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A lipoxygenase has been purified from rabbit reticulocyte‐rich anaemic blood cells. It possesses a molecular weight of 78000 and an isoelectric point of 5.5 and contains 5% neutral sugars and two iron atoms per enzyme molecule. The lipoxygenase has proved to be identical with the inhibitors of respiratory proteins described formerly. The actions of the lipoxygenase on linoleic acid, phospholipids, mitochondrial and erythrocyte membranes and electron transfer particles were studied. A special feature of the reticulocyte lipoxygenase is the suicidal character of its action on lipids. With electron transfer particles the reticulocyte lipoxygenase causes a loss of acid‐labile sulfur which accompanies respiratory inhibition; the strong respiratory inhibition is not exerted by soybean lipoxygenase. The reticulocyte lipoxygenase acts preferably on mitochondrial membranes as compared with cell membranes of the erythrocyte; erythrocyte cytosol moderates the action on mitochondrial membranes. Furthermore, the lipoxygenase reaction can concomitantly and irreversibly inactivate sulfhydryl enzymes as demonstrated with muscle glyceraldehyde‐3‐phosphate dehydrogenase. The occurrence of the lipoxygenase here described is restricted to reticulocytes; very low amounts were observed in bone marrow and no lipoxygenase was detectable in normal blood. During the course of an experimental anaemia the lipoxygenase is produced owing to superinduction in large amounts, which may persist for a long time since they escape inactivation. Preliminary evidence was obtained for the occurrence of other lipoxygenases in tissues of lung, spleen, kidney and also epithelial tumours

A comprehensive kinetic model for lipoxygenase catalysis is proposed which includes the simultaneous occurrence of dioxygenase and hydroperoxidase activities and is based on the assumption of a single binding site for substrate fatty acid and product.The aerobic reaction of purified lipoxygenase from rabbit reticulocytes with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate was studied.The rate constants and the dissociation constants of this enzyme were calculated for the model from progress curves; the model describes correctly the experimental data.The following kinetic features of the reticulocyte enzyme are assumed to apply generally to lipoxygenases. As predicted from the model it was found that at low concentrations of oxygen the regio-and stereospecificities of the dioxygenation are diminished.During the autoactivation phase the steady-state approximation does not hold.Animal lipoxygenases (EC 1.1 3.1 1.12) have acquired particular interest owing to their key role in the biosynthesis of leukotrienes and other biologically active products of their action. So far research on their actions has been focussed on their positional and stereospecificity [l] ; mechanistic studies on their kinetics have hardly been performed. The situation is different with respect to plant enzymes, in particular soybean lipoxygenase, the kinetics of which has been studied extensively [2 -61. We undertook to set up a viable kinetic model for the reticulocyte lipoxygenase which may be considered to be a prototype of the animal lipoxygenases; this enzyme is available in purified form, sufficient amounts and has been the object of some mechanistic work by us [7]. In view of the many properties shared by plant and animal lipoxygenases we scrutinized the models proposed so far for the soybean enzyme as to their applicability to the reticulocyte enzyme and found them to be generally unsatisfactory in two respects.a is difficult to reconcile with the successful and well-tested onesite model for the anaerobic lipohydroperoxidase activity of soybean lipoxygenase [6]. A transition from a one-site catalysis under anaerobic conditions to a two-site catalysis under aerobic ones would imply either the existence of different active sites for hydroperoxidase and dioxygenase activity or the unmasking or forming of a second site by dioxygen.Therefore we tried to elaborate a one-site model which will describe both dioxygenase and concomitant hydroperoxidase activities of lipoxygenase using the kinetic model of Verhagen et al. [6] as starting point. This model and some of its predictions were tested on the reticulocyte lipoxygenase with 9,12(Z,Z)-octadecadienoic acid (linoleic acid) as substrate.The model elaborated is assumed to be applicable to study the kinetic behaviour of other lipoxygenases. MATERIALS AND METHODSLipoxygenase from reticulocytes was isolated and purified as described elsewhere [lo]. A peak fraction of the isoelectricfocussed enzyme was used with a specific activity of 8 s-and a protein content of 3.05mg/ml. Differen...

We have studied the expression of the 15-lipoxygenase gene in various permanent mammalian cell lines in response to interleukins-4 and -13, and found that none of the cell lines tested expressed 5-, 12- or 15-lipoxygenase when cultured under standard conditions. However, when the lung carcinoma cell line A549 was maintained in the presence of either interleukin for 24 h or more, we observed a major induction of 15-lipoxygenase, as indicated by quantification of 15-lipoxygenase mRNA, by immunohistochemistry, by immunoblot analysis and by enzyme activity assays. This effect was 15-lipoxygenases-specific, since expression of 5- and 12-lipoxygenases remained undetectable. The time course of interleukin-4 treatment indicated maximal accumulation of both 15-lipoxygenase mRNA and functional protein after 48 h. Binding studies revealed that A549 cells express about 2100 high-affinity interleukin-4 binding sites per cell. The interleukin-4 mutant Y124D, which is capable of binding to the interleukin-4 receptor but is unable to trigger receptor activation, counteracted the effect of the wild-type cytokine. Other cell lines, including several epithelial cells and various monocytic cell lines expressing comparable numbers of interleukin-4 receptors, did not express 15-lipoxygenase when stimulated with interleukin-4. These data indicate that A549 cells selectively express 15-lipoxygenase when stimulated with interleukins-4 and -13. The activation of the interleukin-4/13 receptor(s) appears to be mandatory, but not sufficient, for 15-lipoxygenase gene expression.