LIPOXYGENASE ACTIVITY ON THE PLASMALEMMA OF SUNFLOWER PROTOPLASTS ANDITS MODULATION

A lipoxygenase (EC 1.13.11.12) activity on the plasmalemma of sunflowe r (Helianthus annuus L.) protoplasts and its possible modulation by re gulatory molecules were investigated. The activity was followed as bot h linolenic acid-dependent conjugated diene formation and oxygen uptak e. Protoplasts exhibited a lipoxygenase activity inhibited by propyl g allate, salicylhydroxamic acid and butylated hydroxytoluene, and stimu lated 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 conc entrations of H2O2 caused a lower extent of stimulation and then becam e inhibitory. The stimulatory effect of CaCl2 was prevented by chelato rs (EGTA or EDTA), a Ca2+ channel blocker (gadolinium oxide) or a Ca2 ionophore (A(23187)). Negligible lipoxygenase was released from proto plasts 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 integri ty, liberating soluble lipoxygenase, strongly increased such an activi ty. However, microsomal membranes obtained from hypocotyls still retai ned a part of this activity which was also recovered in a highly purif ied plasma membrane preparation.