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

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.

The Oncogene PDRG1 Is an Interaction Target of Methionine Adenosyltransferases

“…Additional effects derive from the fact that most of these diseases also present with oxidative stress, and hence with enhanced production of free radicals, nitric oxide and/or changes in the GSH/GSSG ratio. These outcomes result in post-translational modifications reducing MAT I/III activity and/or alterations in the association state [20–23]. MATα2 is protected against these post-translational modifications by the lack of equivalent residues in its sequence [24, 25], but its oligomerization with MATβ is favored by the enhancement of NADP + levels, hence aiding to reduce AdoMet production [10, 25].…”

Acute Liver Injury Induces Nucleocytoplasmic Redistribution of Hepatic Methionine Metabolism Enzymes

“…For dot-blot analysis aliquots (100 ll) of the column fractions were spotted onto nitrocellulose membranes as previously described (54), and incubated with the antibodies of interest using the same dilutions as for western blot.…”

Stalking metal-linked dimers

“…The later elution volume can be attributed to a higher fraction of monomeric species in the diluted protein solution as a result of mass action [54]. Monomer-dimer equilibrium constants can be obtained from the analysis of elution volume data from a series of frontal zone chromatography trials [53][54][55][56][57][58][59][60][61][62][63][64].…”

“…Quantitative data in the form of equilibrium constants can be obtained from the elution volume data gathered in frontal zone chromatography experiments [54,55,57,[61][62][63][64]. The analysis requires the assignment of magnitudes for the elution volumes of monomer, V M , and of polymer (dimer), V P .…”