Biochemical properties of lipoxygenase from opium poppy chloroplasts

Vanko, M.; Rauová, Drahomíra; Bezáková, Lýdia; Holková, Ivana; Bilka, František; Cupáková, M.

doi:10.1007/s10535-011-0207-3

Cited by 1 publication

(5 citation statements)

References 24 publications

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“…For affinity chromatography, LAPA (Sigma-Aldrich) was prepared by binding linoleic acid to ω-aminopropyl agarose using a water-soluble carbodiimide coupling reagent [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride], according to Vanko et al (2012). The LAPA column was eluted stepwise with 10 mM potassium phosphate buffer (pH 6.0), 0.3 M potassium phosphate buffer (pH 6.0), and 0.3 M potassium phosphate buffer (pH 8.5).…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…After removing the organic solvent, the residue was reconstituted in 0.2 mL of mobile phase (methanol/water/acetic acid, 85:15:0.1, v/v/v). Aliquots of 70 µL were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), according to Vanko et al (2012).…”

Section: Analysis Of Lox Reaction Productsmentioning

confidence: 99%

“…In the present work, one LOX enzyme was purified from the cytosolic fraction of opium poppy seedlings. In our previous work, we characterized a different LOX isoenzyme (Vanko et al, 2012). This isoenzyme was isolated from chloroplasts of opium poppy leaves and had 92 kDa due to the presence of N-terminal chloroplast transit peptide sequence (Vanko et al, 2012).…”

Section: Lox Activity During Germination Of Poppy Seeds and Enzyme Pumentioning

confidence: 99%

“…In our previous work, we characterized a different LOX isoenzyme (Vanko et al, 2012). This isoenzyme was isolated from chloroplasts of opium poppy leaves and had 92 kDa due to the presence of N-terminal chloroplast transit peptide sequence (Vanko et al, 2012). Most plant LOX have a relative molecular mass ranging from 94 to 104 kDa (Siedow, 1991).…”

Section: Lox Activity During Germination Of Poppy Seeds and Enzyme Pumentioning

confidence: 99%

“…The amount of total lipids and the distribution of free fatty acids and lipoxygenase activity were determined during poppy seedling development by Bezáková et al (1994). LOX isoenzyme from chloroplasts of opium poppy leaves was purified and characterized (Vanko et al, 2012). In our previous work, we studied the relationship between LOX activity and secondary metabolite biosynthesis after elicitor treatment in opium poppy cultures (Holková et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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Purification and properties of lipoxygenase from opium poppy seedlings(Papaver somniferum L.)

Holková¹,

Bilka²,

Rauová³

et al. 2016

Turk J Biol

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Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) is a key enzyme in the signaling pathway leading to the production of biologically active compounds involved in plant growth development and stress responses. In this study the novel LOX enzyme from opium poppy (Papaver somniferum L.) seedlings was purified to electrophoretic homogeneity and characterized. LOX activity during the germination of opium poppy seeds was analyzed, and the highest LOX activity was determined on the fourth day of germination. Opium poppy LOX was purified from 4-day-old poppy seedlings after ammonium sulfate precipitation followed by hydrophobic chromatography (Phenyl-Sepharose CL-4B), ion exchange chromatography (Q-Sepharose), and affinity chromatography (linoleyl-aminopropyl agarose) for the first time. The relative molecular weight of purified LOX was estimated to be 78 kDa by immunoblotting. The highest enzyme activity occurred at pH 6.5 and 0.75 mM calcium ion concentration. LOX showed preferential activity towards linoleic acid followed by linolenic acid as a substrate. To investigate the positional specificity of the LOX reaction, purified LOX was incubated with linoleic acid, and the products were analyzed by high-performance liquid chromatography. Opium poppy LOX converted linoleic acid to 13-hydroperoxy-(9Z,11E)-octadecadienoic acid (63.5%) and, to a lesser extent, 9-hydroperoxy-(10E,12Z)-octadecadienoic acid (36.5%).

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Section: Enzyme Purificationmentioning

confidence: 99%

Section: Analysis Of Lox Reaction Productsmentioning

confidence: 99%