Role of an Intrasubunit Disulfide in the Association State of the Cytosolic Homo-oligomer Methionine Adenosyltransferase

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 m m dith...

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Bibliographic Details
Published in:The Journal of biological chemistry 2003-02, Vol.278 (9), p.7285-7293
Main Authors: Sanchez-Perez, Gabino F, Gasset, Maria, Calvete, Juan J, Pajares, Maria A
Format: Article
Language:English
Subjects:
Animals
Chromatography
Chromatography, Gel
Crystallography, X-Ray
Cysteine - chemistry
Cytosol - metabolism
Dimerization
Disulfides - metabolism
Dithiothreitol - pharmacology
Dose-Response Relationship, Drug
Glutathione - metabolism
Glutathione Transferase - metabolism
Kinetics
Liver - enzymology
Liver - metabolism
Mass Spectrometry
Methionine Adenosyltransferase - chemistry
Methionine Adenosyltransferase - metabolism
Mutation
Protein Binding
Protein Folding
Protein Structure, Tertiary
Rats
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Thermodynamics
Time Factors
Trypsin - pharmacology
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Summary: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 m m dithiothreitol or 10 m m GSH with 1 m m 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 m m 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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M210177200