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Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
Regulation of plant cytosolic aldolase functions by redox-modifications.
Plant Physiol. Biochem. 49, 946-957 (2011)
From the five genes which code for cytosolic fructose 1,6-bisphosphate aldolases in Arabidopsis thaliana L., the cDNA clone of cAld2 (At2g36460), was heterologously expressed in E. coli and incubated under various oxidizing and reducing conditions. Covalent binding of a GSH moiety to the enzyme was shown by incorporation of biotinylated GSH (BioGEE) and by immunodetection with monoclonal anti-GSH serum. Nitrosylation after incubation with GSNO or SNP was demonstrated using the biotin-switch assay. Mass-spectrometry analysis showed glutathionylation and/or nitrosylation at two different cysteine residues: GSH was found to be attached to C68 and C173, while the nitroso-group was incorporated only into C173. Non-reducing SDS-PAGE conducted with purified wild-type and various Cys-mutant proteins revealed the presence of disulfide bridges in the oxidized enzyme, as described for rabbit muscle aldolase. Incubation of the purified enzyme with GSSG (up to 25 mM) led to partial and reversible inactivation of enzyme activity; NADPH, in the presence of the components of the cytosolic NADP-dependent thioredoxin system, could reactivate the aldolase as did DTT. Total and irreversible inactivation occurred with low concentrations (0.1 mM) of nitrosoglutathione (GSNO). Inactivation was prevented by co-incubation of cAld2 with fructose-1,6-bisphosphate (FBP). Nuclear localization of cAld2 and interaction with thioredoxins was shown by transient expression of fusion constructs with fluorescent proteins in isolated protoplasts.
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Publikationstyp
Artikel: Journalartikel
Dokumenttyp
Wissenschaftlicher Artikel
Schlagwörter
Glutathionylation; Nitrosylation; Cytosolic aldolase; GSNO; GSSG; ROS; Disulfide bridge; Nuclear localization; Thioredoxin h; Arabidopsis thaliana; Split-YFP; Biotin-switch assay
ISSN (print) / ISBN
0981-9428
e-ISSN
1873-2690
Zeitschrift
Plant Physiology and Biochemistry
Quellenangaben
Band: 49,
Heft: 9,
Seiten: 946-957
Verlag
Elsevier
Verlagsort
Amsterdam, Netherlands
Nichtpatentliteratur
Publikationen
Begutachtungsstatus
Peer reviewed
Institut(e)
Institute of Biochemical Plant Pathology (BIOP)