A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants.
Sulfite reductase (SiR; EC 1.8.7.1), an essential enzyme in the sulfate reduction pathway, catalyzes the reduction of sulfite to sulfide, as an intermediate for cysteine biosynthesis. The commonly used kinetic assay for the detection of in vitro SiR activity in plants is based on a coupled reaction, in which the sulfide produced is converted to cysteine through the presence, in the assay medium, of O-acetylserine sulfhydralase (EC 2.5.1.47) and its substrate, O-acetylserine. An improved kinetic assay for SiR activity in crude desalted protein extracts was developed. The improvement was based on pre-treatment of the protein with tungstate, which improved SiR activity in Arabidopsis and tomato leaf by 29 and 12%, respectively, and the addition of NADPH to the reaction medium, which increased SiR activity by 1.6- and 2.8-fold in Arabidopsis and tomato, respectively, in comparison with the current protocols. Despite the availability and reliability of the kinetic assay, there is currently no assay that enables the direct detection of SiR in relatively large numbers of samples. To meet this need, we developed a novel in-gel assay to detect SiR activity in crude extracts. The method is based on the detection of a brownish-black precipitated band of lead sulfide, formed by the reaction of lead acetate with sulfide. The in-gel assay for SiR activity is reliable, sensitive and technically simpler than the kinetic assay, and opens up the possibility for detecting active SiR isoenzymes and splice variants.
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.
Galina
G
Brychkova
Dmitry
D
Yarmolinsky
Yvonne
Y
Ventura
Moshe
M
Sagi
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D005782:Gels; D007527:Isoenzymes / Q000032:analysis; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D012694:Serine / Q000031:analogs & derivatives / Q000378:metabolism; D013379:Substrate Specificity; D017973:Tungsten Compounds / Q000737:chemistry
D005782:Gels; D007527:Isoenzymes; D010940:Plant Proteins; D017973:Tungsten Compounds; D012694:Serine; D009249:NADP; D050862:Oxidoreductases Acting on Sulfur Group Donors; C043943:O-acetylserine; C045951:tungstate