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Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro
Author(s) -
Selinski Jennifer,
Hartmann Andreas,
Höfler Saskia,
DeckersHebestreit Gabriele,
Scheibe Renate
Publication year - 2016
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/ppl.12418
Subject(s) - alternative oxidase , post translational regulation , biochemistry , oxidase test , mutant , respiratory chain , arabidopsis thaliana , cytochrome , arabidopsis , heterologous expression , chemistry , biology , enzyme , gene , recombinant dna
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 O 2 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 O 2 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.