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

被引：0
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作者：
Baudry, Kevin [1 ,2 ,3 ]
Issakidis-Bourguet, Emmanuelle [1 ,2 ]
机构：
[1] Univ Evry, INRAE, Inst Plant Sci Paris Saclay IPS2, Univ Paris Saclay,CNRS, Gif Sur Yvette, France
[2] Univ Paris Cite, CNRS, INRAE, Inst Plant Sci Paris Saclay IPS2, Gif Sur Yvette, France
[3] Boyce Thompson Inst Plant Res, Ithaca, NY USA
来源：
BIO-PROTOCOL | 2023年 / 13卷 / 16期
关键词：
NADP-malate dehydrogenase; NADP-MDH; Enzyme activity assay; Thioredoxin; TRX; R script; Spectrophotometry;
D O I：
10.21769/BioProtoc.4769
中图分类号：
Q [生物科学];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
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.
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页数：13
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