title: Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances.
abstract: Adenosine 5'-phosphosulfate (APS) reductase (APR; EC 1.8.4.9) catalyzes the two-electron reduction of APS to sulfite and AMP, a key step in the sulfate assimilation pathway in higher plants. In spite of the importance of this enzyme, methods currently available for detection of APR activity rely on radioactive labeling and can only be performed in a very few specially equipped laboratories. Here we present two novel kinetic assays for detecting in vitro APR activity that do not require radioactive labeling. In the first assay, APS is used as substrate and reduced glutathione (GSH) as electron donor, while in the second assay APS is replaced by an APS-regenerating system in which ATP sulfurylase catalyzes APS in the reaction medium, which employs sulfate and ATP as substrates. Both kinetic assays rely on fuchsin colorimetric detection of sulfite, the final product of APR activity. Incubation of the desalted protein extract, prior to assay initiation, with tungstate that inhibits the oxidation of sulfite by sulfite oxidase activity, resulted in enhancement of the actual APR activity. The reliability of the two methods was confirmed by assaying leaf extract from Arabidopsis wild-type and APR mutants with impaired or overexpressed APR2 protein, the former lacking APR activity and the latter exhibiting much higher activity than the wild type. The assays were further tested on tomato leaves, which revealed a higher APR activity than Arabidopsis. The proposed APR assays are highly specific, technically simple and readily performed in any laboratory.
authors: Plant Stress Laboratory, The Albert Katz Department of Dryland Biotechnologies, French Associates Institute for Agriculture and Biotechnology of Drylands, Blaustein Institutes for Desert Research, Ben-Gurion University of Negev, Sede Boqer Campus 84990, Israel.
authors: Galina
authors: G
authors: Brychkova
authors: Dmitry
authors: D
authors: Yarmolinsky
authors: Moshe
authors: M
authors: Sagi
mesh_terms: D000250:Adenosine Phosphosulfate / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology*; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism; D057075:Enzyme Assays; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D011868:Radioisotopes / Q000378:metabolism*; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D011868:Radioisotopes; D013431:Sulfates; D000250:Adenosine Phosphosulfate; D050862:Oxidoreductases Acting on Sulfur Group Donors; C019618:adenylylsulfate reductase; D003545:Cysteine