MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF GTP-BINDING PROTEINS

Project: Research project

Project Details

Description

ADP-ribosylation factors (ARFs) are a family of 20-kDa guanine
nucleotide-binding proteins originally identified and purified by their
ability to enhance the ADP-ribosyltransferase activity of cholera toxin.
ARFs are highly conserved proteins found in all eukaryotic cells. At
least six mammalian ARFs genes have been cloned; these can be grouped
into three classes based on protein size, deduced amino acid sequence,
phylogenetic analysis, and gene structure.

Recombinant ARFs from all three classes activate cholera toxin-catalyzed
ADP-ribosylation. Cytosolic or purified ARFs in the presence of GTPrS,
associated with phospholipid or cell membranes. It has been reported
that the amino terminus of ARF is a region critical for its activity in
vitro and probably in vivo. A peptide with the amino acid sequence of
the amino terminus of ARF1 reportedly inhibited ARF function, including
its ability to enhance endosome fusion and Golgi transport as well as to
activate cholera toxin. In this study, recombinant ARF1 (rARF1),
rdelta13ARF1 (recombinant ARF1 lacking the first 13 amino acid) and
rPKA14ARF1 (rARF1 in which the first 14 amino acids were replaced by the
first seven amino acids of the cAMP-dependent protein kinase catalytic
subunit) were used to assess the effect of the amino terminus on the
ability of ARF to enhance ADP-ribosylation of agmatine by the cholera
toxin A subunit. The GTP-dependent ARF activities of rdelta13ARF1 and
rPKA14ARF1 were similar to that ARF1, whereas the GTP requirement for
half-maximal activation of cholera toxin A, was somewhat higher for rARF1
than it was for rdelta13ARF1 and rPKA14ARF1. These results are
consistent with the view that the amino terminus of ARF1 is not critical
for its action as a GTP-dependent activator of cholera toxin.
StatusNot started

Funding

  • National Heart, Lung, and Blood Institute

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