On the nature of the stimulation of the lipoxygenase from rabbit reticulocytes by biological membranes

Abstract

The oxygenation of concentrated emulsions (about 260 microM) of arachidonic acid or linoleic acid catalyzed by the purified lipoxygenase from rabbit reticulocytes is strongly stimulated in the presence of low concentrations of beef heart submitochondrial particles or other membrane preparations. Maximal stimulation was observed at a proportion of about 5 mumoles polyenoic acid per mg of membrane protein. Whereas at a constant ratio of arachidonic acid and membranes the reaction rate obeyed the Michaelis-Menten kinetics with an apparent Km value of 75 microM for arachidonic acid, sigmoid kinetics was observed under conditions of constant concentration of membranes and, consequently, varying proportions of arachidonate and membranes. Under conditions of maximal stimulation binding of reticulocyte lipoxygenase to the membranes was insignificant. Moreover, one-third of the arachidonic acid sedimented during ultracentrifugation of the membranes as judged from experiments with [14C] arachidonic acid. From other sedimentation experiments and from the comparison with soybean lipoxygenase which is not stimulated by membranes it is concluded that the emulsion droplets interacting with the membranes are the substrate of the stimulated reaction. The stimulation may be brought about by facilitating the susceptibility of the fatty acids to reticulocyte lipoxygenase. The membrane-stimulated reaction of reticulocyte lipoxygenase was not inhibited by the antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT) excluding the participation of free radicals. The stimulatory effect of membranes seems not to be related to their susceptibility towards lipoxygenase attack which varied strongly in order mitochondrialmembranes greater than endoplasmic membranes greater than erythrocyte ghosts. The stimulation by membranes resembles that produced by the detergent sodium cholate at concentrations near to the critical micellar concentration. Moreover, the stimulations by membranes and by cholate do not behave synergistically or abolish each other. The stimulatory effect of membranes did not occur at a proportion of 200 nmoles per mg of membrane protein.