In its aconitase-inactive form, IRP-1 (iron regulatory protein-1)/cytosolic aconitase binds to the IRE (iron-responsive element) of several mRNAs to effect post-transcriptional regulation. We have shown previously that IRP-1 has ATPase activity and that binding of ATP suppresses the IRP-1/IRE interaction. In the present study, we characterize the binding activity further. Binding is observed with both [α-32P]ATP and [α-32P]ADP, but not with [γ-32P]ATP. Recombinant IRP-1 binds approximately two molecules of ATP, and positive co-operativity is observed with a Hill coefficient of 1.67±0.36 (EC50=44 μM) commencing at 1 μM ATP. Similar characteristics are observed with both apoprotein and the aconitase form. On binding, ATP is hydrolysed to ADP, and similar binding parameters and co-operativity are seen with ADP, suggesting that ATP hydrolysis is not rate limiting in product formation. The non-hydrolysable analogue AMP-PNP (adenosine 5′-[β,γ-imido]triphosphate) does not induce co-operativity. Upon incubation of IRP-1 with increasing concentrations of ATP or ADP, the protein migrates more slowly on agarose gel electrophoresis, and there is a shift in the CD spectrum. In this new state, adenosine nucleotide binding is competed for by other nucleotides (CTP, GTP and AMP-PNP), although ATP and ADP, but not the other nucleotides, partially stabilize the protein against spontaneous loss of aconitase activity when incubated at 37 °C. A mutant IRP-1(C437S) lacking aconitase activity shows only one ATP-binding site and lacks co-operativity. It has increased IRE-binding capacity and lower ATPase activity (Km=75±17 nmol/min per mg of protein) compared with the wild-type protein (Km=147±48 nmol/min per mg of protein). Under normal cellular conditions, it is predicted that ATP/ADP will maintain IRP-1 in a non-IRE-binding state.
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Research Article|
August 13 2010
Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a non-IRE-binding state
Zvezdana Popovic;
Zvezdana Popovic
1Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8
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Douglas M. Templeton
Douglas M. Templeton
1
1Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, ON, Canada M5S 1A8
1To whom correspondence should be addressed (email doug.templeton@utoronto.ca).
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Publisher: Portland Press Ltd
Received:
January 18 2010
Revision Received:
June 09 2010
Accepted:
June 23 2010
Accepted Manuscript online:
June 23 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 430 (2): 315–324.
Article history
Received:
January 18 2010
Revision Received:
June 09 2010
Accepted:
June 23 2010
Accepted Manuscript online:
June 23 2010
Citation
Zvezdana Popovic, Douglas M. Templeton; Interaction of iron regulatory protein-1 (IRP-1) with ATP/ADP maintains a non-IRE-binding state. Biochem J 1 September 2010; 430 (2): 315–324. doi: https://doi.org/10.1042/BJ20100111
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