Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro

DC FieldValueLanguage
dc.contributor.authorSelinski, Jennifer
dc.contributor.authorHartmann, Andreas
dc.contributor.authorHoefler, Saskia
dc.contributor.authorDeckers-Hebestreit, Gabriele
dc.contributor.authorScheibe, Renate
dc.date.accessioned2021-12-23T15:57:39Z-
dc.date.available2021-12-23T15:57:39Z-
dc.date.issued2016
dc.identifier.issn00319317
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/3047-
dc.description.abstractIn 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 60s 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.
dc.description.sponsorshipDeutsche ForschungsgemeinschaftGerman Research Foundation (DFG); Collaborative Research Center 944 `Physiology and dynamics of cellular microcompartments'; government of Lower Saxonia; Osnabrueck University; We thank Dr Peter Graumann (Marburg) for kindly providing plasmid pSG1164-Venus, Elisabeth Becker, Britta Ballhausen and Marco Kruger for aid in generating plasmids and/or strains and Brigitte Herkenhoff-Hesselmann for expert technical assistance. We greatly acknowledge support from the Deutsche Forschungsgemeinschaft (G. D.-H.) and from the Collaborative Research Center 944 `Physiology and dynamics of cellular microcompartments' (R. S. and G. D.-H.). J. S. thanks the government of Lower Saxonia (Lichtenberg fellowship) and the Osnabrueck University for Frauenforderpool for research funds.
dc.language.isoen
dc.publisherWILEY
dc.relation.ispartofPHYSIOLOGIA PLANTARUM
dc.subjectCYANIDE-RESISTANT
dc.subjectCYTOCHROME-OXIDASE
dc.subjectESCHERICHIA-COLI
dc.subjectEXPRESSION
dc.subjectGENES
dc.subjectINSIGHTS
dc.subjectOXIDOREDUCTASE
dc.subjectPlant Sciences
dc.subjectPLANT-MITOCHONDRIA
dc.subjectPROTEIN
dc.subjectRESPIRATION
dc.titleRefined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro
dc.typejournal article
dc.identifier.doi10.1111/ppl.12418
dc.identifier.isiISI:000379260400002
dc.description.volume157
dc.description.issue3, SI
dc.description.startpage264
dc.description.endpage279
dc.contributor.orcid0000-0002-1247-7282
dc.identifier.eissn13993054
dc.publisher.place111 RIVER ST, HOBOKEN 07030-5774, NJ USA
dcterms.isPartOf.abbreviationPhysiol. Plant.
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.deptidfb05-
crisitem.author.orcid0000-0002-6140-6181-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidDeGa700-
crisitem.author.netidScRe288-
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