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Effects of Hydroxybenzoic Acids on Oxidation of Reduced Nicotinamide Adenine Dinucleotide by Enzymes from Tobacco Leaves
Author(s) -
Lee T. T.
Publication year - 1966
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1966.tb07050.x
Subject(s) - chemistry , hydroxybenzoic acid , peroxidase , enzyme , oxidase test , nicotiana tabacum , horseradish peroxidase , biochemistry , benzoic acid , organic chemistry , gene
Abstract Hydroxyhenzoic acids were tested for their effects on oxidation of the reduced nicotinamide adenine dinucleotide (NADH) in the absence of added H 2 O 2 and Mn 2 * by an enzyme preparation from tobacco leaves (Nicotiana tabacum, var. White Gold). For comparison, a commercial horseradish peroxidase was also used. The rate of NADH oxidation was followed spectruphotometrically at 340 nm. Mono‐ and dihydroxybenzoic acids exerted significant effect on the rate of NADU oxidation, yet their effectiveness was determined by the number and position of the hydroxyl group on the ring. 4‐Hydroxybenzoic acid was very effective in stimulating the reaction. Shifting the hydroxyl from the 4‐ to the 3‐position and from the 3‐ to the 2‐position decreased activity. 2,4‐ And 2,5‐dihydroxybenzoic aeids were more active than the other dihydroxy‐iscuners in stinulating oxidation of NADH. the dihydroxybenzoic acids with the hydroxyls in adjacent positions were less effective, and their activity was affected by other phenolic activators. In the presence of 4‐hydroxybenzoic acid which enhanced oxidation of NADH, 2,4‐ and 2,5‐dihydroxybenzoic acids further stimulated the reaction, but 3,4‐, 2,3‐ and 2,6‐dibydoxybenzoic acids were inhibitory. The inhibition by 3,4‐ and 2,3‐dihydroxybenzoic aciils was non‐competitive. The enzymes extracted by a L‐cysteine‐containing buffer showed lower NADH‐oxidase activity. The enzyme preparation possessed peroxidase activity. The activity of NADH‐oxidase inereased when H 2 O 2 and Mi 2 * were present in addition to 4‐hydroxy‐benzoic acid. The effect of the position and number of hydroxyl substitution on the rate of NADH oxidation by borseradish peroxidase was also significant. This suggests the involvement of peroxidase in the NADH‐oxidase system of tobacco leaves. However, a combination of the inactivated enzyme solution and active horseradish peroxidase with peroxidase activity equivalent to that of the enzyme preparation from tobacco leaves did not reconstitute the NADH‐oxidase activity of tobacco leaves. This and other evidence suggests that the soluble NADH‐oxidizing zyme system of tobacco leaves is more complicated than peroxidase.