CRITICAL ANALYSIS OF PHOSPHOLIPID HYDROLYZING ACTIVITIES IN RIPENING TOMATO FRUITS - STUDY BY SPECTROFLUOROMETRY AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Using the spectrofluorimetric method described by Wittenauer et al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984) Biochem. Biophys. Res. Commun. 118, 894-901] for phospholipase A(2) (PLA(2)) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutant rin 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 in rin 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 (C-6-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) in Advances 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 PLA(2) activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.