{"title":"利用平板阅读器测定植物nadp -苹果酸脱氢酶活性的半通量方法。","authors":"Kevin Baudry, Emmanuelle Issakidis-Bourguet","doi":"10.21769/BioProtoc.4769","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":8938,"journal":{"name":"Bio-protocol","volume":"13 16","pages":"e4769"},"PeriodicalIF":0.0000,"publicationDate":"2023-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/35/73/BioProtoc-13-16-4769.PMC10450749.pdf","citationCount":"0","resultStr":"{\"title\":\"A Semi-throughput Procedure for Assaying Plant NADP-malate Dehydrogenase Activity Using a Plate Reader.\",\"authors\":\"Kevin Baudry, Emmanuelle Issakidis-Bourguet\",\"doi\":\"10.21769/BioProtoc.4769\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>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.</p>\",\"PeriodicalId\":8938,\"journal\":{\"name\":\"Bio-protocol\",\"volume\":\"13 16\",\"pages\":\"e4769\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2023-08-20\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/35/73/BioProtoc-13-16-4769.PMC10450749.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Bio-protocol\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.21769/BioProtoc.4769\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Bio-protocol","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.21769/BioProtoc.4769","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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 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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