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Biochemical properties of lipoxygenase from opium poppy chloroplasts

  • Published:
Biologia Plantarum

Abstract

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (KM = 1.78 mM and Vmax = 11.4 μmol mg−1 min−1) and linolenic acid (KM = 1.27 mM and Vmax = 10.2 μmol mg−1 min−1). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg2+ and Ca2+.

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Abbreviations

GrB:

grinding buffer

HOD:

hydroxyoctadecadienoic acid

HPOD:

hydroperoxyoctadecadienoic acid

KPB:

potassium phosphate buffer

LA:

linoleic acid

LAPA:

linoleyl-aminopropyl agarose

LeA:

linolenic acid

LOD:

limit of detection

LOQ:

limit of quantification

LOX:

lipoxygenase

PMSF:

phenylmethylsulfonyl fluoride

RP HPLC:

reversed-phase highperformance liquid chromatography

SP HPLC:

straight-phase high-performance liquid chromatography

TX-114 :

Triton X-114

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Acknowledgements

This work was supported by grant of Slovak ministry of education VEGA 1/4294/07 and VEGA 1/0089/10.

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Authors and Affiliations

  1. Department of Cellular and Molecular Biology of Drugs, Comenius University, Faculty of Pharmacy, Kalinčiakova 8, 83232, Bratislava, Slovak Republic

    M. Vanko, L. Bezáková, I. Holková & F. Bilka

  2. Toxicological and Antidoping Center, Comenius University, Faculty of Pharmacy, Odbojárov 10, 83232, Bratislava, Slovak Republic

    D. Rauová & M. Cupáková

Authors
  1. M. Vanko
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  2. D. Rauová
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  3. L. Bezáková
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  4. I. Holková
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  5. F. Bilka
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  6. M. Cupáková
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Correspondence to L. Bezáková.

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Vanko, M., Rauová, D., Bezáková, L. et al. Biochemical properties of lipoxygenase from opium poppy chloroplasts. Biol Plant 56, 105–110 (2012). https://doi.org/10.1007/s10535-012-0023-4

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  • DOI: https://doi.org/10.1007/s10535-012-0023-4

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