Ivana HOLKOVA, Frantısek BILKA, Drohomira RAUOVA, Lydia BEZAKOVA

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

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%).

PDF

___

ReferencesAndreou A, Feussner I (2009). Lipoxygenasesstructure and reaction mechanism. Phytochemistry 70: 1504-1510.
Babitha MP, Prakash HS, Shetty HS (2004). Purification and properties of lipoxygenase induced in downy mildew resistant pearl millet seedlings due to infection with Sclerospora graminicola. Plant Sci 166: 31-39.
Beaudoin GAW, Facchini PJ (2014). Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240: 19-32.
Bezáková L, Mistrík I, Kovács P, Penák M (1994). Lipids,fatty acidsand lipoxygenase activityindeveloping poppy seedling, Papaver somniferum L. Biologia 49: 339-345
Bowsher CG, Ferrie BJ, Ghosh S, Todd J, Thompson JE, Rothstein SJ (1992). Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit. Plant Physiol 100: 1802-1807.
Bradford MM (1976). Arapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
Braidot E, Micolini S, Risso A, Macrì F, Vianello A (2003). Immunochemical evidence for the presence of a lipoxygenase 1 in isolated plasma membranes from soybean cotyledons. Plant Sci 164: 9-16.
Chen AO, Whitaker JR (1986). Purification and characterization of a lipoxygenase from immature English peas. J Agric Food Chem 34: 203-211.
Cho K, Han Y, Woo JC, Baudisch B, Klösgen RB, Oh S, Han J, Han O (2011). Cellular localization of dual positional specific maize lipoxygenase-1 in transgenic rice and calcium-mediated membrane association. Plant Sci 181: 242-248.
Christensen SA, Kolomiets MV (2011). The lipid language of plant-fungal interactions. Fungal Genet Biol 48: 4-14.
Daglia M, Aceti C, Giorgetti S, Papetti A, Gazzani G (2005). Purification and characterization of soluble Cichorium intybus var. silvestre lipoxygenase. J Agric Food Chem 53: 6448-6454.
Farmaki T, Sanmartín M, Jiménez P, Paneque M, Sanz C, Vancanneyt LJ, Sánchez-Serrano J (2007). Differential distribution of the lipoxygenase pathway enzymes within potato chloroplasts. J Exp Bot 58: 555-568.
Feussner I, Kühn H, Wasternack C (2001). Lipoxygenase-dependent degradation of storage lipids. Trends Plant Sci 6: 268-273.
Feussner I, Wasternack C (2002). The lipoxygenase pathway. Annu Rev Plant Biol 53: 275-297.
Fornaroli S, Petrussa E, Braidot E, Vianello A, Macrì F (1999). Purification of a plasma membrane-bound lipoxygenase from soybean cotyledons. Plant Sci 145: 1-10.
Gardner HW (1989). Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism. Biochim Biophys Acta 1001: 274-281.
Gerhardt B, Fischer K, Balkenhohl TJ, Pohnert G, Kühn H, Wasternack C, Feussner I (2005). Lipoxygenase-mediated metabolism of storage lipids in germinating sunflower cotyledons and β-oxidation of (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid by the cotyledonary glyoxysomes. Planta 220: 919-930.
Hilbers MP, Kerkhoff B, Finazzi-Agrò A, Veldink GA, Vliegenthart JFG (1995). Heterogeneity and developmental changes of lipoxygenase in etiolated lentil seedlings. Plant Sci 111: 169-180.
Holková I, Bezáková L, Bilka F, Balažová A, Vanko M, Blanáriková V (2010). Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures. Plant Physiol Biochem 48: 887-892.
Huang FC, Kutchan TM (2000). Distribution of morphinan and benzo[c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum. Phytochemistry 53: 555-564.
Jacobo-Velázquez DA, Hernández-Brenes C, Cisneros-Zevallos L, Benavides J (2010). Partial purification and enzymatic characterization of avocado (Persea americana Mill, cv. Hass) lipoxygenase. Food Res Int 43: 1079-1085.
Kim ES, Choi E, Kim Y, Cho K, Lee A, Shim J, Rakwal L, Agrawal GK, Han O (2003). Dual positional specificity and expression of non-traditional lipoxygenase induced by wounding and methyl jasmonate in maize seedlings. Plant Mol Biol 52: 1203-1213.
Kuo JM, Hwang A, Yeh DB, Pan MH, Tsai ML, Pan BS (2006). Lipoxygenase from banana leaf: purification and characterization of an enzyme that catalyzes linoleic acid oxygenation at the 9-position. J Agric Food Chem 54: 3151-3156.
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. Liavonchanka A, Feussner I (2006). Lipoxygenases: occurrence, functions and catalysis. J Plant Physiol 163: 348-357.
Liptáková Ľ, Huttová J, Mistrík I, Tamás L (2013). Enhanced lipoxygenase activity is involved in the stress response but not in the harmful lipid peroxidation and cell death of short-term cadmium-treated barley. J Plant Physiol 170: 646-652.
Lorenzi V, Maury J, Casanova J, Berti L (2006). Purification, product characterization and kinetic properties of lipoxygenase from olive fruit (Olea europaea L.). Plant Physiol Biochem 44: 450-454.
Mishra S, Triptahi V, Singh S, Phukan UJ, Gupta MM, Shanker K, Shukla RK (2013). Wound induced transcriptional regulation of benzylisoquinoline pathway and characterization of wound inducible PsWRKY transcription factor from Papaver somniferum.
PLoS ONE 8: e52784. doi:10.1371/journal.pone.0052784. Mosblech A, Feussner I, Heilmann I (2009). Oxylipins: structurally diverse metabolites from fatty acid oxidation. Plant Physiol Biochem 47: 511-517.
Porta H, Rocha-Sosa M (2002).Plant lipoxygenases. Physiological and molecular features. Plant Physiol 130: 15-21.Ritchie RJ, Prvan T (1996). Current statistical methods for estimating the Km and Vmax of Michaelis-Menten kinetics. Biochem Educ 24: 196-206.
Sellhorn GE, Youn B, Webb BN, Gloss LM, Kang CH, Grimes HD (2011). Biochemical characterization, kinetic analysis and molecular modeling of recombinant vegetative lipoxygenases from soybean. Int J Biol 3: 44-62.
Siedow JN (1991). Plant lipoxygenase: structure and function. Annu Rev Plant Physiol Plant Mol Biol 42: 145-188.Suurmeijer CNSP, Pérez-Gilabert M, van der Hijden HTWM, Veldink GA, Vliegenthart JFG (1998). Purification, product characterization and kinetic properties of soluble tomato lipoxygenase. Plant Physiol Biochem 36: 657-663.
Szymanowska U, Jakubczyk A, Baraniak B, Kur A (2009). Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.). Food Chem 116: 906-910.
Tatulian SA, Steczko J, Minor W (1998). Uncovering a calcium-regulated membrane-binding mechanism for soybean lipoxygenase-1. Biochemistry 37: 15481-15490.
Vanko M, Rauová D, Bezáková L, Holková I, Bilka F, Cupáková M (2012). Biochemical properties of lipoxygenase from opium poppy chloroplasts. Biol Plantarum 56: 105-110.
Wang R, Shen W, Liu L, Jiang L, Liu Y, Su N, Wan J (2008). A novel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack. Plant Mol Biol 66: 401-414.
Yadav MK, Bhatla SC (2011). Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method. Plant Physiol Biochem 49: 230-234.
Zichová M, vajdlenka E, Penák M (1996). The content and distribution of precursors and alkaloids in developing poppy seedlings. Chem Listy 90: 687-688.
Ziegler J, Facchini PJ, Geissler R, Schmidt J, Ammer C, Kramell R, Voigtländer S, Gesell A, Pienkny S, Brandt W (2009). Evolution of morphine biosynthesis in opium poppy. Phytochemistry 70: 1696-1707.