Skip to main content
SpringerLink
Log in

Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves

  • Published:
Protoplasma Aims and scope Submit manuscript

Summary

The NAD(P)H:quinone oxidoreductase activity of tobacco leaves is catalyzed by a soluble flavoprotein [NAD(P)H-QR] and membrane-bound forms of the same enzyme. In particular, the activity associated with the plasma membrane cannot be released by hypoosmotic and salt washing of the vesicles, suggesting a specific binding. The products of the plasma-membrane-bound quinone reductase activity are fully reduced hydroquinones rather than semi-quinone radicals. This peculiar kinetic property is common with soluble NAD(P)H-QR, plasma-membrane-bound NAD(P)H:quinone reductase purified from onion roots, and animal DT-diaphorase. These and previous results demonstrate that soluble and plasma-membrane-bound NAD(P)H:quinone reductases are strictly related flavo-dehydrogenases which seem to replace DT-diaphorase in plant tissues. Following purification to homogeneity, the soluble NAD(P)H-QR from tobacco leaves was digested. Nine peptides were sequenced, accounting for about 50% of NAD(P)H-QR amino acid sequence. Although one peptide was found homologous to animal DT-diaphorase and another one to plant monodehydroascorbate reductase, native NAD(P)H-QR does not seem to be structurally similar to any known flavoprotein.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

MDAR:

monodehydroascorbate reductase

PM:

plasma membrane

NAD(P)H-QR:

NAD(P)H:quinone oxidoreductase

DPI:

diphenylene iodonium

DQ:

duroquinone

CoQ2 :

coenzyme Q2

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local aligment search tool. J Mol Biol 215: 403–410

    Google Scholar 

  • Asard H, Horemans N, Caubergs RJ (1995) Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions. Protoplasma 184: 36–41

    Google Scholar 

  • Bagnaresi P, Basso B, Pupilo P (1997) The NADH Fe3+-chelate reductases of tomato roots. Planta 202: 427–434

    Google Scholar 

  • Bérczi A, Møller IM (1998) NADH-monodehydroascorbate oxidoreductase is one of the redox enzymes in spinach leaf plasma membranes. Plant Physiol 116: 1029–1036

    Google Scholar 

  • —, Fredlund KM, Møller IM (1995) Purification and characterization of an NADH hexacyanoferrate(III) reductase from spinach leaf plasma membrane. Arch Biochem Biophys 320: 65–72

    Google Scholar 

  • Besandoun A, Weinstein D (1976) Assay of proteins in the presence of interfering materials. Anal Biochem 70: 241–250

    Google Scholar 

  • Beyer RE, Segura-Aguilar J, Di Bernardo S, Cavazzoni M, Fato R, Fiorentini D, Galli MC, Setti M, Landi L, Lenaz G (1996) The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc Natl Acad Sci USA 93: 2528–2532

    Google Scholar 

  • Brock BJ, Rieble S, Gold MH (1995) Purification and characterization of a 1,4-benzoquinone reductase from the basidiomycetePhanaerochete chrysosporium. Appl Environ Microbiol 61: 3076–3081

    Google Scholar 

  • Campbell WH (1996) Nitrate reductase biochemistry comes of age. Plant Physiol 111: 355–361

    Google Scholar 

  • Do TQ, Schultz JR, Clarke CF (1996) Enhanced sensitivity of ubiquinone-deficient mutants ofSaccharomyces cerevisiae to products of autoxidized polyunsaturated fatty acids. Proc Natl Acad Sci USA 93: 7534–7539

    Google Scholar 

  • Ernster L (1987) DT-diaphorase: a historical review. Chem Scr 27A: 1–13

    Google Scholar 

  • Fredlund KM, Struglics A, Widell S, Askerlund P, Kader JC, Møller IM (1994) Comparison of the stereospecificity and immunoreactivity of NADH-ferricyanide reductases in plant membranes. Plant Physiol 106: 1103–1106

    Google Scholar 

  • Fujiara M, Ishida Y, Nimura N, Toyama A, Kinoshita T (1987) Post-column fluorometric detection system for liquid Chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent. Anal Biochem 16: 672–678

    Google Scholar 

  • Guerrini F, Lombini A, Bizarri M, Pupillo P (1994) The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells. J Exp Bot 45: 1227–1233

    Google Scholar 

  • Iyanagi T (1987) On the mechanism of one- and two-electron transfer by flavin enzymes. Chem Scr 27A: 31–36

    Google Scholar 

  • —, Yamazaki I (1970) One-electron transfer reactions in biochemical systems V: difference in the mechanism of quinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase). Biochim Biophys Acta 216: 282–294

    Google Scholar 

  • Kunze M, Riedel J, Lange V, Hurwitz R, Tischner R (1997) Evidence for the presence of GPI-anchored PM-NR in leaves ofBeta vulgaris and for PM-NR in barley leaves. Plant Physiol Biochem 35: 507–512

    Google Scholar 

  • Larsson C, Sommarin M, Widell S (1994) Isolation of highly purified plant plasma membranes and separation of inside-out and right-side out vesicles. Methods Enzymol 228: 451–469

    Google Scholar 

  • Li R, Bianchet MA, Talalay P, Amzel LM (1995) The three dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. Proc Natl Acad Sci USA 92: 8846–8850

    Google Scholar 

  • Luster DG, Buckhout TJ (1989) Purification and identification of a plasma membrane associated electron transport protein from maize (Zea mays) roots. Plant Physiol 91: 1014–1019

    Google Scholar 

  • Lüthje S, Döring O, Heuer S, Luethen H, Böttger M (1997) Oxidoreductases in plant plasma membranes. Biochim Biophys Acta 1331: 81–102

    Google Scholar 

  • Matsudaira P (1993) A practical guide to protein and peptide purification for microsequencing. Academic Press, New York

    Google Scholar 

  • Prochaska HJ, De Long MJ, Talalay P (1985) On the mechanism of induction of cancer protective enzymes: a unifying proposal. Proc Natl Acad Sci USA 82: 8232–8236

    Google Scholar 

  • Quail PH (1979) Plant cell fractionation. Annu Rev Plant Physiol 30: 425–484

    Google Scholar 

  • Rescigno A, Sollai F, Masala S, Porcu MC, Sanjust E, Rinaldi AC, Curreli N, Grifi D, Rinaldi A (1995) Purification and characterization of an NAD(P)H:quinone oxidoreductase fromGlycine max seedlings. Prep Biochem 25: 57–67

    Google Scholar 

  • Serrano A, Cordoba F, Gonzales-Reyes JA, Navas P, Villalba JM (1994) Purification and characterization of two distinct NAD(P)H dehydrogenases from onion (Allium cepa L.) root plasma membrane. Plant Physiol 106: 87–96

    Google Scholar 

  • Siegel D, Bolton EM, Burr JA, Liebler DC, Ross D (1997) The reduction of α-tocopherolquinone by human NAD(P)H:quinone oxidoreductase: the role of α-tocopherolhydroquinone as a cellular antioxidant. Mol Pharmacol 52: 300–305

    Google Scholar 

  • Sparia F, Tedeschi G, Trost P (1996) NAD(P)H:(quinone-acceptor) oxidoreductase of tobacco leaves is an FMN containing flavoenzyme. Plant Physiol 112: 249–258

    Google Scholar 

  • —, Bagnaresi P, Scagliarini S, Trost P (1997) NADH.Fe(III)-chelate reductase of maize roots is an active cytochromeb 5 reductase. FEBS Lett 414: 571–575

    Google Scholar 

  • Stöhr C, Tischner R, Ward MR (1993) Characterization of the plasma membrane bound nitrate reductase inChlorella saccharophila (Krüger) Nadson. Planta 191: 79–85

    Google Scholar 

  • Trost P, Bonora P, Scagliarini S, Pupillo P (1995) Purification and properties of NAD(P)H:(quinone-acceptor) oxidoreductase of sugarbeet cells. Eur J Biochem 234: 452–458

    Google Scholar 

  • —, Foscarini S, Preger V, Bonora P, Vitale L, Pupillo P (1997) Dissecting the diphenylene iodonium-sensitive NAD(P)H:quinone oxidoreductase ofCucurbita plasma membrane. Plant Physiol 114: 737–746

    Google Scholar 

  • Valenti V, Guerrini F, Pupillo P (1990) NAD(P)H-duroquinone reductase in the plant plasma membrane. J Bot 223: 183–192

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, I-40126, Bologna, Italy

    F. Sparia, P. Pupillo & P. Trost

  2. Institute of Veterinary Biochemistry, University of Milano, Milano

    G. Tedeschi

Authors
  1. F. Sparia
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. G. Tedeschi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. Pupillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. Trost
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sparia, F., Tedeschi, G., Pupillo, P. et al. Molecular characterization of NAD(P)H:quinone oxidoreductase of tobacco leaves. Protoplasma 205, 52–58 (1998). https://doi.org/10.1007/BF01279293

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01279293

Keywords

Search

Navigation