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J Biol Chem. 2003 Feb 28;278(9):7285-93. Epub 2002 Dec 20.

Role of an intrasubunit disulfide in the association state of the cytosolic homo-oligomer methionine adenosyltransferase.

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1
Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Cientificas (CSIC)-Universidad Autónoma de Madrid, Arturo Duperier 4, 28029 Madrid, Spain.

Abstract

Recombinant rat liver methionine adenosyltransferase has been refolded into fully active tetramers (MAT I) and dimers (MAT III), using as a source chaotrope-solubilized aggregates resulting from specific washes of inclusion bodies. The conditions of refolding, dialysis in the presence of 10 mm dithiothreitol or 10 mm GSH with 1 mm GSSG, allowed the production of both isoforms, the nature of the redox agent determining the capacity of the final product (MAT I/III) to interconvert. Refolding in the presence of 10 mm dithiothreitol yielded mainly MAT III in a concentration-dependent equilibrium with the homotetramer MAT I. However, refolding in the presence of the redox pair GSH/GSSG resulted in a stable MAT I and III mixture. Blockage of dimer-tetramer interconversion has been found related to the production of a single intramolecular disulfide in methionine adenosyltransferase during the GSH/GSSG folding process. The residues involved in this disulfide have been identified by mass spectrometry and using a set of single cysteine mutants as cysteines 35 and 61. In addition, a kinetic intermediate in the MAT I dissociation to MAT III has been detected. The physiological importance of these results is discussed in light of the structural and regulatory data available.

PMID:
12496263
DOI:
10.1074/jbc.M210177200
[Indexed for MEDLINE]
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