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. 1982 Jan;69(1):72–76. doi: 10.1104/pp.69.1.72

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

Yoke Wah Kow 1,2, Douglas A Smyth 1,3, Martin Gibbs 1
PMCID: PMC426148  PMID: 16662188

Abstract

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, α,α′-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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AKAZAWA T., CONN E. E. The oxidation of reduced pyridine nucleotides by peroxidase. J Biol Chem. 1958 May;232(1):403–415. [PubMed] [Google Scholar]
  2. Anderson L. E., Duggan J. X. Light modulation of glucose-6-phosphate dehydrogenase: partial characterization of the light inactivation system and its effects on the properties of the chloroplastic and cytoplasmic forms of the enzyme. Plant Physiol. 1976 Aug;58(2):135–139. doi: 10.1104/pp.58.2.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arnon D. I. COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. Plant Physiol. 1949 Jan;24(1):1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beevers H. The Oxidation of Reduced Diphosphopyridine Nucleotide by an Ascorbate System from Cucumber. Plant Physiol. 1954 May;29(3):265–269. doi: 10.1104/pp.29.3.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Groden D., Beck E. H2O2 destruction by ascorbate-dependent systems from chloroplasts. Biochim Biophys Acta. 1979 Jun 5;546(3):426–435. doi: 10.1016/0005-2728(79)90078-1. [DOI] [PubMed] [Google Scholar]
  8. Heldt W. H., Werdan K., Milovancev M., Geller G. Alkalization of the chloroplast stroma caused by light-dependent proton flux into the thylakoid space. Biochim Biophys Acta. 1973 Aug 31;314(2):224–241. doi: 10.1016/0005-2728(73)90137-0. [DOI] [PubMed] [Google Scholar]
  9. Kachru R. B., Anderson L. E. Inactivation of pea leaf phosphofructokinase by light and dithiothreitol. Plant Physiol. 1975 Feb;55(2):199–202. doi: 10.1104/pp.55.2.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaiser W. The effect of hydrogen peroxide on CO2 fixation of isolated intact chloroplasts. Biochim Biophys Acta. 1976 Sep 13;440(3):476–482. doi: 10.1016/0005-2728(76)90035-9. [DOI] [PubMed] [Google Scholar]
  11. Kelly G. J., Latzko E. Soluble ascorbate peroxidase: detection in plants and use in vitamim C estimation. Naturwissenschaften. 1979 Dec;66(12):617–619. doi: 10.1007/BF00405128. [DOI] [PubMed] [Google Scholar]
  12. Nishimura M., Graham D., Akazawa T. Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts. Plant Physiol. 1976 Sep;58(3):309–314. doi: 10.1104/pp.58.3.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Robinson J. M., Smith M. G., Gibbs M. Influence of Hydrogen Peroxide upon Carbon Dioxide Photoassimilation in the Spinach Chloroplast: I. HYDROGEN PEROXIDE GENERATED BY BROKEN CHLOROPLASTS IN AN "INTACT" CHLOROPLAST PREPARATION IS A CAUSAL AGENT OF THE WARBURG EFFECT. Plant Physiol. 1980 Apr;65(4):755–759. doi: 10.1104/pp.65.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shigeoka S., Nakano Y., Kitaoka S. Metabolism of hydrogen peroxide in Euglena gracilis Z by L-ascorbic acid peroxidase. Biochem J. 1980 Jan 15;186(1):377–380. doi: 10.1042/bj1860377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shigeoka S., Nakano Y., Kitaoka S. Purification and some properties of L-ascorbic-acid-specific peroxidase in Euglena gracilis Z. Arch Biochem Biophys. 1980 Apr 15;201(1):121–127. doi: 10.1016/0003-9861(80)90495-6. [DOI] [PubMed] [Google Scholar]
  16. Spalding M. H., Edwards G. E. Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum. Plant Physiol. 1980 Jun;65(6):1044–1048. doi: 10.1104/pp.65.6.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]

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