A Semi-throughput Procedure for Assaying Plant NADP-malate Dehydrogenase Activity Using a Plate Reader

Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a redox regulated enzyme playing an important role in plant redox homeostasis. Leaf NADP-MDH activation level is considered a proxy for the chloroplast redox status. NADP-MDH enzyme activity is commonly assayed spectrophotometrically by f...

Descripción completa

Detalles Bibliográficos
Autores principales: Baudry, Kevin, Issakidis-Bourguet, Emmanuelle
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Bio-Protocol 2023
Materias:
Methods Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10450749/
https://www.ncbi.nlm.nih.gov/pubmed/37638298
http://dx.doi.org/10.21769/BioProtoc.4769
Descripción
Sumario:Chloroplast NADP-dependent malate dehydrogenase (NADP-MDH) is a redox regulated enzyme playing an important role in plant redox homeostasis. Leaf NADP-MDH activation level is considered a proxy for the chloroplast redox status. NADP-MDH enzyme activity is commonly assayed spectrophotometrically by following oxaloacetate-dependent NADPH oxidation at 340 nm. We have developed a plate-adapted protocol to monitor NADP-MDH activity allowing faster data production and lower reagent consumption compared to the classic cuvette format of a spectrophotometer. We provide a detailed procedure to assay NADP-MDH activity and measure the enzyme activation state in purified protein preparations or in leaf extracts. This protocol is provided together with a semi-automatized data analysis procedure using an R script.

Ejemplares similares