title: Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro.
abstract: In isolated membranes, posttranslational regulation of quinol oxidase activities can only be determined simultaneously for all oxidases - quinol oxidases as well as cytochrome c oxidases - because of their identical localization. In this study, a refined method to determine the specific activity of a single quinol oxidase is exemplarily described for the alternative oxidase (AOX) isoform AOX1A from Arabidopsis thaliana and its corresponding mutants, using the respiratory chain of an Escherichia coli cytochrome bo and bd-I oxidase double mutant as a source to provide electrons necessary for O2 reduction via quinol oxidases. A highly sensitive and reproducible experimental set-up with prolonged linear time intervals of up to 60 s is presented, which enables the determination of constant activity rates in E. coli membrane vesicles enriched in the quinol oxidase of interest by heterologous expression, using a Clark-type oxygen electrode to continuously follow O2 consumption. For the calculation of specific quinol oxidase activity, activity rates were correlated with quantitative signal intensity determinations of AOX1A present in a membrane-bound state by immunoblot analyses, simultaneously enabling normalization of specific activities between different AOX proteins. In summary, the method presented is a powerful tool to study specific activities of individual quinol oxidases, like the different AOX isoforms, and their corresponding mutants upon modification by addition of effectors/inhibitors, and thus to characterize their individual mode of posttranslational regulation in a membranous environment.
authors.affiliation: Division of Plant Physiology, Department of Biology/Chemistry, University of Osnabrueck, D-49069, Osnabrueck, Germany.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Selinski
authors.affiliation: Division of Plant Physiology, Department of Biology/Chemistry, University of Osnabrueck, D-49069, Osnabrueck, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Hartmann
authors.affiliation: Division of Plant Physiology, Department of Biology/Chemistry, University of Osnabrueck, D-49069, Osnabrueck, Germany.
authors.forename: Saskia
authors.initials: S
authors.lastname: Höfler
authors.affiliation: Division of Microbiology, Department of Biology/Chemistry, University of Osnabrueck, D-49069, Osnabrueck, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Deckers-Hebestreit
authors.affiliation: Division of Plant Physiology, Department of Biology/Chemistry, University of Osnabrueck, D-49069, Osnabrueck, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Scheibe
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003580:Cytochromes / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007527:Isoenzymes; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D000099188:Alternative Oxidase
chemical_list: D003580:Cytochromes; D003576:Electron Transport Complex IV; D007527:Isoenzymes; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010100:Oxygen; D010940:Plant Proteins; D000099188:Alternative Oxidase; C020094:duroquinol oxidase