Hydroxamate‐activated peroxidases in potato tuber callus. Interaction with the determination of the cytochrome and the alternative pathways

In potato (Solatium tuberosum L. cv. Bintje and Dore) callus a very active hydrox-amate-stimulated NADH-dependent O2-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 mM gives half-maximal stimulation. Maximal stimulation is observed with 1–3 mM benzhydroxamate (BHAM) and 1–15 mM 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 (Km near 10 mM). 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 O2-consuming and a H2O2-consuming reaction. In both reactions one NAD(P)H is consumed. In the first reaction H2O2 is formed which can be consumed in the second reaction, resulting in an overall stoichiometry of 2 NADH consumed for each O2 molecule and in the production of H2O. 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 O2-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 O2-uptake in the presence of exogenous NADH. In vivo the peroxidase does not appear to function in O2-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.

[1]  I. Møller,et al.  Salicylhydroxamic acid‐stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots , 1986 .

[2]  H. Lambers,et al.  Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements. , 1986, Plant physiology.

[3]  L. Plas,et al.  Compartmentation of Alternative Oxidase in Plant Mitochondria , 1986 .

[4]  I. Møller,et al.  Oxygen consumption by purified plasmalemma vesicles from wheat roots , 1985 .

[5]  F. L. Crane,et al.  Oxidation of reduced pyridine nucleotides by plasma membranes of soybean hypocotyl. , 1985, Biochemical and biophysical research communications.

[6]  J. Owens,et al.  Peroxidase Activity in Relation to Suberization and Respiration in White Spruce (Picea glauca [Moench] Voss) Seedling Roots. , 1985, Plant physiology.

[7]  K. Espelie,et al.  Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solanum tuberosum). , 1985, Archives of biochemistry and biophysics.

[8]  T. Le,et al.  Localization and properties of cell wall enzyme activities related to the final stages of lignin biosynthesis , 1985 .

[9]  C. Kevers,et al.  Soluble, Membrane and Wall Peroxidases, Phenylalanine Ammonia-lyase, and Lignin Changes in Relation to Vitrification of Carnation Tissues Cultured in vitro , 1985 .

[10]  M. J. Wagner,et al.  Influence of osmotic stress on the respiration of potato tuber callus , 1984 .

[11]  T. Blacquière,et al.  Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity. , 1984, Plant physiology.

[12]  K. Stich,et al.  Investigation of hydrogen peroxide formation in plants , 1984 .

[13]  J. L. Brooks.  Stimulation of peroxidase reactions by hydroxamates. , 1983, Biochemical and biophysical research communications.

[14]  M. J. Wagner,et al.  Regulation of the activity of the alternative oxidase in callus forming discs from potato tubers , 1983 .

[15]  L. Plas,et al.  Respiration rate and redox state of nad during wound-induced differentiation of potato tuber tissue (Solanum tuberosum L.) , 1982 .

[16]  L. H. van der Plas,et al.  Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs. , 1982, Plant physiology.

[17]  G. Laties.  The Cyanide-Resistant, Alternative Path in Higher Plant Respiration , 1982 .

[18]  M. J. Wagner,et al.  Influence of ethanol on alternative oxidase in mitochondria from callus‐forming potato tuber discs , 1980 .

[19]  F. G. Brinkman,et al.  Localization of Dehydrogenases in Peroxisomes and Mitochondria during Wound-induced Differentiation of Potato Tuber Tissue , 1979 .

[20]  H. Blum,et al.  Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids. , 1978, Biochimica et biophysica acta.

[21]  J. T. Bahr,et al.  Cyanide-insensitive respiration. I. The steady states of skunk cabbage spadix and bean hypocotyl mitochondria. , 1973, The Journal of biological chemistry.

[22]  B. T. Storey,et al.  Specific inhibition of the cyanide-insensitive respiratory pathway in plant mitochondria by hydroxamic acids. , 1971, Plant physiology.