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Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

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Lipids

Abstract

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A2 (PLA2) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C6-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA2 activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.

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Abbreviations

C6-NBD-PC:

1-acyl-2-{6[(7-nitro-2,1,3 benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine

LOX:

lipoxygenase

PLA:

phospholipase A

PLC:

phospholipase C

PLD:

phospholipase D

SP-HPLC:

straight-phase high-performance liquid chromatography

TLC:

thin-layer chromatography

UV:

ultraviolet

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Authors and Affiliations

  1. Laboratoire de Physiologie des Organes Végétaux après Récolte et Université Pierre et Marie Curie, 92190, Meudon, France

    Marie-Aude Rouet-Mayer, Elizabeth Simond-Côte, Jean Daussant & Claudine Thévenot

  2. Institute of Chemical Technology, 166 28, Prague, Czech Republic

    Olga Valentova

Authors
  1. Marie-Aude Rouet-Mayer
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  2. Olga Valentova
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  3. Elizabeth Simond-Côte
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  4. Jean Daussant
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  5. Claudine Thévenot
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Rouet-Mayer, MA., Valentova, O., Simond-Côte, E. et al. Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography. Lipids 30, 739–746 (1995). https://doi.org/10.1007/BF02537801

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  • DOI: https://doi.org/10.1007/BF02537801

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