EP - 17772
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 25
ER -