Lipoxygenase activity on the plasmalemma of sunflower protoplasts and its modulation

A lipoxygenase (EC 1.13.11.12) activity on the plasmalemma of sunflower (Helianthus annuus L.) protoplasts and its possible modulation by regulatory molecules were investigated. The activity was followed as both linolenic acid-dependent conjugated diene formation and oxygen uptake. Protoplasts exhibited a lipoxygenase activity inhibited by propyl gallate, salicylhydroxamic acid and butylated hydroxytoluene, and stimulated by the addition of CaCl2, H2O2 and ATP. The stimulation by CaCl2 and H2O2 was evident up to 1.5 mM and 5 nM, respectively. Higher concentrations of H2O2 caused a lower extent of stimulation and then became inhibitory. The stimulatory effect of CaCl2 was prevented by chelators (EGTA or EDTA), a Ca2+ channel blocker (gadolinium oxide) or a Ca2+ ionophore (A23187). Negligible lipoxygenase was released from protoplasts after 6 h incubation in 3.6 mM MES-Tris (pH 5.6), 0.5 M glycine betaine and 1 mM CaCl2, whereas mechanical disruption of cell integrity, liberating soluble lipoxygenase, strongly increased such an activity. However, microsomal membranes obtained from hypocotyls still retained a part of this activity which was also recovered in a highly purified plasma membrane preparation.