Plant Physiol. Drug Metab Dispos
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Plant Physiology 69:72-76 (1982)
© 1982 American Society of Plant Biologists

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Oxidation of Reduced Pyridine Nucleotide by a System Using Ascorbate and Hydrogen Peroxide from Plants and Algae 1

Yoke Wah Kow2, Douglas A. Smyth3 and Martin Gibbs

Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

A NAD(P)H oxidizing system (NAAP) was detected and partially purified from leaves of spinach and Sedum praealtum, seeds and leaves of pea and cells of green and red algae which oxidized NAD(P)H in the presence of ascorbate and H2O2.

The partially-purified spinach system had substrate Km values of 5 micromolar for NADH, 50 micromolar for H2O2, and 300 micromolar for L-ascorbic acid at the pH optimum of 6.8. NADH was a better electron donor than NADPH. Among other electron donors, isoascorbic acid had considerable activity but hydroquinone and resorcinol had only weak activities. The enzyme was inhibited by cyanide, {alpha},{alpha}'-dipyridyl, and mono-and di-thiol reagents. Inhibition by thiol-reagents was partially restored by Fe2+ as was enzymic activity lost following dialysis against buffer.

Subcellular localization studies with spinach and S. praealtum leaves indicated that a portion of the cell's NAAP was in the chloroplast fraction. Photosynthetic conditions resulted in a decrease in this activity solubilized from spinach and S. praealtum chloroplasts. The presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or Fe2+ in the incubation medium eliminated the light-mediated inhibition of NAAP.

NAAP may function in the recycling of NAD(P)H generated in the dark within the chloroplast. Inasmuch as all preparations of NAAP contained ascorbate peroxidase activity, the data do not rule out the possibility that NAAP is the same protein as ascorbate peroxidase or, alternatively, a combination of ascorbate peroxidase and some other enzyme.


2 Present address: Department of Biochemistry, University of Wisconsin, Madison, WI 53706.

3 Present address: Department of Agronomy and Soils, Washington State University, Pullman, WA 99164.

1 Supported by National Science Foundation Grant PCM 79-22612 and Department of Energy Grant EY-76-S-3231-14.







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Copyright © 1982 by the American Society of Plant Biologists