Oxidative modification converts low-density lipoprotein (LDL) into its atherogenic form and appears to be a necessary precondition for LDL uptake by macrophages during foam cell formation. Cellular lipoxygenases have been implicated in this process. We studied the interaction of purified mammalian lipoxygenases with human LDL in v i m and found that the arachidonate 15-lipoxygenases of rabbit and man are capable of oxygenating lipoproteins as indicated by oxygen uptake and by the formation of thiobarbituric-acid-reactive substances. Furthermore, oxygenated polyenoic fatty acids, such as 13-hydro(pero)xy-9Z, 11 E-octadecadienoic acid and 15-hydro(pero)xy-5,8,11,13 (Z,Z, Z,E)-eicosatetraenoic acid were detected in the lipid compartment of various lipoproteins classes after lipoxygenase treatment. More than 90% of the oxygenated polyenoic fatty acids were found in the ester-lipid fraction, particularly in the cholesterol esters, whereas only small amounts of free hydro(pero)xy polyenoic fatty acids were detected. Lipoxygenase-catalyzed oxygenation of LDL is not restricted to the lipid compartment but also leads to a cooxidative modification of the apoproteins as indicated by changes in the electrophoretic mobility and by the formation of carbonyl derivatives of amino acid side chains. The possible biological significance of lipoxygenase-induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.Atherosclerosis is a multifactoral disease, the pathogenesis of which is not fully understood [l-31. It has been shown by morphological studies that the accumulation of lipid-loaded foam cells in the subendothelial space leads to the formation of fatty streaks, which are generally accepted as the early atherosclerotic lesions [l -41. Foam cells develop from monocyte-derived macrophages [5, 61 or from smooth muscle cells [7] by taking up modified low-density lipoproteins (LDL) via scavenger-receptor(s)-mediated pathways. The occurrence of oxidatively modified proteins in atherosclerotic lesions [8, 91 and the fact that oxidatively modified LDL is rapidly taken up by macrophages [lo] suggested an involvement of oxidative processes in the pathogenesis of atherosclerosis. However, the mechanism of oxidative modification in vivo remains unclear. In-vitro studies with cell-free systems indicated that copper-mediated oxidation converts LDL into its atherogenic form [ll] The oxygenation of lipoproteins by mammalian lipoxygenases has not been studied so far. The soybean lipoxygenase which largely differs from mammalian lipoxygenases with respect to its protein chemical and enzymic properties is capable of oxidizing LDL only in the presence of phospholipase A, which provides free polyenoic fatty acids by cleaving lipoprotein phospholipids [20]. This result is not surprising since the soybean lipoxygenase has been shown to effectively oxygenate ester lipids only in the presence of detergents [21]. However, the rabbit reticulocyte lipoxygenase is capable of oxygenating complex ester lipids such ...

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

Mammalian lipoxygenases have been implicated in inflammation and atherosclerosis and, thus, lipoxygenase inhibitors may be of pharmacological interest. In cells, lipoxygenases occur in a catalytically silent ground state that requires activation to become active. We found that the seleno-organic drug ebselen [2-phenyl-1, 2-benzisoselenazol-3(2H)-one], which exhibits anti-inflammatory properties, irreversibly inhibited pure rabbit 15-lipoxygenase, with an IC50 in the nM range when preincubated with the enzyme in the absence of fatty acid substrates. Subsequent dialysis, gel filtration, or substrate addition did not restore the enzyme activity, and experiments with [14C]ebselen indicated a covalent linkage of the drug. The presence of sulfhydryl compounds in the incubation mixture prevented both enzyme labeling and inactivation, but we did not see any reactivation when sulfhydryl compounds were added afterward. X-ray absorption studies indicated that ebselen did alter the geometry of the iron ligand sphere, and the data are consistent with an iron complexation by the drug. When fatty acid substrate was present during lipoxygenase-ebselen interaction, the inhibitory potency was strongly reduced and a competitive mode of action was observed. These data suggest that ebselen inactivated the catalytically silent ground-state lipoxygenase irreversibly by covalent linkage and alteration of the iron ligand sphere. In contrast, it functions as a competitive inhibitor of the catalytically active enzyme species. The pharmacological relevance of ebselen as a potential in vivo lipoxygenase inhibitor will be discussed.