Abstract
Opium poppy (Papaver somniferum L.) is an ancient medicinal plant producing pharmaceutically important benzylisoquinoline alkaloids. In the present work we focused on the study of enzyme lipoxygenase (LOX, EC 1.13.11.12) from opium poppy cultures. LOX is involved in lipid peroxidation and lipoxygenase oxidation products of polyunsaturated fatty acids have a significant role in regulation of growth, development and plant defense responses to biotic or abiotic stress. The purpose of this study was to isolate and characterize LOX enzyme from opium poppy callus cultures. LOX was purified by ammonium sulfate precipitation and then followed by hydrophobic chromatography using Phenyl-Sepharose CL-4B and hydroxyapatite chromatography using HA Ultrogel sorbent. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting revealed that LOX from opium poppy cultures was a single monomeric protein showing the relative molecular weight of 83 kDa. 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 in two steps, firstly with reverse phase (120-5 Nucleosil C18 column) and secondly with normal phase (Zorbax Rx-SIL column). LOX converted linoleic acid primarily to 13-hydroperoxy-(9Z,11E)-octadecadienoic acids (78%) and to a lesser extent 9-hydroperoxy-(10E,12Z)-octadecadienoic acids (22%). Characterization of LOX from opium poppy cultures provided valuable information in understanding LOX involvement in regulation of signaling pathways leading to biosynthesis of secondary metabolites with significant biological activity.
Highlights
Opium poppy, Papaver somniferum L., is one of the world’s oldest medicinal plants producing valuable benzylisoquinoline alkaloids (BIAs)
[1].isConsidering the importance in signaling andofpossible in regulation signaling processes and possible regulation of alkaloid biosynthesis we from focused on purification of of alkaloid biosynthesis we focused on purification of LOX
LOX enzyme was purified from opium poppy (Papaver somniferum L.) callus cultures using material was fractionated into a 100,000× g soluble fraction and a membrane fraction
Summary
Papaver somniferum L., is one of the world’s oldest medicinal plants producing valuable benzylisoquinoline alkaloids (BIAs). It remains the only commercial source for the narcotic analgesics; morphine, codeine, and semi-synthetic derivatives such as oxycodone and naltrexone [1]. The content of morphine alkaloids or benzo[c]phenanthridine alkaloid sanguinarine, with anti-microbial and potential anti-cancer properties, has made opium poppy one of the most valuable plants in the pharmaceutical industry. Lipoxygenases (LOXs, linoleate:oxygen oxidoreductases, EC 1.13.11.12) belong to a class of fatty acid dioxygenases occurring both in the plant and animal kingdom. Arachidonic acid (AA, 20:4) is a preferred substrate for animal LOX enzymes.
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