title: Regulation of plant cytosolic aldolase functions by redox-modifications.
abstract: 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.
authors.affiliation: Department of Plant Physiology, University of Osnabrueck, D-49069 Osnabrueck, Germany. lindek@mpi-marburg.mpg.de
authors.forename: Karina
authors.initials: K
authors.lastname: van der Linde
authors.forename: Nora
authors.initials: N
authors.lastname: Gutsche
authors.forename: Hans-Martin
authors.initials: HM
authors.lastname: Leffers
authors.forename: Christian
authors.initials: C
authors.lastname: Lindermayr
authors.forename: Bernd
authors.initials: B
authors.lastname: Müller
authors.forename: Simone
authors.initials: S
authors.lastname: Holtgrefe
authors.forename: Renate
authors.initials: R
authors.lastname: Scheibe
mesh_terms: D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000201:enzymology* / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005635:Fructosediphosphates / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D005635:Fructosediphosphates; D011994:Recombinant Proteins; D005634:Fructose-Bisphosphate Aldolase; C029063:fructose-1,6-diphosphate