Fluorimetric assay of tobacco leaf dehydrogenases with resazurin.
A versatile fluorimetric assay based on the reduction of resazurin to resorufin demonstrated high specific activities for a number of important pyridine nucleotide-linked dehydrogenases in tobacco leaves. The Michaelis constant for the important photosynthetic enzyme, D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (EC 1.2.1.13), determined by the fluorimetric method, was considerably lower than constants determined by conventional extraction and assay methods reported for the enzyme from other plants. The sensitivity of the fluorimetric method enabled the use of dilute enzyme preparations with resultant low background and high substrate specificity. Inclusion of the anti-oxidant diethyldithiocarbamate in the extraction medium preserved the enzymes during extraction. Primary amines inhibited competitively, and phenazine methosulfate non-competitively each of the eight dehydrogenases tested with the fluorimetric assay. The Mn2+ dependence of NADP-linked dehydrogenases specific for isocitrate and malate was confirmed. The method is rapid, requires a simple combination of ingredients and should be useful for surveying dehydrogenase activity in leaves.
D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis; D005456:Fluorescent Dyes; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000032:analysis; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000032:analysis; D008345:Manganese / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis* / Q000037:antagonists & inhibitors; D010078:Oxazines; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis; D010947:Plants, Toxic; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014026:Nicotiana / Q000201:enzymology*; D014325:Tromethamine / Q000494:pharmacology; D014966:Xanthenes
D005456:Fluorescent Dyes; D010078:Oxazines; D014966:Xanthenes; D014325:Tromethamine; C005843:resazurin; D008345:Manganese; D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase; D010734:Phosphogluconate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase