Premium
Hydroxamate‐activated peroxidases in potato tuber callus. Interaction with the determination of the cytochrome and the alternative pathways
Author(s) -
Plas Linus H. W.,
Gude Henk,
Wagner Marijke J.
Publication year - 1987
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.1399-3054.1987.tb08693.x
Subject(s) - peroxidase , nad+ kinase , sephadex , callus , chemistry , stimulation , enzyme , biochemistry , stereochemistry , redox , biology , organic chemistry , botany , neuroscience
In potato ( Solatium tuberosum L. cv. Bintje and Doré) callus a very active hydrox‐amate‐stimulated NADH‐dependent O 2 ‐uptake develops during the growth of the callus, which is caused by a peroxidase. More than 95% of the peroxidase activity is found in the 40000 g supernatant. The total activity may be as high as 1000 times the respiratory acitivity of the callus tissue. At least two fractions, obtained by Sephadex gel filtration, can be distinguished showing this peroxidase activity, one of about 15 kDa and one > 50 kDa. The main properties of both fractions are: a) Hydroxamate at 0.2–0.5 m M gives half‐maximal stimulation. Maximal stimulation is observed with 1–3 m M benzhydroxamate (BHAM) and 1–15 m M salicylhydroxamate (SHAM). Higher concentrations, especially of BHAM, give less or no stimulation. b) Hydroxamates are not consumed during the reaction. c) Both NADH and NADPH can serve as the electron donor for the reaction. The affinity for NAD(P)H is very low (K m near 10 m M ). In the absence of hydroxamates NAD(P)H is only slowly oxidized, with an even lower affinity. d) The peroxidase can carry out two reactions: an O 2 ‐consuming and a H 2 O 2 ‐consuming reaction. In both reactions one NAD(P)H is consumed. In the first reaction H 2 O 2 is formed which can be consumed in the second reaction, resulting in an overall stoichiometry of 2 NADH consumed for each O 2 molecule and in the production of H 2 O. e) The reaction is completely blocked by cyanide, superoxide dismutase (EC 1.15.1.1) and (excess) catalase (EC 1.11.1.6), but not by antimycin A or azide. This peroxidase‐mediated O 2 ‐uptake might interfere with respiratory measurements. In experiments with isolated mitochondria this interference can be prevented by the addition of catalase to the reaction mixture. The use of high concentrations of hydroxamate is not allowed because of inhibitory effects on the cytochrome pathway. In intact callus tissue hydroxamates only stimulate O 2 ‐uptake in the presence of exogenous NADH. In vivo the peroxidase does not appear to function in O 2 ‐uptake, probably because of its localization (at least partly in the cell wall) and/or its low affinity for NADH. The use of hydroxamates in the determination of cytochrome and alternative pathway activity is discussed.