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Lipoxygenase-2 oxygenates storage lipids in embryos of germinating barley

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Author: Holtman, W.L. · Vredenbregt-Heistek, J.C. · Schmitt, N.F. · Feussner, I.
Type:article
Date:1997
Institution: Centraal Instituut voor Voedingsonderzoek TNO TNO Voeding
Source:European Journal of Biochemistry, 248, 452-458
Identifier: 56381
Keywords: Barley · Lipoxygenase isoenzyme · Oxygenation · Storage lipid · Isoenzyme · Lipoxygenase · Enzyme activity · Enzyme specificity · Germination · High performance liquid chromatography · Lipid oxidation · Phytochemistry · Priority journal · Chromatography, High Pressure Liquid · Germination · Hordeum · Isoenzymes · Kinetics · Lipid Metabolism · Lipids · Lipoxygenase · Substrate Specificity · Hordeum vulgare subsp. vulgare

Abstract

Besides the pre-existing lipoxygenase (LOX-1) present in quiescent grains, a new lipoxygenase (LOX-2) is induced in embryos of germinating barley [Holtman, W. L., Van Duijn, G., Sedee, N. J. A. and Douma, A. C. (1996) Plant Physiol. 111, 569-576]. The fact that LOX-1 and LOX-2 form different products after incubation with linoleic acid, the (9S)- and (13S)-hydroperoxides, respectively [Van Aarle, P. G. M., De Barse, M. M. J., Veldink, G. A. and Vliegenthart, J. E G. (1991) FEES Lett. 280, 159-162; Doderer, A., Kokkelink, I., Van der Veen, S., Valk, B. E., Schram, A. W. and Douma, A. C. (1992) Biochim. Biophys. Acta 1120, 97-104], and differ in temporal expression, suggests different physiological functions for the two isoenzymes at the onset of germination. We aimed to obtain more information about these functions by studying the substrate and product specificities of both isoenzymes. Analyses of the products formed from linoleic acid confirmed that LOX-1 oxygenated at C9, and LOX-2 at C13. When testing more complex substrates, it was found that both LOX-1 and LOX-2 were capable of metabolizing esterified fatty acids. K(m) values from both isoenzymes for free fatty acids were much lower than for esterified fatty acids (7-35-fold for LOX-1 versus 2-8-fold for LOX-2). interestingly LOX-1 showed significantly higher K(m) values for esterified fatty acids than did LOX-2. This was reflected by analyses of the products formed from di- and tri-linoleoylglycerol; LOX-2 formed higher amounts of oxygenated polyunsaturated fatty acids within the esterified lipids than did LOX-1, with a corresponding larger extent of oxygenation. In order to identify potential endogenous substrates, we analyzed free and esterified lipids in total lipid extracts from barley after different periods of germination for LOX-derived products. The results indicated that esterified fatty acids were preferentially metabolized by LOX-2 activity. Analysis of the positional specificity within the lipids after alkaline hydrolysis revealed that only (13S)-hydroxy derivatives were formed, indicating the in vivo action of LOX-2. These data show that LOX-2 is capable of oxygenating storage lipids and suggest that during the onset of germination LOX-2 may be involved in oxygenation of esterified polyunsaturated fatty acids in barley seeds. We suggest that the oxygenation of these lipids precedes the onset of their catabolism and that the degradation product, (9Z,11E,13S)-13-hydroxy-octadecadienoic acid, serves as an endogenous substrate for β-oxidation and therefore as a carbon source for the growing barley embryo. Chemicals/CAS: Isoenzymes; Lipids; Lipoxygenase, EC 1.13.11.12